2.2.1 Through-space multidimensional correlation spectroscopy

Through-space correlation experiments rely on distance-dependent internuclear dipolar couplings (D_IS ∝ γ _I γ _S/r³). Observed correlations can be short or long range, depending on the chosen pulse sequence and experimental parameters (e.g., mixing time). Early through-space correlation experiments were optimized for MAS frequencies of 10–30 kHz. With advances in probe technology and faster spinning speeds, methods have been developed to achieve efficient polarization transfer at higher MAS rates. Common through-space homonuclear correlation experiments optimized for the slower spinning regime (10–30 kHz) include dipolar-assisted rotational resonance (DARR) (Takegoshi et al. Reference Takegoshi, Nakamura and Terao2001), RF-assisted diffusion (RAD) (Morcombe et al. Reference Morcombe, Gaponenko, Byrd and Zilm2004), proton-driven spin diffusion (PDSD) (Szeverenyi et al. Reference Szeverenyi, Sullivan and Maciel1982), and dipolar recoupling enhanced by amplitude modulation (DREAM) (Verel et al. Reference Verel, Ernst and Meier2001) to obtain ¹³C–¹³C correlations. Some applications of note on biological assemblies include PDSD for resonance assignments and structure determination of the type III secretion system (T3SS) needle (Demers et al. Reference Demers, Habenstein, Loquet, Vasa, Giller, Becker, Baker, Lange and Sgourakis2014; Loquet et al. Reference Loquet, Lv, Giller, Becker and Lange2011), BacA filament (Shi et al. Reference Shi, Fricke, Lin, Chevelkov, Wegstroth, Giller, Becker, Thanbichler and Lange2015; Vasa et al. Reference Vasa, Lin, Shi, Habenstein, Riedel, Kuhn, Thanbichler and Lange2015), HET-s amyloid (Wasmer et al. Reference Wasmer, Lange, Van Melckebeke, Siemer, Riek and Meier2008), and DARR for detection of the Pf1 bacteriophage DNA signals (Sergeyev et al. Reference Sergeyev, Day, Goldbourt and Mcdermott2011) and characterization of the human immunodeficiency virus 1 (HIV-1) capsid and CA-SP1 maturation intermediate (Han et al. Reference Han, Ahn, Concel, Byeon, Gronenborn, Yang and Polenova2010, Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). For determination of heteronuclear NCA, NCO, NCACX, and NCOCX correlations, ¹⁵N–¹³C double cross-polarization (DCP), first presented by Schaefer et al. (Reference Schaefer, Mckay and Stejskal1979), is commonly employed. Baldus et al. developed frequency-selective DCP (known as SPECIFIC-CP) (Baldus et al. Reference Baldus, Petkova, Herzfeld and Griffin1998) for selective NCA or NCO excitation, as demonstrated for resonance assignments of SH3 (Pauli et al. Reference Pauli, Baldus, Van Rossum, De Groot and Oschkinat2001). SPECIFIC-CP has been shown to be broadly applicable (Luca et al. Reference Luca, Heise and Baldus2003). Other recoupling sequences such as dipolar insensitive nuclei enhanced by polarization transfer (INEPT) for selective C–H excitation at both moderate (De Vita & Frydman, Reference De Vita and Frydman2001; Wickramasinghe et al. Reference Wickramasinghe, Shaibat, Jones, Casabianca, De Dios, Harwood and Ishii2008) and fast (Holland et al. Reference Holland, Cherry, Jenkins and Yarger2010) spinning speeds have been also reported.

At MAS frequencies faster than 30 kHz, the conventional spin diffusion-based experiments for recording homonuclear correlations are no longer efficient. Under these conditions, DREAM and fpRFDR (finite pulse rf driven recoupling (Ishii, Reference Ishii2001)) are efficient for recording one- and two-bond correlations. Another family of experiments that is particularly useful for recording long-range ¹³C–¹³C distance restraints is COmbined R2 _n ^ν -Driven (CORD) dipolar recoupling sequences, where the magnetization transfer is driven by rotor-synchronized R2 _n ^ν symmetry-based recoupling (Hou et al. Reference Hou, Yan, Sun, Han, Byeon, Ahn, Concel, Samoson, Gronenborn and Polenova2011a, Reference Hou, Yan, Trebosc, Amoureux and Polenova2013a; Lu et al. Reference Lu, Guo, Hou and Polenova2015b). The RN _n ^ν and CN _n ^ν symmetry recoupling schemes were originally presented by Levitt and co-workers (Carravetta et al. Reference Carravetta, Eden, Zhao, Brinkmann and Levitt2000). CORD utilizes a super-cycled R2 _n ^ν recoupling to achieve broadband homonuclear correlations with high polarization transfer efficiency at both moderate and fast MAS rates while not suffering from dipolar truncation effects. Proton-assisted recoupling (PAR) is another method that performs well at fast MAS to obtain long distance ¹³C–¹³C (De Paepe et al. Reference De Paepe, Lewandowski, Loquet, Bockmann and Griffin2008; Lewandowski et al. Reference Lewandowski, De Paepe, Eddy, Struppe, Maas and Griffin2009b) or ¹⁵N–¹⁵N (Lewandowski et al. Reference Lewandowski, De Paepe, Eddy and Griffin2009a) correlations. PAR is based on third spin assisted recoupling (TSAR), in which two spins are connected via dipolar couplings with a third spin leading to zero quantum (ZQ) polarization transfer. Distances of ~6–7 Å can be observed with PAR and CORD.

Beyond DCP, several methods have been developed for the acquisition of long range ¹⁵N–¹³C correlations. PAIN-CP (proton-assisted insensitive nuclei cross-polarization) is a third-spin-assisted heteronuclear polarization transfer (Agarwal et al. Reference Agarwal, Sardo, Scholz, Bockmann, Ernst and Meier2013; De Paepe et al. Reference De Paepe, Lewandowski, Loquet, Eddy, Megy, Bockmann and Griffin2011; Lewandowski et al. Reference Lewandowski, De Paepe and Griffin2007) first presented by Griffin and co-workers, which like its homonuclear counterpart discussed above, utilizes neighboring proton spins to enhance magnetization transfer efficiency with appropriate choice of ¹³C, ¹⁵N, and ¹H rf fields. Transferred echo double resonance (TEDOR) (Hing et al. Reference Hing, Vega and Schaefer1992) is a REDOR (rotational echo double resonance (Gullion & Schaefer, Reference Gullion and Schaefer1989)) derived scheme that can also be used to detect ¹⁵N–¹³C distances up to ~8 Å. In REDOR-based pulse sequences, the dipolar coupling between two spins is reintroduced by a train of rotor-synchronized 180° pulses (Gullion & Schaefer, Reference Gullion and Schaefer1989). The resulting dephasing of magnetization is proportional to the magnitude of the dipolar coupling (and hence distance between the two spins). A variation of the TEDOR pulse sequence developed by Jaroniec et al. (Reference Jaroniec, Filip and Griffin2002), z-filtered TEDOR, is shown in Fig. 2e . The inclusion of a z-filter is needed to eliminate artifacts due to ¹³C–¹³C J couplings in uniformly labeled systems. TEDOR-derived distance restraints have been applied to a range of systems including structure determination of microcrystalline GB1 by Rienstra and co-workers (Nieuwkoop et al. Reference Nieuwkoop, Wylie, Franks, Shah and Rienstra2009) and L7Ae-bound Box C/D RNA by Carlomagno and co-workers (Marchanka et al. Reference Marchanka, Simon, Althoff-Ospelt and Carlomagno2015). Pulse sequences, schematics, and model compound spectra for several through-space correlation methods are presented in Fig. 2.

Fig. 2. (a) Schematic representation for homonuclear and heteronuclear third spin assisted recoupling, a second-order mechanism, which uses the dipolar couplings with a third spin to achieve magnetization transfer (De Paepe et al. Reference De Paepe, Lewandowski, Loquet, Eddy, Megy, Bockmann and Griffin2011). (b) Pulse sequence for 2D ¹⁵N–¹³C PAIN–CP heteronuclear correlation experiment (De Paepe et al. Reference De Paepe, Lewandowski, Loquet, Eddy, Megy, Bockmann and Griffin2011). (c) 2D homonuclear PAR pulse sequence (De Paepe et al. Reference De Paepe, Lewandowski, Loquet, Bockmann and Griffin2008). (d) ¹⁵N–¹³C correlation spectra of MLF: (top) DCP, (bottom) PAIN–CP, demonstrating the more efficient magnetization transfer of PAIN-CP (Lewandowski et al. Reference Lewandowski, De Paepe and Griffin2007). (e) Pulse sequence for ¹⁵N–¹³C heteronuclear z-filtered TEDOR correlations (Jaroniec et al. Reference Jaroniec, Filip and Griffin2002). Shaded portions indicate z-filters incorporated to eliminate artifacts arising ¹³C–¹³C J couplings in uniformly labeled samples. (a, b) Reprinted with permission from De Paepe et al. (Reference De Paepe, Lewandowski, Loquet, Eddy, Megy, Bockmann and Griffin2011). Copyright 2011 AIP Publishing. (c) Reprinted with permission from De Paepe et al. (Reference De Paepe, Lewandowski, Loquet, Bockmann and Griffin2008). Copyright 2008 AIP Publishing. (d) Reprinted with permission from Lewandowski et al. (Reference Lewandowski, De Paepe and Griffin2007). Copyright 2007 American Chemical Society. (e) Reprinted with permission from Jaroniec et al. (Reference Jaroniec, Filip and Griffin2002). Copyright 2002 American Chemical Society.

2.2.2 Through-bond multidimensional correlation spectroscopy

Complementary to through-space, dipolar-based correlation experiments, scalar-based through-bond transfer mechanisms can be exploited to obtain inter-nuclear correlations. Through-bond experiments utilize the electron-mediated J coupling between neighboring atoms. This transfer mechanism can be especially valuable in cases of dynamics (Heise et al. Reference Heise, Hoyer, Becker, Andronesi, Riedel and Baldus2005) and at fast MAS frequencies, situations where dipolar couplings are partially or fully averaged. J-based experiments are also ideal at faster spinning frequencies due to the lower required decoupling power, and can allow for the necessary longer coherence evolution times (Bertini et al. Reference Bertini, Emsley, Felli, Laage, Lesage, Lewandowski, Marchetti, Pierattelli and Pintacuda2011a). Experiments such as heteronuclear (Elena et al. Reference Elena, Lesage, Steuernagel, Bockmann and Emsley2005) or homonuclear (Linser et al. Reference Linser, Fink and Reif2008) INEPT (Morris & Freeman, Reference Morris and Freeman1979), homonuclear total through-bond correlation spectroscopy (TOBSY) (Hardy et al. Reference Hardy, Verel and Meier2001), homonuclear constant-time uniform sign cross-peak COSY (CTUC-COSY), (Chen et al. Reference Chen, Olsen, Elliott, Boettcher, Zhou, Rienstra and Mueller2006), as well as solid-state INADEQUATE (Lesage et al. Reference Lesage, Auger, Caldarelli and Emsley1997) and refocused INADEQUATE (Lesage et al. Reference Lesage, Bardet and Emsley1999) have been used to complement dipolar-based correlation spectroscopy in the study of protein assemblies (Fig. 3). With these methods, sufficient sensitivity is attained despite the relatively small size of the J-couplings (e.g. 50 Hz ¹³C–¹³C J coupling versus 2 kHz dipolar coupling). TOBSY experiments utilize the POST-C7 symmetry sequence (Hohwy et al. Reference Hohwy, Jakobsen, Eden, Levitt and Nielsen1998) to achieve efficient, scalar-based polarization transfer. CTUC-COSY offers excellent sensitivity by converting both zero-quantum and double-quantum magnetization, and has been applied to detect dynamic regions of the Y145Stop human prion protein (Helmus et al. Reference Helmus, Surewicz, Surewicz and Jaroniec2010) and α-Synuclein fibrils (Comellas et al. Reference Comellas, Lemkau, Nieuwkoop, Kloepper, Ladror, Ebisu, Woods, Lipton, George and Rienstra2011), as well as to obtain pure one-bond correlations in ¹³C–¹³C spectra of CAP-Gly (Sun et al. Reference Sun, Siglin, Williams and Polenova2009). INADEQUATE experiments in the solid state use double-quantum coherence transfer identical to solution NMR. More recent modifications of solid-state INADEQUATE have included the addition of a z-filter (Cadars et al. Reference Cadars, Sein, Duma, Lesage, Pham, Baltisberger, Brown and Emsley2007) and FSLG (frequency-switched Lee–Goldberg) homonuclear ¹H–¹H decoupling (Baltisberger et al. Reference Baltisberger, Musapelo, Sutton, Reynolds and Gurung2011) to reduce artifacts, and development of band-selective INADEQUATE using the spin state selective excitation (S³E) scheme, which has been demonstrated at 60 kHz MAS (Bertini et al. Reference Bertini, Emsley, Felli, Laage, Lesage, Lewandowski, Marchetti, Pierattelli and Pintacuda2011a). The use of scalar transfers in proton-detected experiments at fast MAS has recently been demonstrated in the solid state as well including ¹³C–¹³C INEPT transfer for resonance assignments of superoxide dismutase (SOD) (Knight et al. Reference Knight, Webber, Pell, Guerry, Barbet-Massin, Bertini, Felli, Gonnelli, Pierattelli, Emsley, Lesage, Herrmann and Pintacuda2011). Pintacuda and co-workers reported the application of ‘out-and-back’ ¹³C–¹³C scalar-based transfer for resonance assignments with fast MAS (frequencies of 60 kHz and higher (Barbet-Massin et al. Reference Barbet-Massin, Pell, Jaudzems, Franks, Retel, Kotelovica, Akopjana, Tars, Emsley, Oschkinat, Lesage and Pintacuda2013)). These proton-detected 3D experiments can be applied to both fully protonated samples as well as perdeuterated samples with 100% H^N back exchange, and were demonstrated on AP205 bacteriophage as well as numerous other diverse classes of proteins (Barbet-Massin et al. Reference Barbet-Massin, Pell, Retel, Andreas, Jaudzems, Franks, Nieuwkoop, Hiller, Higman, Guerry, Bertarello, Knight, Felletti, Le Marchand, Kotelovica, Akopjana, Tars, Stoppini, Bellotti, Bolognesi, Ricagno, Chou, Griffin, Oschkinat, Lesage, Emsley, Herrmann and Pintacuda2014b).

Fig. 3. Scalar-based correlation experiments frequently used in the solid state. (a) Heteronuclear ¹H−¹³C INEPT pulse sequence (Elena et al. Reference Elena, Lesage, Steuernagel, Bockmann and Emsley2005), (b) homonuclear ¹³C–¹³C TOBSY pulse sequence (Hardy et al. Reference Hardy, Verel and Meier2001), (c) homonuclear ¹³C–¹³C INADEQUATE pulse sequences, (top) solid-state INADEQUATE, (bottom) refocused INADEQUATE (Lesage et al. Reference Lesage, Bardet and Emsley1999). (d) ¹H–¹³C INEPT (black) and ¹³C–¹³C INEPT-TOBSY spectra (green) of HET-s amyloids (Wasmer et al. Reference Wasmer, Schutz, Loquet, Buhtz, Greenwald, Riek, Bockmann and Meier2009). (e) Direct (black) and CP (orange) INADEQUATE spectra of CA-SP1 tubular assemblies (Han et al. Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). (a) Adapted with permission from Elena et al. (Reference Elena, Lesage, Steuernagel, Bockmann and Emsley2005). Copyright 2005 American Chemical Society. (b) Adapted with permission from Hardy et al. (Reference Hardy, Verel and Meier2001). Copyright 2001 Elsevier. (c) Reprinted with permission from Lesage et al. (Reference Lesage, Bardet and Emsley1999). Copyright 1999 American Chemical Society. (d) Reprinted with permission from Wasmer et al. (Reference Wasmer, Schutz, Loquet, Buhtz, Greenwald, Riek, Bockmann and Meier2009). Copyright 2009 Elsevier. (e) Reprinted with permission from Han et al. (Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). Copyright 2013 American Chemical Society.

2.2.3 Proton detection and fast MAS

In contrast to solution NMR where dipolar couplings are averaged out by molecular tumbling, the strong ¹H–¹H dipolar couplings present in solid-state NMR (SSNMR) samples lead to very broad ¹H lines. As a consequence, MAS NMR experiments have customarily been acquired with direct detection of low γ nuclei such as ¹³C and ¹⁵N, which greatly limits sensitivity. Proton detection takes advantage of the high gyromagnetic ratio of protons for increased sensitivity and with advances in hardware is increasingly applied in SSNMR. Early work by Reif, Griffin, and Zilm demonstrated that with perdeuteration to reduce ¹H–¹H dipolar couplings and 100% amide ¹H-back exchange, proton-detected HETCOR experiments could be applied in the solid state and that the anticipated sensitivity gains are realized, while dipolar truncation is avoided (Paulson et al. Reference Paulson, Morcombe, Gaponenko, Dancheck, Byrd and Zilm2003; Reif & Griffin, Reference Reif and Griffin2003; Reif et al. Reference Reif, Jaroniec, Rienstra, Hohwy and Griffin2001). Subsequent work demonstrated that increased ¹H resolution can be achieved with higher levels of deuteration (i.e., only 10–40% ¹H back exchange) (Akbey et al. Reference Akbey, Lange, Franks, Linser, Rehbein, Diehl, Van Rossum, Reif and Oschkinat2010) and/or faster MAS frequencies (Chevelkov et al. Reference Chevelkov, Rehbein, Diehl and Reif2006; Samoson et al. Reference Samoson, Tuherm and Gan2001). Linser et al. first demonstrated the application of ¹H detection to amyloids and membrane proteins (Linser et al. Reference Linser, Dasari, Hiller, Higman, Fink, Del Amo, Markovic, Handel, Kessler, Schmieder, Oesterhelt, Oschkinat and Reif2011b). With the advent of fast MAS (⩾40 kHz), proton-detection even on fully protonated proteins, first demonstrated by Rienstra and co-workers (Zhou et al. Reference Zhou, Shah, Cormos, Mullen, Sandoz and Rienstra2007a), has become feasible with improvements in resolution and sensitivity scaling with the MAS rate (Agarwal et al. Reference Agarwal, Penzel, Szekely, Cadalbert, Testori, Oss, Past, Samoson, Ernst, Bockmann and Meier2014; Lewandowski et al. Reference Lewandowski, Dumez, Akbey, Lange, Emsley and Oschkinat2011a; Marchetti et al. Reference Marchetti, Jehle, Felletti, Knight, Wang, Xu, Park, Otting, Lesage, Emsley, Dixon and Pintacuda2012). Further, the sensitivity gains of ¹H detection enable the use of very small sample amounts (Agarwal et al. Reference Agarwal, Penzel, Szekely, Cadalbert, Testori, Oss, Past, Samoson, Ernst, Bockmann and Meier2014; Dannatt et al. Reference Dannatt, Taylor, Varga, Higman, Pfeil, Asilmovska, Judge and Watts2015). Recent works of note in the application of proton detection include studies of RNA–protein interfaces by Asami et al. (Reference Asami, Rakwalska-Bange, Carlomagno and Reif2013), structure determination of SOD by Knight et al. (Reference Knight, Pell, Bertini, Felli, Gonnelli, Pierattelli, Herrmann, Emsley and Pintacuda2012), and measurements of heteronuclear dipolar couplings in Pf1 bacteriophage by Opella and co-workers (Park et al. Reference Park, Yang, Opella and Mueller2013). Additional capabilities of ¹H detection include obtaining direct information on hydrogen-bond length from ¹H chemical shifts (Zhou & Rienstra, Reference Zhou and Rienstra2008). Pintacuda and co-workers have recently demonstrated a suite of 3D proton-detected experiments to enable rapid data acquisition and assignment, with data sets of sufficient quality for the automated assignment routines to be applicable (Barbet-Massin et al. Reference Barbet-Massin, Pell, Retel, Andreas, Jaudzems, Franks, Nieuwkoop, Hiller, Higman, Guerry, Bertarello, Knight, Felletti, Le Marchand, Kotelovica, Akopjana, Tars, Stoppini, Bellotti, Bolognesi, Ricagno, Chou, Griffin, Oschkinat, Lesage, Emsley, Herrmann and Pintacuda2014b). They demonstrated the use of these sequences on several challenging systems, including assemblies of AP205 bacteriophage and Measles virus (MeV) nucleocapsid (Barbet-Massin et al. Reference Barbet-Massin, Felletti, Schneider, Jehle, Communie, Martinez, Jensen, Ruigrok, Emsley, Lesage, Blackledge and Pintacuda2014a, Reference Barbet-Massin, Pell, Retel, Andreas, Jaudzems, Franks, Nieuwkoop, Hiller, Higman, Guerry, Bertarello, Knight, Felletti, Le Marchand, Kotelovica, Akopjana, Tars, Stoppini, Bellotti, Bolognesi, Ricagno, Chou, Griffin, Oschkinat, Lesage, Emsley, Herrmann and Pintacudab).

2.2.4 Protein structure determination by MAS NMR

Structure determination by SSNMR was first demonstrated on small peptides oriented in lipid bilayers with static methods by Cross and co-workers (Ketchem et al. Reference Ketchem, Lee, Huo and Cross1996; Wang et al. Reference Wang, Kim, Kovacs and Cross2001) and Opella and co-workers (Opella et al. Reference Opella, Marassi, Gesell, Valente, Kim, Oblatt-Montal and Montal1999) MAS NMR structure determination of a protein was first reported by Oschkinat and co-workers for the α-spectrin SH3 domain (Castellani et al. Reference Castellani, Van Rossum, Diehl, Schubert, Rehbein and Oschkinat2002). Technical and methodological advances have enabled the application of MAS NMR to structure determination of increasingly complex systems (Fig. 4a ). MAS NMR is particularly valuable for the high-resolution structure determination of supramolecular assemblies, which are often insoluble or non-crystalline. Protein structure determination by MAS NMR requires determination of a sufficient number of quantitative or semi-quantitative structural restraints including distance restraints obtained from homonuclear correlation and HETCOR spectra, using long-range magnetization transfer techniques described above, which can probe interatomic distances of up to ~7 Å (Fig. 4b ). ¹³C–¹³C distances are the most frequently utilized and often make use of selectively labeled samples for spectral simplification and semi-quantitative crosspeak intensity analysis. Additional distance restraints that have been utilized include ¹⁵N–¹³C (Nieuwkoop et al. Reference Nieuwkoop, Wylie, Franks, Shah and Rienstra2009), ¹⁵N–¹⁵N (Hu et al. Reference Hu, Qiang, Bermejo, Schwieters and Tycko2012; Lewandowski et al. Reference Lewandowski, De Paepe, Eddy and Griffin2009a), and increasingly ¹H–¹H (Andreas et al. Reference Andreas, Jaudzems, Stanek, Lalli, Bertarello, Le Marchand, Cala-De Paepe, Kotelovica, Akopjana, Knott, Wegner, Engelke, Lesage, Emsley, Tars, Herrmann and Pintacuda2016; Linser et al. Reference Linser, Bardiaux, Higman, Fink and Reif2011a; Zhou et al. Reference Zhou, Shea, Nieuwkoop, Franks, Wylie, Mullen, Sandoz and Rienstra2007b) distances. Very recently, Pintacuda and co-workers presented the first protein structures determined on fully protonated samples with ¹H detection (Andreas et al. Reference Andreas, Jaudzems, Stanek, Lalli, Bertarello, Le Marchand, Cala-De Paepe, Kotelovica, Akopjana, Knott, Wegner, Engelke, Lesage, Emsley, Tars, Herrmann and Pintacuda2016). They acquired RFDR-based ¹H–¹H distance restraints with ⩾100 kHz MAS to determine the structures of two proteins: GB1 and the AP205 nucleocapsid assembly. Less than 0·5 mg of U–¹⁵N, ¹³C protein and 2 weeks of experiment time and ‘unsupervised’ structure determination were sufficient to derive the protein structures.

Fig. 4. (a) PDB structures determined by solid-state NMR each year. Blue indicates structures determined by MAS NMR alone while orange indicates structures determined with an integrated approach, including methods such as electron microscopy or solution NMR in addition to SSNMR data. Year 2015 includes structures deposited through February 2016. (b) Contact map of MT-associated CAP-Gly illustrating all intra- and inter-residue correlations observed from MAS NMR distance restraints used in the structure calculation (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). (b) Adapted with permission from Yan et al. (Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). Copyright 2015 National Academy of Sciences.

In addition to inter-atomic distance restraints, anisotropic spin interactions including dipolar and chemical shift tensor magnitudes and orientations are a powerful tool for protein structure determination. These interactions exhibit secondary structure, orientation, and amino acid type dependence that can be exploited in structure determination, demonstrated extensively by Rienstra and co-workers on GB1 (Franks et al. Reference Franks, Wylie, Schmidt, Nieuwkoop, Mayrhofer, Shah, Graesser and Rienstra2008; Wylie et al. Reference Wylie, Schwieters, Oldfield and Rienstra2009, Reference Wylie, Sperling, Nieuwkoop, Franks, Oldfield and Rienstra2011). In structure calculations, dipolar couplings and CSA tensors can constrain backbone torsion angles (Ladizhansky et al. Reference Ladizhansky, Jaroniec, Diehl, Oschkinat and Griffin2003; Rienstra et al. Reference Rienstra, Tucker-Kellogg, Jaroniec, Hohwy, Reif, Mcmahon, Tidor, Lozano-Perez and Griffin2002) as demonstrated on Aβ _11–25 with recoupling of chemical shift anisotropy (ROCSA) measurements (Chan & Tycko, Reference Chan and Tycko2003). Recent applications include the use of ¹H–¹⁵N and ¹H–¹³Cα dipolar couplings determined from separated local field (SLF) measurements (Das et al. Reference Das, Lin and Opella2013) as orientation restraints (with dihedral angles derived from isotropic chemical shifts) in determining the structure of CXCR1, a chemokine receptor involved in inflammatory response ((Park et al. Reference Park, Das, Casagrande, Tian, Nothnagel, Chu, Kiefer, Maier, De Angelis, Marassi and Opella2012), Fig. 5). Anisotropic spin interactions can also provide valuable dynamics information, as further discussed in Section 2.3. Additional structural restraints that may be incorporated into a structure calculation include predicted torsion angles based on backbone chemical shifts from TALOS (Shen & Bax, Reference Shen and Bax2015; Shen et al. Reference Shen, Delaglio, Cornilescu and Bax2009), hydrogen bonding, and paramagnetic relaxation enhancements (PREs, (Nadaud et al. Reference Nadaud, Helmus, Hofer and Jaroniec2007)). PREs utilize the enhanced R₁ relaxation of residues in close proximity to a paramagnetic center as a structural restraint and were applied to structure determination of SOD (Knight et al. Reference Knight, Pell, Bertini, Felli, Gonnelli, Pierattelli, Herrmann, Emsley and Pintacuda2012) and the membrane protein Anabaena sensory rhodopsin (ASR) (Wang et al. Reference Wang, Munro, Shi, Kawamura, Okitsu, Wada, Kim, Jung, Brown and Ladizhansky2013). Beyond intra-subunit restraints, long-range distances, such as those acquired in zf-TEDOR experiments, can contribute inter-subunit restraints for structure determination of supramolecular assemblies (Nieuwkoop & Rienstra, Reference Nieuwkoop and Rienstra2010).

Fig. 5. Structure determination of CXCR1 with dipolar couplings as a structural restraint (Park et al. Reference Park, Das, Casagrande, Tian, Nothnagel, Chu, Kiefer, Maier, De Angelis, Marassi and Opella2012). (a) CO–Cα correlations from NCACX 3D. (b) Strip plots from SLF measurements, indicating the ¹H–¹⁵N dipolar coupling strength at a given ¹³Cα chemical shift, corresponding to the residues indicated. (c) ¹H–¹⁵N dipolar coupling versus residue number. The ‘wave’ pattern (cyan) is a feature of the transmembrane helices. (d) 10 lowest energy structures of CXCR1. Adapted with permission from Park et al. (Reference Park, Das, Casagrande, Tian, Nothnagel, Chu, Kiefer, Maier, De Angelis, Marassi and Opella2012). Copyright 2012 Nature Publishing Group.

Structural restraints are incorporated in simulated annealing calculations in a program such as Xplor-NIH (Schwieters et al. Reference Schwieters, Kuszewski, Tjandra and Clore2003, Reference Schwieters, Kuszewski and Clore2006) or CYANA (Guntert, Reference Guntert2004), with optimization protocols, which include molecular dynamics (MD) and Monte Carlo simulations. Recently, multiple laboratories have demonstrated the use of de novo structure prediction based on isotropic chemical shifts and amino acid sequence with CS-ROSETTA (Das et al. Reference Das, Andre, Shen, Wu, Lemak, Bansal, Arrowsmith, Szyperski and Baker2009; Shen et al. Reference Shen, Lange, Delaglio, Rossi, Aramini, Liu, Eletsky, Wu, Singarapu, Lemak, Ignatchenko, Arrowsmith, Szyperski, Montelione, Baker and Bax2008), without requiring distance restraints. CS-ROSETTA has been incorporated into structure determination of the biological assemblies T3SS (Demers et al. Reference Demers, Habenstein, Loquet, Vasa, Giller, Becker, Baker, Lange and Sgourakis2014; Loquet et al. Reference Loquet, Sgourakis, Gupta, Giller, Riedel, Goosmann, Griesinger, Kolbe, Baker, Becker and Lange2012) and the M13 bacteriophage (Morag et al. Reference Morag, Sgourakis, Baker and Goldbourt2015). (Whether this approach is generally applicable to a wide range of systems remains to be investigated.) Rosetta enables the modeling of symmetric macromolecular assemblies and has been a key development for the atomic-resolution structure determination of these large and complex systems (DiMaio et al. Reference Dimaio, Leaver-Fay, Bradley, Baker and Andre2011).

The capabilities of MAS NMR for structure determination have been further expanded in recent years by the application of integrated structure determination, wherein MAS NMR restraints are combined with other biophysical methods, such as EM, solution NMR, and MD simulations. While exact approaches may differ, in general, the secondary structure as determined from secondary chemical shifts or high-resolution monomeric structure is mapped into the lower resolution electron density map (typically by rigid body modeling), and this structure is further refined with simulated annealing, using structure restraints such as cryo-EM structure factors and NMR distance restraints. Fig. 6e illustrates the iterative protocol. Lower-resolution microscopy can provide information on the symmetry and macromolecular organization, while MAS NMR data provide atomic-level structural details including inter-subunit contacts. This approach is proving to be particularly auspicious for the study of macromolecular assemblies including structure determination of αB-crystallin (with small-angle X-ray scattering (SAXS), MD (Jehle et al. Reference Jehle, Rajagopal, Bardiaux, Markovic, Kuhne, Stout, Higman, Klevit, Van Rossum and Oschkinat2010)), T3SS (with cryo-EM (Demers et al. Reference Demers, Habenstein, Loquet, Vasa, Giller, Becker, Baker, Lange and Sgourakis2014; Loquet et al. Reference Loquet, Sgourakis, Gupta, Giller, Riedel, Goosmann, Griesinger, Kolbe, Baker, Becker and Lange2012)), DsbB (with X-ray crystallography, MD (Tang et al. Reference Tang, Nesbitt, Sperling, Berthold, Schwieters, Gennis and Rienstra2013)), FimA (with solution NMR, STEM (scanning transmission electron microscopy) (Habenstein et al. Reference Habenstein, Loquet, Hwang, Giller, Vasa, Becker, Habeck and Lange2015)), and the mouse ASC inflammasome (with cryo-EM, Fig. 6 (Sborgi et al. Reference Sborgi, Ravotti, Dandey, Dick, Mazur, Reckel, Chami, Scherer, Huber, Bockmann, Egelman, Stahlberg, Broz, Meier and Hiller2015)).

Fig. 6. Combined use of MAS NMR and cryo-EM to determine the structure of the mouse ASC inflammasome (ASC-PYD) (Sborgi et al. Reference Sborgi, Ravotti, Dandey, Dick, Mazur, Reckel, Chami, Scherer, Huber, Bockmann, Egelman, Stahlberg, Broz, Meier and Hiller2015). (a) Electron density map determined by cryo-EM. (b) Strips from ¹³C–¹³C–¹³C 3D. (c) Strips from ¹³C–¹³C 2D (top) and CHHC 2D (bottom). (d) Secondary chemical shift plot, indicating the predominantly α-helical content of the protein. (e) Flow chart illustrating the protocol for structure refinement. MAS NMR data contributions are shaded yellow and cryo-EM data are shaded green. (f) Cryo-EM density reconstruction superimposed with a monomer of ASC-PYD. (g, h) Superposition of the 20 lowest energy structures of the filament and monomer. Positions of 10 arbitrary residues as determined by structure refinement are shown in orange. (i) Inter-residue interactions in a monomer of ASC-PYD. Orange lines indicate ambiguous distance restraints between Tyr 60, Leu 68 (orange) and neighboring residues (gray). Reprinted with permission from Sborgi et al. (Reference Sborgi, Ravotti, Dandey, Dick, Mazur, Reckel, Chami, Scherer, Huber, Bockmann, Egelman, Stahlberg, Broz, Meier and Hiller2015). Copyright 2015 National Academy of Sciences.

2.3.1 Microsecond to nanosecond timescale dynamics

Dynamic processes on the microsecond-to-nanosecond timescale include backbone fluctuations and rotation of side chain methyl groups. These motions can be observed with ¹H–¹⁵N and ¹H–¹³C dipolar as well as ¹³C, ¹⁵N, and ¹H chemical shift anisotropy (CSA) tensor measurements, and R₁ relaxation experiments. Dynamic processes on this timescale result in narrowing of the tensors below their rigid-limit value (Torchia & Szabo, Reference Torchia and Szabo1985). The rigid limit ¹H–¹⁵N^H and ¹H–¹³C ^α dipolar coupling constants are 11.34 kHz (Yao et al. Reference Yao, Vogeli, Ying and Bax2008) and 22.7 kHz (Alkaraghouli & Koetzle, Reference Alkaraghouli and Koetzle1975), respectively. Reduced ¹H–¹³C and ¹H–¹⁵N dipolar coupling strengths due to dynamics are also reflected in peak intensities in ¹H–¹³C or ¹H–¹⁵N cross polarization experiments as demonstrated by the differential ¹H–¹⁵N CP buildup for the soluble and transmembrane domains of the N-terminal FMN-binding domain of NADPH-cytochrome P450 oxidoreductase, a redox partner of cytochrome P450 (Huang et al. Reference Huang, Yamamoto, Zhang, Popovych, Hung, Im, Gan, Waskell and Ramamoorthy2014). For further characterization of microsecond-to-nanosecond dynamics, several methods that rely on quantitative measurement of T₁ relaxation, dipolar couplings, and CSA tensors have been developed. Reducing interference from strong ¹H–¹H couplings and spin diffusion have been important components in the development of methods to study microsecond to nanosecond dynamics.

Longitudinal spin-lattice relaxation (R ₁) is used to probe protein backbone mobility on the nanosecond timescale. Pseudo-quantitative R ₁ measurements were first conducted on Crh by Emsley and co-workers (Giraud et al. Reference Giraud, Bockmann, Lesage, Penin, Blackledge and Emsley2004). Quantitative R ₁ measurements have subsequently been applied to several microcrystalline systems including GB1 (Lewandowski et al. Reference Lewandowski, Sein, Sass, Grzesiek, Blackledge and Emsley2010), SH3 (Zinkevich et al. Reference Zinkevich, Chevelkov, Reif, Saalwachter and Krushelnitsky2013), and ubiquitin (Haller & Schanda, Reference Haller and Schanda2013; Schanda et al. Reference Schanda, Meier and Ernst2010), as well as the transmembrane protein ASR (Good et al. Reference Good, Wang, Ward, Struppe, Brown, Lewandowski and Ladizhansky2014), the metalloprotein SOD (Knight et al. Reference Knight, Pell, Bertini, Felli, Gonnelli, Pierattelli, Herrmann, Emsley and Pintacuda2012), and an amyloid-like fragment of the yeast prion protein Sup35p (Lewandowski et al. Reference Lewandowski, Van Der Wel, Rigney, Grigorieff and Griffin2011c). While ¹⁵N R₁ measurements are relatively straightforward, ¹³C R ₁ measurements require fast MAS (>60 kHz, (Lewandowski et al. Reference Lewandowski, Sein, Sass, Grzesiek, Blackledge and Emsley2010)) or selective labeling (Asami et al. Reference Asami, Porter, Lange and Reif2015), in order to reduce ¹³C–¹³C proton driven spin diffusion.

Dipolar chemical shift correlation (DIPSHIFT), first presented by Griffin and co-workers (Munowitz et al. Reference Munowitz, Griffin, Bodenhausen and Huang1981, Reference Munowitz, Aue and Griffin1982) and extended to slower dynamics by DeAzevedo et al. (Reference DeAzevedo, Saalwachter, Pascui, De Souza, Bonagamba and Reichert2008) is a common technique for the measurement of ¹H–¹⁵N and ¹H–¹³C dipolar couplings and characterization of microsecond-to-nanosecond timescale dynamics. In traditional DIPSHIFT experiments, the magnetization evolves under the influence of the heteronuclear dipolar coupling, while ¹H–¹H couplings are suppressed with phase-modulated Lee–Goldburg decoupling (PMLG) (Vinogradov et al. Reference Vinogradov, Madhu and Vega1999). Alternatively, DISPSHIFT-based RN-symmetry recoupling experiments can be used for the measurement of ¹H–¹⁵N and ¹H–¹³C dipolar couplings (Fig. 7, (Hou et al. Reference Hou, Byeon, Ahn, Gronenborn and Polenova2011b)). These sequences selectively reintroduce heteronuclear dipolar couplings while reducing interference from homonuclear dipolar couplings (Carravetta et al. Reference Carravetta, Eden, Zhao, Brinkmann and Levitt2000). In addition, these pulse sequences are suitable for fast MAS frequencies and can be used in fully protonated systems (Hou et al. Reference Hou, Byeon, Ahn, Gronenborn and Polenova2011b). In these R symmetry sequences, the heteronuclear dipolar coupling is reintroduced with a rotor-synchronized RN _n ^ν radio frequency pulse train applied on the proton channel. An NCA, NCO, or ¹³C–¹³C correlation dimension is incorporated for site-specific determination of dynamics. Recently a modification of the RN-DIPSHIFT experiment, phase-alternating R-symmetry (PARS), was developed (Hou et al. Reference Hou, Lu, Vega and Polenova2014). This sequence incorporates a phase-shifted RN symmetry block applied on ¹H, with π pulses applied on the X channel and efficiently suppresses effects from the ¹H CSA. Further, the series of X channel pulses refocuses the chemical shift, eliminating the need for a Hahn echo and giving the experiment inherently higher sensitivity than RN-DIPSHIFT. Under fast MAS conditions (⩾60 kHz), cross-polarization with variable contact time (CPVC) is another promising approach for characterization of motions on these timescales (Paluch et al. Reference Paluch, Pawlak, Amoureux and Potrzebowski2013, Reference Paluch, Trebosc, Nishiyama, Potrzebowski, Malon and Amoureux2015b; Zhang et al. Reference Zhang, Damron, Vosegaard and Ramamoorthy2015). This approach has been recently demonstrated for recording motions in aromatic groups in GB1 and dynein light-chain LC8 proteins (Paluch et al. Reference Paluch, Pawlak, Jeziorna, Trebosc, Hou, Vega, Amoureux, Dracinsky, Polenova and Potrzebowski2015a).

Fig. 7. RN-symmetry based sequences for the measurement of dipolar and chemical shift anisotropy lineshapes. (Hou et al. Reference Hou, Lu, Vega and Polenova2014) (a) conventional RN-based DIPSHIFT, (b) ¹H CSA recoupling with or without heteronuclear decoupling, (c) PARS, (d) constant time PARS, (e) 3D PARS for dipolar lineshapes measurements. Reproduced with permission from Hou et al. (Reference Hou, Lu, Vega and Polenova2014). Copyright 2014 AIP Publishing.

CN (Chan & Tycko, Reference Chan and Tycko2003) and RN (Hou et al. Reference Hou, Paramasivam, Byeon, Gronenborn and Polenova2010, Reference Hou, Byeon, Ahn, Gronenborn and Polenova2012, Reference Hou, Paramasivam, Yan, Polenova and Vega2013b) symmetry sequences for measurement of chemical shift tensors have also been established (dubbed ROCSA and RNCSA, respectively). Like their dipolar counterparts, these experiments can be used under fast MAS and in fully protonated, uniformly ¹³C enriched systems, with effective suppression of homonuclear dipolar couplings. Several variations of these RN symmetry pulse sequences for the study of microsecond-to-nanosecond dynamics are presented in Fig. 7. RN-DIPSHIFT, PARS, and RNCSA experiments have been successfully applied to a range of supramolecular assemblies, with several studies highlighted below.

2.3.2 Millisecond to microsecond timescale dynamics

Biological processes on the millisecond-to-microsecond timescale include domain motions and enzyme catalysis. Rotating frame (R_1ρ ) and transverse (R₂*) spin-lattice relaxation and ¹⁵N–¹³C dipolar couplings are sensitive to dynamics on this timescale. Quantitative spin-lattice relaxation methods can measure exchange rates, population distributions, and chemical shift differences among exchanging sites. An important consideration for relaxation-based dynamics studies is interference from relaxation mechanisms unrelated to dynamics. These issues can be overcome by the use of deuteration and/or fast MAS (Lewandowski et al. Reference Lewandowski, Sass, Grzesiek, Blackledge and Emsley2011b; Quinn & McDermott, Reference Quinn and Mcdermott2012; Tollinger et al. Reference Tollinger, Sivertsen, Meier, Ernst and Schanda2012).

Rotating frame-based experiments measure relaxation resulting from spatial reorientation of a CSA or dipolar tensor and are sensitive to microsecond dynamics. Lewandowski et al. quantified site-specific backbone dynamics of GB1 with ¹⁵N and ¹³C R_1ρ relaxation measurements (Fig. 8a,b , (Lamley et al. Reference Lamley, Lougher, Sass, Rogowski, Grzesiek and Lewandowski2015; Lewandowski et al. Reference Lewandowski, Sass, Grzesiek, Blackledge and Emsley2011b)). The method has been recently applied to the membrane protein ASR by Ladizhansky and co-workers (Good et al. Reference Good, Wang, Ward, Struppe, Brown, Lewandowski and Ladizhansky2014). Krushelnitsky and co-workers presented a suite of relaxation studies on SH3 over a range of timescales that included R_1ρ relaxation, as well as ¹H–¹⁵N dipolar couplings and R₁ relaxation (Zinkevich et al. Reference Zinkevich, Chevelkov, Reif, Saalwachter and Krushelnitsky2013). As technology and methodology in the field of MAS NMR continue to advance, these experiments are being applied to increasingly complex systems.

Fig. 8. Methods for millisecond to microsecond timescale dynamics measurements. (a) Backbone amide ¹⁵N R_1ρ relaxation dispersion curves for select GB1 residues (Lewandowski et al. Reference Lewandowski, Sass, Grzesiek, Blackledge and Emsley2011b). (b) Residue-specific ¹⁵N R₁ and R_1ρ relaxation rates for GB1. (c) Dipolar CODEX pulse sequence (Krushelnitsky et al. Reference Krushelnitsky, Deazevedo, Linser, Reif, Saalwachter and Reichert2009). (d) Residue-specific intensity ratios for dipolar CODEX measurements of SH3. (e) Peak intensity ratio as a function of CODEX mixing time for select SH3 residues. Residues that lack slow dynamics (e.g., Gln 50) exhibit no mixing time dependence. (a,b) Reprinted with permission from Lewandowski et al. (Reference Lewandowski, Sass, Grzesiek, Blackledge and Emsley2011b). Copyright 2011 American Chemical Society. (c-e) Reprinted with permission from Krushelnitsky et al. (Reference Krushelnitsky, Deazevedo, Linser, Reif, Saalwachter and Reichert2009). Copyright 2009 American Chemical Society.

Carr–Purcell–Meiboom–Gill (CPMG) measurements, sensitive to sub-millisecond motions, have been well established for the study of protein dynamics in solution state NMR (Epstein et al. Reference Epstein, Benkovic and Wright1995; Farrow et al. Reference Farrow, Muhandiram, Singer, Pascal, Kay, Gish, Shoelson, Pawson, Formankay and Kay1994; Lorieau et al. Reference Lorieau, Louis and Bax2010; Mandel et al. Reference Mandel, Akke and Palmer1995; Meiboom & Gill, Reference Meiboom and Gill1958; Volkman et al. Reference Volkman, Lipson, Wemmer and Kern2001; Zhang et al. Reference Zhang, Sun, Watt and Al-Hashimi2006). These experiments have recently been extended to applications in the solid state, as demonstrated by Schanda and co-workers on microcrystalline ubiquitin (Tollinger et al. Reference Tollinger, Sivertsen, Meier, Ernst and Schanda2012). In this study, differential line broadening of zero- and double-quantum coherences as an initial indicator of dynamics was also utilized (Dittmer & Bodenhausen, Reference Dittmer and Bodenhausen2004). Dipolar evolution time from REDOR dephasing curves can also serve as a measure of millisecond timescale dynamics. In this case, reduced effective ¹⁵N–¹³C dipolar couplings at dynamic sites result in the absence of REDOR dephasing, as observed for the important linker residue Tyr145 in HIV-1 capsid assemblies (Byeon et al. Reference Byeon, Hou, Han, Suiter, Ahn, Jung, Byeon, Gronenborn and Polenova2012). Reduced ¹⁵N–¹³C dipolar couplings and hence the presence of millisecond timescale dynamics can also be reflected in reduced N–C cross-peak intensities (Helmus et al. Reference Helmus, Surewicz, Nadaud, Surewicz and Jaroniec2008).

Exchange experiments such as center-band only detection of exchange (CODEX) can monitor slow dynamics on the timescale of milliseconds-to-seconds. In a CODEX experiment, dipolar (Krushelnitsky et al. Reference Krushelnitsky, Deazevedo, Linser, Reif, Saalwachter and Reichert2009; Li & McDermott, Reference Li and Mcdermott2009) or CSA (DeAzevedo et al. Reference DeAzevedo, Hu, Bonagamba and Schmidt-Rohr1999) recoupling is applied before and after a mixing period. Dynamic residues will undergo only partial refocusing after the mixing period and exhibit reduced peak intensities. CODEX has been applied to characterize site-specific backbone and side-chain dynamics of α-spectrin SH3 (Fig. 8c–e , (Krushelnitsky et al. Reference Krushelnitsky, Deazevedo, Linser, Reif, Saalwachter and Reichert2009)), as well as small organic molecules (Li & McDermott, Reference Li and Mcdermott2012).

2.4.1 Chemical shift perturbations

Analysis of chemical shift perturbations, including intensity changes and line broadening, between free and bound states of a system of interest can indicate sites affected by the presence of a binding partner as demonstrated in early work of ligand binding to Bcl-xL with MAS NMR from Zech and McDermott (Zech et al. Reference Zech, Olejniczak, Hajduk, Mack and Mcdermott2004). Chemical shift perturbations indicate not only direct protein–protein (Liu et al. Reference Liu, Perilla, Ning, Lu, Hou, Ramalho, Himes, Zhao, Bedwell, Byeon, Ahn, Gronenborn, Prevelige, Rousso, Aiken, Polenova, Schulten and Zhang2016) or protein–ligand interactions (Schutz et al. Reference Schutz, Soragni, Hornemann, Aguzzi, Ernst, Bockmann and Meier2011), but also allosteric structural changes that occur as a result of binding. An extension of chemical shift perturbations, changes in the CSA tensor magnitude can also serve as a probe of intermolecular interactions, such as applied to studies of cytb(5) in complex with cytP4502B4 (Pandey et al. Reference Pandey, Vivekanandan, Ahuja, Huang, Im, Waskell and Ramamoorthy2013). Further, MAS NMR methods exist to characterize the bona fide intermolecular binding interface. For example, PRE in sites distal to the spin label can also be used as an indicator of intermolecular interactions (Wang et al. Reference Wang, Munro, Kim, Jung, Brown and Ladizhansky2012). Dipolar edited correlation techniques have been particularly productive for the study of direct intermolecular interactions in biological macromolecular assemblies.

2.4.2 Dipolar-edited correlation spectroscopy

One approach to identifying intermolecular interfaces is the use of differential isotopic labeling of the binding partners, where one binding partner is ¹³C or ¹³C,¹⁵N labeled and the other is ¹⁵N labeled, first demonstrated by Polenova (Marulanda et al. Reference Marulanda, Tasayco, Mcdermott, Cataldi, Arriaran and Polenova2004; Yang et al. Reference Yang, Tasayco and Polenova2008) and Baldus (Etzkorn et al. Reference Etzkorn, Bockmann, Lange and Baldus2004; Weingarth & Baldus, Reference Weingarth and Baldus2013). Long range polarization transfer across the intermolecular interface is achieved using heteronuclear mixing sequences such as REDOR (Gullion & Schaefer, Reference Gullion and Schaefer1989) or TEDOR (Hing et al. Reference Hing, Vega and Schaefer1992), NHHC (Lange et al. Reference Lange, Luca and Baldus2002), PAIN (Lewandowski et al. Reference Lewandowski, De Paepe and Griffin2007), or a combination of these methods such as REDOR–PAINCP (Fig. 9, panel 2 (Yang et al. Reference Yang, Tasayco and Polenova2008)). These techniques have been applied for the observation of intermonomer interactions in microcrystalline Crh (Etzkorn et al. Reference Etzkorn, Bockmann, Lange and Baldus2004) and structural characterization of α-synuclein amyloid fibrils (Lv et al. Reference Lv, Kumar, Giller, Orcellet, Riedel, Fernandez, Becker and Lange2012) with NHHC correlation experiments, PAIN-derived intermolecular correlations in thioredoxin reassemblies (Yang et al. Reference Yang, Tasayco and Polenova2008), and quaternary structure of GB1 crystals with TEDOR (Nieuwkoop & Rienstra, Reference Nieuwkoop and Rienstra2010). An alternate approach is to use complementary, selective ¹³C labeling such as a mixture of [1-¹³C]glucose and [2-¹³C]glucose labeled protein to observe intermolecular ¹³C–¹³C correlations. This scheme has been applied to α-synuclein fibrils (Loquet et al. Reference Loquet, Giller, Becker and Lange2010).

Fig. 9. Two methods for the study of intermolecular interactions in protein assemblies. (Panel 1) MELODI–HETCOR (a) MELODI–HETCOR pulse sequence. (b–d) LG-HETCOR ¹H–¹⁵N spectra of an Arg-rich membrane-embedded peptide (b) no REDOR dephasing, (c) only ¹H–¹³C REDOR dephasing, (d) both ¹H–¹³C and ¹H–¹⁵N REDOR dephasing. (Li et al. Reference Li, Su, Luo and Hong2010) (Panel 2) REDOR-PAINCP (e) pulse sequence for REDOR–PAINCP experiment. (f) 2D ¹⁵N–¹³C REDOR–PAINCP spectra of thioredoxin. (g) Observed intermolecular correlations plotted onto the structure of thioredoxin. (Yang et al. Reference Yang, Tasayco and Polenova2008) (a–d) Adapted with permission fron Li et al. (Reference Li, Su, Luo and Hong2010). Copyright 2010 American Chemical Society. (e–g) Adapted with permission from Yang et al. (Reference Yang, Tasayco and Polenova2008). Copyright 2008 American Chemical Society.

A considerable drawback to the approach described above is the requirement that both species are isotopically labeled. Many systems of interest cannot be readily prepared with isotopic labels. Thus methods to observe binding interfaces where one binding partner is natural abundance are essential. These interface correlations can be achieved in REDOR-filter-based experiments. In these experiments, REDOR dephasing is applied to ¹H–¹³C and/or ¹H–¹⁵N dipolar couplings to eliminate magnetization arising from directly bonded protons. Subsequent ¹H polarization transfer arises from the source without ¹³C/¹⁵N labels and magnetization is transferred to the magnetically active nuclei of the isotopically labeled protein at the binding interface. This approach has been demonstrated for the study of natural abundance rhodopsin in complex with ¹³C-labeled 11-cis-retinal (Kiihne et al. Reference Kiihne, Creemers, De Grip, Bovee-Geurts, Lugtenburg and De Groot2005) and assemblies of U-²H,¹³C,¹⁵N-CAP-Gly with natural abundance polymerized microtubules (MTs) assemblies (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). A significant drawback of this approach in the latter study is the presence of residual deuteration at ca. 1%, resulting in unwanted intramolecular transfers. To overcome this limitation, double-REDOR filter approach (dREDOR) was demonstrated on assemblies of U-¹³C,¹⁵N-CAP-Gly with natural abundance polymerized MTs (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). In this method, a double ¹H–¹³C/¹H–¹⁵N REDOR filter is applied to dephase the ¹H signals from the U-¹³C,¹⁵N-CAP-Gly, with the subsequent transfer of ¹H magnetization from the MTs back to U-¹³C,¹⁵N-CAP-Gly across the intermolecular interface.

In addition to the observation of protein–protein or protein–ligand interactions, REDOR-filter-based experiments, such as MELODI-HETCOR (Fig. 9, panel 1 (Yao et al. Reference Yao, Schmidt-Rohr and Hong2001)) have also been applied to the study of water–protein interactions, including the identification of a hydrated arginine for an antimicrobial peptide in a lipid bilayer (Li et al. Reference Li, Su, Luo and Hong2010) and the characterization of Pf1 bacteriophage hydration (Purusottam et al. Reference Purusottam, Rai and Sinha2013; Sergeyev et al. Reference Sergeyev, Bahri, Day and Mcdermott2014).

In a variation of these dipolar edited methods, Asami et al. identified residues of the protein L7Ae that form the binding interface with box C/D RNA in the protein-RNA complex using ²H, ¹⁵N-labeled protein and ¹H, ¹³C, ¹⁵N- or ²H, ¹³C, ¹⁵N-labeled RNA. In this experiment, protein residues interacting with ¹H-RNA exhibited line broadening and weaker peak intensities relative to the corresponding peaks in the ²H-RNA complex due to ¹H–dipolar interactions (Asami et al. Reference Asami, Rakwalska-Bange, Carlomagno and Reif2013).

Beyond qualitative identification of intermolecular interfaces, the distance dependence of dipolar couplings (1/r³) can be used to quantify protein–protein and protein–ligand intermolecular distances. In addition to applications in structure calculation and dynamics studies, REDOR dephasing or TEDOR build-up curves can be used to quantify intermolecular distances. Protein–protein intermolecular distances can lend insight into higher-order structural features, as has been demonstrated for amyloid fibrils by Tycko, Dobson, and others (Fitzpatrick et al. Reference Fitzpatrick, Debelouchina, Bayro, Clare, Caporini, Bajaj, Jaroniec, Wang, Ladizhansky, Muller, Macphee, Waudby, Mott, De Simone, Knowles, Saibil, Vendruscolo, Orlova, Griffin and Dobson2013; Petkova et al. Reference Petkova, Yau and Tycko2006; Van der Wel et al. Reference Van Der Wel, Lewandowski and Griffin2010) (Fig. 10) and membrane protein architecture by Griffin and co-workers (Andreas et al. Reference Andreas, Reese, Eddy, Gelev, Ni, Miller, Emsley, Pintacuda, Chou and Griffin2015). REDOR distance measurements are also used for the studies of ligand–protein interactions, including binding of amantadine and its derivatives in the M2 channel by Cross (Wright et al. Reference Wright, Batsomboon, Dai, Hung, Zhou, Dudley and Cross2016) and Hong (Cady et al. Reference Cady, Schmidt-Rohr, Wang, Soto, Degrado and Hong2010) and retinal binding in bacteriorhodopsin (Helmle et al. Reference Helmle, Patzelt, Ockenfels, Gartner, Oesterhelt and Bechinger2000). REDOR-derived distances can also indicate changes in protein or nucleic acid conformation upon ligand binding, demonstrated for tat peptide-bound TAR RNA of HIV-1 (Olsen et al. Reference Olsen, Edwards, Deka, Varani, Sigurdsson and Drobny2005).

Fig. 10. Application of REDOR distance measurements to a selectively labeled amyloid protofilament revealed anti-parallel stacking of the β-sheets. (a) 2D ¹⁵N–¹³C ZF-TEDOR spectrum, (b) 2D ¹³C–¹³C PDSD spectrum, (c) cross-section of anti-parallel β-sheets, red and blue lines indicate intermolecular distances measured, (d) REDOR dephasing curve of residues Y105 and S115, indicating head-to-tail arrangement of the protofilament. Reproduced with permission from Fitzpatrick et al. (Reference Fitzpatrick, Debelouchina, Bayro, Clare, Caporini, Bajaj, Jaroniec, Wang, Ladizhansky, Muller, Macphee, Waudby, Mott, De Simone, Knowles, Saibil, Vendruscolo, Orlova, Griffin and Dobson2013). Copyright 2013 National Academy of Sciences.

Various classes of supramolecular assemblies studied by MAS NMR include amyloid systems (reviewed in (Comellas & Rienstra, Reference Comellas and Rienstra2013; Tycko, Reference Tycko2011)), the Shigella type-III secretion system (TSS3) (Demers et al. Reference Demers, Habenstein, Loquet, Vasa, Giller, Becker, Baker, Lange and Sgourakis2014; Loquet et al. Reference Loquet, Sgourakis, Gupta, Giller, Riedel, Goosmann, Griesinger, Kolbe, Baker, Becker and Lange2012, Reference Loquet, Habenstein, Chevelkov, Vasa, Giller, Becker and Lange2013a), the Escherichia coli pilus protein FimA (Habenstein et al. Reference Habenstein, Loquet, Hwang, Giller, Vasa, Becker, Habeck and Lange2015), and the MAVS (mitochondrial antiviral signaling) protein (He et al. Reference He, Luhrs and Ritter2015, Reference He, Bardiaux, Ahmed, Spehr, Konig, Lunsdorf, Rand, Luhrs and Ritter2016). This review focuses on two particular classes of supramolecular assemblies: cytoskeletal proteins and viruses.

3.1.1 Structure of CAP-Gly domain of dynactin

Dynactin, an activator of the dynein motor assembly, is a protein complex involved in intracellular transport (Caviston & Holzbaur, Reference Caviston and Holzbaur2006). Dynactin regulates dynein transport along MTs, and mutations within its p150^Glued subunit lead to neurodegenerative disorders, such as Huntington's disease, Charcot–Marie Tooth disease, amyotropic lateral sclerosis, distal spinal bulbar muscular atrophy, and Perry syndrome (Chen et al. Reference Chen, Xu, Cooper and Liu2014). Within the p150^Glued subunit of dynactin, CAP-Gly (cytoskeleton-associated protein glycine-rich) is an 89 residue MT-binding domain (Vaughan et al. Reference Vaughan, Miura, Henderson, Byrne and Vaughan2002; Waterman-Storer et al. Reference Waterman-Storer, Karki and Holzbaur1995). Dynactin CAP-Gly is the first MAP assembled with MTs, whose structure and dynamics have been investigated in depth by MAS NMR, yielding atomic-resolution insights unavailable from other techniques and establishing a proof of principle for investigations of other cytoskeleton-associated assemblies (Ahmed et al. Reference Ahmed, Sun, Siglin, Polenova and Williams2010; Sun et al. Reference Sun, Siglin, Williams and Polenova2009; Yan et al. Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a, Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a, Reference Yan, Zhang, Hou, Ahmed, Williams and Polenovab). Recently, the atomic-level resolution structure of CAP-Gly bound to polymeric MTs was reported (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a) (PDB ID code 2MPX), the first structure of any cytoskeleton-associated protein assembled with cytoskeleton.

Fig. 11. Transmission electron microscopy of cytoskeleton-associated proteins for MAS NMR experiments. (a, b) ²H,¹³C,¹⁵N CAP-Gly/MT complex before and after MAS. Adapted with permission from Yan et al. (Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). Copyright 2015 National Academy of Sciences. (c) ¹³C, ¹⁵N BacA. Filament bundles are indicated by arrows, sheets are indicated by asterisks, and single filaments are indicated by arrowheads. Adapted with permission from Vasa et al. (Reference Vasa, Lin, Shi, Habenstein, Riedel, Kuhn, Thanbichler and Lange2015). Copyright 2015 National Academy of Sciences.

To determine the structure of CAP-Gly in complex with MTs, three different isotopic labeling schemes were used: U-¹⁵N,¹³C; U-¹⁵N, [2-¹³C]glucose; and U-¹⁵N, [1,6-¹³C]glucose. The structure was determined using hundreds of medium- and long-range distance restraints collected from ¹³C–¹³C CORD and ¹⁵N–¹³C PAIN-CP experiments, as well as hydrogen-bonding restraints and torsion angles from TALOS+. The equivalent resolution in the structure was 1.9–2.5 Å with a tightly constrained ensemble of lowest-energy conformers. A very similar approach was previously used to determine the structure of free CAP-Gly (PDB ID code 2M02) (Yan et al. Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a). The structure of CAP-Gly assembled on MTs indicates that, while the overall secondary structure is retained, the flexible loops of CAP-Gly have remarkably different conformations when associated with MTs (Fig. 12). Loop β ₃/β ₄ adopts a more open conformation in the free state of CAP-Gly, and rearranges to a more closed conformation when bound to MTs. The different sidechain orientations of residues in this loop may play a role in CAP-Gly's structural plasticity and ability to interact with different binding partners.

Fig. 12. (a) Structure of CAP-Gly bound to polymerized MTs (purple, 2MPX) and free CAP-Gly (orange, 2M02), both determined with MAS NMR, and CAP-Gly bound to EB1 (green, 2HKQ). (b) Expansion of loop regions of CAP-Gly in the three systems, indicating the differences in loop position and side-chain orientation for CAP-Gly in its three different states (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). (c) Chemical shift perturbations for several residues in CAP-Gly indicating multiple conformers of free CAP-Gly (black) that collapse to a single conformer in complex with EB1 (Yan et al. Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a), (a) and (b) Adapted with permission from Yan et al. (Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). Copyright 2015 National Academy of Sciences. (c) Adapted with permission from Yan et al. (Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a). Copyright 2013 Elsevier.

EB1 is another MAP that, like dynactin, localizes at the plus end of the growing MT. EB1 is thought to have a role in MT dynamics; specifically it may promote MT elongation (Rogers et al. Reference Rogers, Rogers, Sharp and Vale2002). EB1 interacts with the p150^Glued subunit of dynactin and it is hypothesized that the two proteins form a plus end complex to regulate MT dynamics (Ligon et al. Reference Ligon, Shelly, Tokito and Holzbaur2003). The p150^Glued subunit may play a role in recruiting EB1 to the MTs. Residues of CAP-Gly that are perturbed by binding of EB1 were identified by chemical shift changes. Chemical shift perturbations indicate that free CAP-Gly exists in multiple conformers, but is conformationally homogeneous when bound to MTs (Yan et al. Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a).

3.1.2 Interface of CAP-Gly with MTs

The main challenge for NMR characterization of MTs and their assemblies with associated proteins is that to date there have been no efficient isotopic labeling protocols established for tubulin. This precludes in-depth structural characterization of MTs and limits the approaches for determination of intermolecular interfaces formed by MTs and their binding partners. To overcome this challenge, the application of dREDOR filters was explored to characterize the intermolecular interfaces that dynactin's CAP-Gly forms with MTs and EB1 (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). In these experiments, ¹H–¹³C and ¹H–¹⁵N REDOR filters were simultaneously applied to dephase all ¹H magnetization arising from U-¹³C,¹⁵N CAP-Gly. Subsequently, polarization was transferred from ¹H of natural abundance MTs or EB1 to their binding interface on the surface of CAP-Gly. A ¹³C-¹³C CORD dimension was included to enable site-specific assignment of the binding interface. Figure 13 shows dREDOR–CORD and dREDOR–HETCOR spectra of CAP-Gly in complex with MTs (b), EB1 (c), and the intermolecular interfaces as determined by dREDOR (a left, green) and chemical shift perturbations (a right, orange/purple). The results confirmed that loop β ₃/β ₄ including the GKNDG motif comprises the primary binding interface with MTs. CAP-Gly interacts with its binding partners on the flat side of the protein, where most of the surface-exposed hydrophobic residues are located. dREDOR experiments of the CAP-Gly/EB1 complex were consistent with the known binding interface for this complex, which has been determined previously (Hayashi et al. Reference Hayashi, Wilde, Mal and Ikura2005; Honnappa et al. Reference Honnappa, Okhrimenko, Jaussi, Jawhari, Jelesarov, Winkler and Steinmetz2006; Yan et al. Reference Yan, Hou, Sehwieters, Ahmed, Williams and Polenova2013a), validating the approach for characterizing the CAP-Gly/MT interface.

Fig. 13. (a) Intermolecular interfaces of CAP-Gly with MT and EB1 determined with dREDOR (left, green), and observed chemical shift perturbations (right, purple/orange). For chemical shift perturbations, purple residues indicate large shifts >1 ppm, orange indicates shifts between 0.5 and 1 ppm. (b) dREDOR–HETCOR and dREDOR–CORD spectra of U-¹³C,¹⁵N CAP-Gly bound to MT and (c) in complex with EB1. (Yan et al. Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a) Reproduced with permission from Yan et al. (Reference Yan, Guo, Hou, Zhang, Lu, Williams and Polenova2015a). Copyright 2015 National Academy of Sciences.

3.1.3 Dynamics of CAP-Gly

MT-associated motors and their activators possess conformational plasticity, which is essential for their ability to bind to and slide along the MTs (Howard, Reference Howard2001; Vale, Reference Vale2003). Conformational plasticity is directly related to internal flexibility, and therefore, knowledge of dynamics is essential for understanding the biological function of MAP assemblies. The dynamics of CAP-Gly free, in complex with EB1, and bound to polymeric MTs have been characterized using MAS NMR, over a range of functionally relevant timescales from nanoseconds to milliseconds (Yan et al. Reference Yan, Zhang, Hou, Ahmed, Williams and Polenova2015b). Global dynamics were probed through a temperature series of 1D- and 2D ¹³C spectra (Fig. 14a-c ). Site-specific dynamics were characterized using ¹H–¹⁵N and ¹H–¹³C DOPs (Fig. 14d ) (Hou et al. Reference Hou, Byeon, Ahn, Gronenborn and Polenova2011b). As indicated by the temperature series in Fig. 14a–c , free and MT-bound CAP-Gly are dynamic across the entire range of timescales under investigation, and the motions are strongly temperature dependent. In contrast, the CAP-Gly in complex with EB1 is largely rigid on these timescales and its spectra are not temperature dependent. From the measurement of DOPs, it was found that the loops of CAP-Gly are mobile in the free protein as well as in complex with MTs. However, consistent with 1D and 2D spectra, the dynamics of CAP-Gly are notably attenuated when in complex with EB1. Remarkably, the loops of CAP-Gly show an increase in fast timescale backbone fluctuations (nanosecond-to-microsecond), but a decrease in slower dynamics (microsecond-to-millisecond) upon binding to MTs (Fig. 14d ). It was proposed that these observed changes in dynamics have a critical function in CAP-Gly/MT interactions. The combined structural and dynamics studies of CAP-Gly highlight the structural plasticity of this protein and the essential role this flexibility plays in CAP-Gly's ability to adopt different conformations and interact with different binding partners.

Fig. 14. ¹⁵N–¹³C SPECIFIC CP NCA 1D spectra indicating temperature dependence of global conformational dynamics of (a) free CAP-Gly, (b) CAP-Gly/EB1 complex, (c) CAP-Gly bound to MTs. (d) DOPs of free CAP-Gly at −2 °C (purple), and MT-bound CAP-Gly at −2 °C (green) and −19 °C (black). The micro-to-nanosecond timescale dynamics of MT-bound CAP-Gly are enhanced at −2° in comparison to the free protein. Adapted with permission from Yan et al. (Reference Yan, Zhang, Hou, Ahmed, Williams and Polenova2015b). Copyright 2015 The American Society for Biochemistry and Molecular Biology.

4.1.1 Structural characterization of HIV-1 capsid assemblies

CA is a 231 residue, predominantly α-helical protein with independently folding N-terminal (NTD, residues 1–143) and C-terminal (CTD, residues 148–231) domains, connected by an inter-domain linker (Deshmukh et al. Reference Deshmukh, Schwieters, Grishaev, Ghirlando, Baber and Clore2013; Gres et al. Reference Gres, Kirby, Kewalramani, Tanner, Pornillos and Sarafianos2015). Early studies (Chen & Tycko, Reference Chen and Tycko2010; Han et al. Reference Han, Ahn, Concel, Byeon, Gronenborn, Yang and Polenova2010) revealed that most residues in both NTD and CTD are relatively rigid, and CA assemblies retain the secondary and tertiary structure determined from solution NMR and x-ray crystallography studies. Upon maturation, the viral capsid morphology changes from spherical to conical. Mature conical HIV-1 capsids are built from hexameric and pentameric CA assemblies, whose stoichiometry in the final capsid is variable, and so is the capsid's cone shape. Four kinds of intermolecular contacts are critical for capsid morphology and stability both in vivo and in vitro: intra-hexameric NTD–NTD and NTD–CTD, and inter-hexameric CTD–CTD interfaces around pseudo twofold and pseudo threefold axes (Fig. 18c – e ) (Byeon et al. Reference Byeon, Meng, Jung, Zhao, Yang, Ahn, Shi, Concel, Aiken, Zhang and Gronenborn2009; Pornillos et al. Reference Pornillos, Ganser-Pornillos, Kelly, Hua, Whitby, Stout, Sundquist, Hill and Yeager2009). The capsid's structural polymorphism is also observed in vitro, where the morphology can be controlled by assembly conditions. CA can assemble into cones, tubes, and spheres (Barklis et al. Reference Barklis, Mcdermott, Wilkens, Fuller and Thompson1998; Ehrlich et al. Reference Ehrlich, Liu, Scarlata, Chu and Carter2001; Ganser-Pornillos et al. Reference Ganser-Pornillos, Cheng and Yeager2007; Gross et al. Reference Gross, Hohenberg and Krausslich1997; Han et al. Reference Han, Ahn, Concel, Byeon, Gronenborn, Yang and Polenova2010, Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). MAS NMR studies of spheres, cones, and tubes indicated no major differences in secondary or tertiary structure among the three morphologies (Bayro et al. Reference Bayro, Chen, Yau and Tycko2014; Chen & Tycko, Reference Chen and Tycko2010; Han et al. Reference Han, Ahn, Concel, Byeon, Gronenborn, Yang and Polenova2010, Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013), but no 3D structure is available so atomic-level details of structural organization of these in vitro assemblies remain poorly understood.

HIV-1 exhibits significant sequence variability and sequence-dependent viral infectivity (Price et al. Reference Price, Fletcher, Schaller, Elliott, Lee, Kewalramani, Chin, Towers and James2012). To date, two HIV-1 strains have been studied by MAS NMR: HXB2 and NL4-3 (Byeon et al. Reference Byeon, Hou, Han, Suiter, Ahn, Jung, Byeon, Gronenborn and Polenova2012; Bayro et al. Reference Bayro, Chen, Yau and Tycko2014; Chen & Tycko, Reference Chen and Tycko2010; Han et al. Reference Han, Ahn, Concel, Byeon, Gronenborn, Yang and Polenova2010, Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013; Lu et al. Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a). The wild-type sequences of these proteins differ by only four amino acids, but this variation causes considerable conformational changes across the CA sequence (Han et al. Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). These structural perturbations may play a role in the differing viral infectivity of the two strains. Viral infectivity is also regulated through the interactions with host factors. Cyclophilin A (CypA) is one such host factor protein that modulates HIV-1 infectivity and uncoating through direct interactions with the HIV-1 capsid's CypA-binding loop located in the NTD. The mechanism of CypA is complex and poorly understood (Colgan et al. Reference Colgan, Yuan, Franke and Luban1996; Luban et al. Reference Luban, Bossolt, Franke, Kalpana and Goff1993). A recent study yielded the structure of CypA in complex with the assembled HIV-1 capsid at 8 Å resolution (Liu et al. Reference Liu, Perilla, Ning, Lu, Hou, Ramalho, Himes, Zhao, Bedwell, Byeon, Ahn, Gronenborn, Prevelige, Rousso, Aiken, Polenova, Schulten and Zhang2016). It was discovered, using cryo-EM-guided all-atom MD simulations and MAS NMR spectroscopy that CypA simultaneously interacts with two CA subunits in different hexamers. This binding mechanism established through the integrated cryo-EM/MAS NMR/MD approach provided insights into the mechanism of HIV-1 capsid stabilization by and recruitment of CypA to promote the viral infectivity.

Additional mutations in the various regions of CA sequence have been shown to affect the assembly morphology and efficiency, capsid stability, and interactions with host factors (Bocanegra et al. Reference Bocanegra, Rodriguez-Huete, Fuertes, Del Alamo and Mateu2012; Forshey et al. Reference Forshey, Von Schwedler, Sundquist and Aiken2002; Gres et al. Reference Gres, Kirby, Kewalramani, Tanner, Pornillos and Sarafianos2015; Jiang et al. Reference Jiang, Ablan, Derebail, Hercik, Soheilian, Thomas, Tang, Hewlett, Nagashima, Gorelick, Freed and Levin2011; Manocheewa et al. Reference Manocheewa, Swain, Lanxon-Cookson, Rolland and Mullins2013; McCarthy et al. Reference McCarthy, Schmidt, Kirmaier, Wyand, Newman and Johnson2013; Qi et al. Reference Qi, Yang and Aiken2008; Yang & Aiken, Reference Yang and Aiken2007). For example, mutations in the linker region of CA render the capsid assembly inefficient or abolish it completely (Jiang et al. Reference Jiang, Ablan, Derebail, Hercik, Soheilian, Thomas, Tang, Hewlett, Nagashima, Gorelick, Freed and Levin2011). On the contrary, the E45A mutation makes a ‘hyperstable’ capsid, which does not disassemble, thus dramatically reducing the viral infectivity (Forshey et al. Reference Forshey, Von Schwedler, Sundquist and Aiken2002; Hulme et al. Reference Hulme, Kelley, Okocha and Hope2015; Yang et al. Reference Yang, Shi, Byeon, Ahn, Sheehan, Meiler, Gronenborn and Aiken2012). Mutations in the CypA-binding loop, such as A92E and G94D exhibit drastically reduced viral infectivity in the presence of the normally required CypA. Full infectivity can be restored by inhibition of CypA by cyclosporin. MAS NMR analysis of these mutants revealed that the mutations induce conformational changes, as well as changes in the dynamics, vide infra (Lu et al. Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a).

To get insights into the structural changes occurring upon formation of the hexagonal CA lattice (predominant symmetry in the cones), Bayro et al. used MAS NMR-derived structural restraints in tubular CA assemblies for refinement against solution NMR and x-ray crystallography structures of monomeric CA (Bayro et al. Reference Bayro, Chen, Yau and Tycko2014). ¹⁵N–¹⁵N backbone distances, determined by ¹⁵N–¹⁵N dipolar recoupling (¹⁵N-BARE), have been used by Tycko and co-workers to derive backbone torsion angles and secondary structure (Hu et al. Reference Hu, Qiang, Bermejo, Schwieters and Tycko2012). In α-helices and tight turns, ¹⁵N–¹⁵N distances are short, leading to rapid signal decay, while the decay is slower in loop regions (Fig. 19a ). Deviations of experimental ¹⁵N-BARE curves of CA tubular assemblies from simulated curves derived from crystal or solution NMR structures indicated differences in secondary structure. Using the ¹⁵N–¹⁵N distances and TALOS torsion angle predictions as structural restraints, structure refinement was carried out against CA coordinates derived from solution NMR (monomer, 2LF4 (Shin et al. Reference Shin, Tzou and Krishna2011)) and crystal structures (hexamer, 3MGE (Pornillos et al. Reference Pornillos, Ganser-Pornillos, Banumathi, Hua and Yeager2010)). Figure 19b–d shows regions of the CA structure that adopt a more extended conformation in tubular assemblies than in their respective initial structures. Among these regions that undergo changes are the 3₁₀ helix near the start of the CTD, as well as the loops between helices 3 and 4, and helices 10 and 11. These perturbations were attributed to conformational changes arising from higher-order oligomerization and/or lattice curvature.

Fig. 19. (a) ¹⁵N–¹⁵N BARE curves for selected CA residues. Circles indicate experimental curves for CA tubular assemblies. Simulated curves correspond to the following structures: (- -) 2LF4, (−) 3MGE, (−) 2KOD. (b) Initial (red) and final (blue) structure refinement against 2LF4, indicating the change in the conformation of the 3₁₀ helix. (c, d) Initial (orange) and final (blue) structure refinement against 3MGE, indicating the change in conformation of loop 3/4 and loop 10/11, respectively. Adapted with permission from Bayro et al. (Reference Bayro, Chen, Yau and Tycko2014). Copyright 2014 Elsevier.

Very recently Bayro and Tycko characterized the structure of tubular assemblies of the capsid pseudo-twofold inter-hexameric interface (Bayro & Tycko, Reference Bayro and Tycko2016) using mixed and selective labeling schemes and dipolar-based distance measurements, with additional restraints derived from the cryo-EM structure determined by Zhang and co-workers (Zhao et al. Reference Zhao, Perilla, Yufenyuy, Meng, Chen, Ning, Ahn, Gronenborn, Schulten, Aiken and Zhang2013). In this study, quantitative W184 to M185 intermolecular distances across the dimerization interface were obtained with ¹⁵N–¹³C REDOR dephasing experiments. A series of experiments were performed to characterize intra-residue distances of W184 and M185 in order to constrain the side-chain conformations, including ¹⁵N–¹³C REDOR distances, ¹³C–¹³C BroBaRR (BroadBand Rotational Resonance) distances for M185 (Chan & Tycko, Reference Chan and Tycko2004), and tensor correlation experiments (Dabbagh et al. Reference Dabbagh, Weliky and Tycko1994) of W184 to determine the angle between the backbone Cα–N bond vector and sidechain Cδ1–Nε1 bond vector. The distances and angles determined differed somewhat from prior solution NMR (Byeon et al. Reference Byeon, Meng, Jung, Zhao, Yang, Ahn, Shi, Concel, Aiken, Zhang and Gronenborn2009) and X-ray crystallography studies (Gres et al. Reference Gres, Kirby, Kewalramani, Tanner, Pornillos and Sarafianos2015; Worthylake et al. Reference Worthylake, Wang, Yoo, Sundquist and Hill1999), suggesting the structure of the dimerization interface in tubular assemblies differs from that in crystals or CA in solution. Further intra-CA monomer distance constraints were obtained from ¹³C–¹³C correlations with long mixing (700 ms). With an integrated structure calculation approach described above, they were able to determine the structure of the inter-hexameric dimer interface in CA tubular assemblies, and further demonstrated that this interface is well ordered in this morphology.

4.1.2 Conformational dynamics of HIV-1 capsid assemblies by MAS NMR

Protein dynamics have indispensible functions in many stages of the HIV-1 lifecycle, including assembly, release, and maturation (Freed, Reference Freed2015). While CA is relatively rigid in the individual α-helices, residues in loops and the flexible linker, which are key functional regions of the protein, are dynamic, giving rise to conformational plasticity, which is directly connected to capsid morphology, interactions with host factors, and viral infectivity.

Lu et al. have characterized the site-specific dynamics of several capsid constructs, including the HXB2 and NL4-3 strains discussed above, as well as CypA-bound CA and the A92E and G94D ‘escape mutants’ (Lu et al. Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a). These mutants have approximately 10% the infectivity of the wild-type virus; however, infectivity can be restored upon CypA inhibition (Ylinen et al. Reference Ylinen, Schaller, Price, Fletcher, Noursadeghi, James and Towers2009). The CypA loop of CA (residues 84–100) undergoes conformational changes upon binding of CypA as well as in the A92E and G94D ‘escape’ mutants. ¹H–¹⁵N and ¹H–¹³C dipolar tensors and resonance intensities reported on CA dynamics on the nano- through millisecond timescales. The CypA loop was observed to exhibit an unprecedented degree of mobility over the entire range of timescales under study. As expected, the motions are attenuated upon CypA binding. A completely unexpected finding was that the dynamic profiles of the escape mutants A92E and G94D closely resembled that of CA in complex with CypA: mobility of CypA loop is dramatically attenuated due to the mutations. Furthermore, NMR parameters derived from all-atom MD trajectories were in remarkable agreement with experimental results, corroborating that the dynamics are modulated by the mutations in the CypA loop. Figure 20 demonstrates (a) DOPs and (b) ¹H–¹⁵N dipolar lineshapes of residues in the CypA loop with (c) peak intensities and (d) DOPs mapped to the CA structure. Taken together, these results indicate that that the capsid escapes the CypA dependence by a dynamic allostery mechanism and highlight the key role of conformational dynamics in the HIV-1 CA function.

Fig. 20. (a) ¹H–¹⁵N DOPs and (b) lineshapes for residues in the CypA loop in WT CA (HXB2), cyclophilinA-bound CA (HXB2), WT CA (NL4–3), CA A92E (NL4–3), and CA G94D (NL4–3), listed from top to bottom. (c) Peak intensities observed in an NCACX correlation spectrum for each of the five constructs. (d) DOPs (top) and peak intensities (bottom) mapped onto the structure of CA. Adapted with permission from Lu et al. (Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a). Copyright 2015 National Academy of Sciences.

Conformational plasticity of HIV-1 CA is essential for its assembly into the viral capsid core. As discussed above, the hinge that links the NTD and CTD has been shown to play a key role in HIV-1 core stability (Byeon et al. Reference Byeon, Meng, Jung, Zhao, Yang, Ahn, Shi, Concel, Aiken, Zhang and Gronenborn2009; Jiang et al. Reference Jiang, Ablan, Derebail, Hercik, Soheilian, Thomas, Tang, Hewlett, Nagashima, Gorelick, Freed and Levin2011). The dynamic origin and mechanism of CA's pleiomorphic assembly was investigated by an integrated MAS and solution NMR approach, using conical assemblies of U–¹³C, ¹⁵N–Tyr-labeled samples of full length CA, of individual CTD constructs, and solutions of U-¹³C,¹⁵N-CA and CTD (Byeon et al. Reference Byeon, Hou, Han, Suiter, Ahn, Jung, Byeon, Gronenborn and Polenova2012). Tyrosine is an ideal probe of linker dynamics as functionally important Y145 is located in the linker; CA has only three additional Tyr residues located in rigid regions of the protein, which provide internal controls in the NMR characterization. To observe dynamics on the millisecond timescale, ¹³C–¹⁵N REDOR dephasing experiments were performed. The dephasing profiles for the three Tyr residues located in the helical regions of CA are consistent with them being rigid. On the contrary, the Y145 signals could not be dephased due to the complete averaging of the ¹³C–¹⁵N dipolar couplings, which is a clear manifestation of dynamics occurring on the timescale of the order of ~10 ms (Gullion & Schaefer, Reference Gullion and Schaefer1989). To probe the dynamics of the interdomain linker on the microsecond to nanosecond timescale, ¹H–¹⁵N dipolar and ¹⁵N CSA lineshapes were acquired. All Tyr residues, including Y145 showed close to rigid limit dipolar and CSA parameters, establishing that Y145 is rigid on the corresponding timescales. On the basis of these results it was concluded that millisecond timescale conformational dynamics of the interdomain linker is essential for the CA assembly. Solution NMR experiments revealed that, while these millisecond timescale motions of the linker are essential for opening up the conformational space, the number of accessible conformers is finite, and their populations are controlled through electrostatic intermolecular interactions between side chains of W184 and E175. This integrated approach established a molecular switch mechanism by which dynamics and electrostatic interactions work together to permit the formation of varied capsid morphologies in the mature HIV (Byeon et al. Reference Byeon, Hou, Han, Suiter, Ahn, Jung, Byeon, Gronenborn and Polenova2012).

In another study, Tycko and co-workers observed that while CA is generally rigid, there are regions of crucial biological function that exhibit static or dynamic disorder (Bayro et al. Reference Bayro, Chen, Yau and Tycko2014). Using scalar-based ¹³C–¹³C TOBSY (Hardy et al. Reference Hardy, Verel and Meier2001) experiments, dynamically disordered regions of the protein were identified. Residues attributed to the interdomain linker, loop 8/9, and the C-terminal tails were observed in this data set, indicating that these residues are dynamically disordered. To distinguish residues that are mobile on the sub-millisecond timescale, ¹H T₂ filtered NCA data sets were acquired. In this experiment, a ¹H T₂ filter is incorporated and signals decay according to the local ¹H–¹H dipolar coupling strength. Thus, peak intensities of rigid residues are weakened to 20–30% of their peak intensity, while dynamic residues retain more than 40% of their peak intensity. ¹H T₂ filtered experiments indicated that many dynamic residues are in loops and/or solvent exposed, with the most mobile residues being in the CypA loop (Fig. 21), in agreement with the DOP measurements by Lu et al. (Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a). The N-terminal segment was also observed to be mobile.

Fig. 21. (a) NCA spectrum of CA tubular assemblies, with sufficiently resolved peaks labeled. (b) ¹H T₂ filtered NCA spectrum with 168 µs spin echo. Label colors correspond to peak intensity from ¹H T₂ filtered NCA experiment: 15–30%, dark blue; 31–40%, light blue; 41–55%, green; 56–85%, magneta. Reprinted with permission from Bayro et al. (Reference Bayro, Chen, Yau and Tycko2014). Copyright 2014 Elsevier.

4.1.3 MAS NMR of HIV-1 maturation intermediates

Understanding the viral maturation process has been of interest both from the fundamental science standpoint and for the development of small molecule maturation inhibitors as a venue for HIV-1 treatment. Despite intense research into the maturation mechanism, many key questions remain open, including how the conical CA capsids form from the immature Gag lattice. Two hypotheses have emerged to explain how capsid reorganization takes place upon maturation: (i) during maturation, the capsid gradually changes shape from spherical to conical, and (ii) cleavage of SP1 triggers disassembly of the immature lattice and subsequent de novo reassembly of the mature capsid core. Recently, a small molecule BVM was discovered, which was shown to inhibit viral maturation by abolishing the final step, the cleavage of the SP1 peptide from the CA-SP1 maturation intermediate (Fig. 22, panel 1) (Adamson et al. Reference Adamson, Sakalian, Salzwedel and Freed2010; de Marco et al. Reference De Marco, Muller, Glass, Riches, Krausslich and Briggs2010; Fontana et al. Reference Fontana, Keller, Urano, Ablan, Steven and Freed2016; Nguyen et al. Reference Nguyen, Feasley, Jackson, Nitz, Salzwedel, Air and Sakalian2011). Studies on the BVM interaction with the Gag and maturation intermediates by EM and biochemical methods suggest that the second hypothesis is correct, i.e. capsid formation takes place through de novo assembly rather than by the gradual lattice remodeling (Keller et al. Reference Keller, Huang, England, Waki, Cheng, Heymann, Craven, Freed and Steven2013). These EM studies are limited by resolution, and to gain atomic-level insights on the final step of HIV-1 maturation, MAS NMR spectroscopy was pursued. In a recent investigation, the SP1 peptide was characterized in tubular assemblies of two strains of the CA-SP1 maturation intermediate: wild type HXB2 and NL4-3 A92E mutant (Han et al. Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). Dipolar and scalar-based correlation experiments (Fig. 22, panel 2) revealed that the SP1 peptide in the assembled state is dynamically disordered and does not adopt a stable helical structure proposed on the basis of early solution NMR experiments on isolated SP1 peptide in organic solvents (Datta et al. Reference Datta, Temeselew, Crist, Soheilian, Kamata, Mirro, Harvin, Nagashima, Cachau and Rein2011). The presence of the SP1 peptide affects the conformation and dynamics of CA protein: residues 226–231 of the CTD directly preceding the SP1 peptide, and which are mobile on microsecond timescale in CA assemblies, become more rigid, with the motions occurring on millisecond timescale in the CA-SP1 tubes. Most surprisingly, the dynamics of the CypA loop is modulated by the presence of the SP1 peptide: the loop becomes more flexible. Taken together, the results of this investigation support the de novo reassembly hypothesis (Han et al. Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013; Keller et al. Reference Keller, Huang, England, Waki, Cheng, Heymann, Craven, Freed and Steven2013; Lu et al. Reference Lu, Hou, Zhang, Suiter, Ahn, Byeon, Perilla, Langmead, Hung, GOR'KOV, Gan, Brey, Aiken, Zhang, Schulten, Gronenborn and Polenova2015a).

Fig. 22. (Panel 1) Sequence of Gag polyprotein cleavage during maturation. (Panel 2) (a–d) Direct-DARR, (e–j) INADEQUATE, and (k) CP-DARR spectra of CA (orange) and CA-SP1 (black) NL4–3 strain assembled into tubes. Selected regions show the presence of SP1 peaks not observed in the CA spectra. Reprinted with permission from Han et al. (Reference Han, Hou, Suiter, Ahn, Byeon, Lipton, Burton, Hung, GOR'KOV, Gan, Brey, Rice, Gronenborn and Polenova2013). Copyright 2013 American Chemical Society.

To summarize, MAS NMR is a powerful emerging method for atomic-level characterization of HIV-1 viral assemblies. Recent work has demonstrated the significance of sequence-dependent conformational changes and dynamics of the HIV-1 capsid assemblies, as well as brought to light the importance of dynamic allostery in HIV maturation, infectivity, and interactions with host factor proteins.

4.2.1 Structural characterization of bacteriophage capsid proteins

McDermott and co-workers first characterized the intact Pf1 bacteriophage by MAS NMR, where resonance assignments and secondary structure determination were performed and the feasibility of this approach for atomic-level analysis was established (Goldbourt et al. Reference Goldbourt, Gross, Day and Mcdermott2007b). Pf1 undergoes a cooperative phase transition at 10 °C (Thiriot et al. Reference Thiriot, Nevzorov and Opella2005), with the high- and low-temperature forms having slightly different helical symmetries. Goldbourt et al. mapped chemical shift perturbations observed in ¹³C–¹³C correlation spectra to the structure of Pf1 (Goldbourt et al. Reference Goldbourt, Day and Mcdermott2010). Most chemical shift perturbations appeared in the side chains of residues in the hydrophobic region of the protein, at the interface between subunits. They postulated that adjustments in these hydrophobic regions enabled the temperature transition of what is an overall rigid structure.

Hydration waters play critical roles in biological function, including maintenance of structure and facilitating protein dynamics. McDermott and co-workers (Sergeyev et al. Reference Sergeyev, Bahri, Day and Mcdermott2014), and Sinha and co-workers (Purusottam et al. Reference Purusottam, Rai and Sinha2013) used ¹H–¹⁵N and ¹H–¹³C HETCOR spectroscopy, including MEdium-to-LOng Distance (MELODI) HETCOR (Yao & Hong, Reference Yao and Hong2001) to probe the hydration state of Pf1. REDOR dephasing pulses (Gullion & Schaefer, Reference Gullion and Schaefer1989) were applied during HETCOR experiments to dephase magnetization arising from directly bonded protons, such that observed magnetization is known to come from direct contact with water. McDermott and co-workers also utilized water-selective T₂′-filtering for spectral simplification in some HETCOR experiments and a 3D MELODI HETCOR experiment which incorporates a train of ¹³C and ¹⁵N REDOR dephasing pulses on both ¹³C and ¹⁵N with a DARR ¹³C–¹³C mixing component which allowed for the assignment of numerous protein residues in contact with water, as shown in Fig. 23. SD-HETCOR experiments (HETCOR with ¹H spin diffusion (Kumashiro et al. Reference Kumashiro, Schmidt-Rohr, Murphy, Ouellette, Cramer and Thompson1998)) were also included to allow for the observation of hydration waters on the encapsulated DNA. Hydration sites include surface exposed residues at the N-terminus, as well as C-terminal residues, which contact the interior cavity of the phage, including Arg 44 and Lys 45, which participate in protein–DNA interactions, as discussed below. These results indicate that hydration waters are essential in the protein–DNA interactions, which stabilize the capsid structure. The data also revealed hydrophilic groves in the coat protein, which allow for water exchange between the interior of the phage and the exterior surface.

Fig. 23. (a) ¹H-¹³C MELODI-HETCOR of Pf1. (b) ¹³C–¹³C slice of a ¹H–¹³C–¹³C 3D spectrum at the water frequency. (c) Pf1 subunit; residues interacting with water are shaded purple. Hydrated residues are concentrated at the N- and C-termini. Reprinted with permission from Sergeyev et al. (Reference Sergeyev, Bahri, Day and Mcdermott2014). Copyright 2014 AIP Publishing.

Goldbourt and co-workers reported the structure of the capsid protein from intact M13 bacteriophage (Morag et al. Reference Morag, Sgourakis, Baker and Goldbourt2015). This is the first structure of an intact filamentous virus capsid solved by MAS NMR. The intra- and inter-subunit distance restraints were established using selectively labeled samples (Fig. 24a ) (Morag et al. Reference Morag, Abramov and Goldbourt2011). The ‘fold-and-dock’ protocol of CS-ROSETTA (Das et al. Reference Das, Andre, Shen, Wu, Lemak, Bansal, Arrowsmith, Szyperski and Baker2009; Shen et al. Reference Shen, Lange, Delaglio, Rossi, Aramini, Liu, Eletsky, Wu, Singarapu, Lemak, Ignatchenko, Arrowsmith, Szyperski, Montelione, Baker and Bax2008) containing 35 M13 subunits to capture all unique interactions was used (DiMaio et al. Reference Dimaio, Leaver-Fay, Bradley, Baker and Andre2011). This protocol is designed to determine optimum structures of higher-order oligomers. M13 and other Ff class bacteriophages have fivefold subunit symmetry around the virion axis with largely α-helical secondary structure. Many inter-subunit interactions were observed in hydrophobic regions, highlighting the importance of the hydrophobic pockets for subunit packing. Each monomer was found to participate in four hydrophobic pockets, with participating residues distributed across the monomer (Fig. 24c ). Many of these key aromatic/hydrophobic residues are conserved among filamentous bacteriophages. The structure also reveals hydrogen bonding and electrostatic interactions that further contribute to capsid stability.

Fig. 24. (a) ¹³C–¹³C CORD spectrum of [1,3–¹³C]glycerol, U-¹⁵N M13 bacteriophage with 500 ms mixing time. Intra-residue contacts are labeled in black. Inter-residue contacts within the same subunit are labeled in green. Inter-residue contacts between residues in different subunits are labeled in blue. Select inter-subunit correlations are shown on the structure. (b) Sideview of the NMR-ROSETTA model of M13 containing 35 subunits, the minimum number of subunits required to contain all unique interactions. (c) Hydrophobic pockets formed by several subunits. Adapted with permission from Morag et al. (Reference Morag, Sgourakis, Baker and Goldbourt2015). Copyright 2015 National Academy of Sciences.

4.2.2 Characterization of bacteriophage capsid dynamics

Lorieau et al. probed backbone dynamics in Pf1 by Lee–Goldburg cross-polarization experiments (Lorieau et al. Reference Lorieau, Day and Mcdermott2008). ¹H–¹³C DOPs were measured for several C _α , C _β , C _γ , and C _δ sites. The backbone of Pf1 was found to be remarkably rigid. As would be expected, many solvent-exposed side chains exhibited reduced order parameters. Significantly, Arg 44 and Lys 45 side chains, which are directed into the phage cavity, were also observed to be highly mobile. It was postulated that the dynamics of these two residues is essential for mediating DNA–protein interactions. Opella and co-workers also measured ¹H–¹⁵N dipolar lineshapes of this system with ¹H-detected experiments at 50 kHz MAS (Park et al. Reference Park, Yang, Opella and Mueller2013).

4.2.3 Characterization of protein–nucleic acid interactions in bacteriophages

Nucleic acids are an integral component of viruses (Speir & Johnson, Reference Speir and Johnson2012). Recently several labs have demonstrated that MAS NMR can characterize nucleic acids (Cherepanov et al. Reference Cherepanov, Glaubitz and Schwalbe2010; Huang et al. Reference Huang, Bardaro, Varani and Drobny2012) and protein–nucleic acid interactions (Asami et al. Reference Asami, Rakwalska-Bange, Carlomagno and Reif2013; Asami & Reif, Reference Asami and Reif2013; Marchanka et al. Reference Marchanka, Simon, Althoff-Ospelt and Carlomagno2015, Reference Marchanka, Simon and Carlomagno2013) including studies of protein–DNA interactions in intact bacteriophages (Morag et al. Reference Morag, Abramov and Goldbourt2014; Sergeyev et al. Reference Sergeyev, Day, Goldbourt and Mcdermott2011; Yu & Schaefer, Reference Yu and Schaefer2008).

In contrast to other bacteriophages, Pf1 has an unusually high nucleotide-to-capsid subunit ratio of 1:1 (Day et al. Reference Day, Marzec, Reisberg and Casadevall1988) and a highly extended conformation. (Other inoviruses have ratios of between 2–2.5 and 1.) Using dynamic nuclear polarization (DNP, (Ni et al. Reference Ni, Daviso, Can, Markhasin, Jawla, Swager, Temkin, Herzfeld and Griffin2013)) for sensitivity enhancement, Sergeyev et al. were able to detect nucleotide resonances in the intact Pf1 bacteriophage (Sergeyev et al. Reference Sergeyev, Day, Goldbourt and Mcdermott2011). The reported chemical shifts for the deoxyribose moieties capture the unusual DNA conformation of Pf1. As compared with average B-form DNA chemical shifts, C2′, C3′, C4′, and C5′ shifts in Pf1 appeared downfield, indicating a C2′-endo/gauche conformation with an anti glycosidic bond orientation. Nucleotide base chemical shifts, such as those of TC7 and CC5 fall outside the averages reported in the BMRB (Ulrich et al. Reference Ulrich, Akutsu, Doreleijers, Harano, Ioannidis, Lin, Livny, Mading, Maziuk, Miller, Nakatani, Schulte, Tolmie, Kent Wenger, Yao and Markley2008) indicating that Pf1 DNA does not form typical base-pairing interactions.

Goldbourt, Morag, and Abramov used ¹³C–¹³C and ¹H-mediated ³¹P–¹³C correlation experiments to characterize capsid–ssDNA interactions in the fd bacteriophage (Abramov et al. Reference Abramov, Morag and Goldbourt2011; Morag et al. Reference Morag, Abramov and Goldbourt2014). Initial studies demonstrated that well-resolved spectra with narrow peaks could be attained for the wild-type coat protein, enabling resonance assignments and further structural characterization (Abramov et al. Reference Abramov, Morag and Goldbourt2011). Prior structural studies with static SSNMR, cryo-EM, and fiber diffraction relied on the rigid Y21M mutant (Zeri et al. Reference Zeri, Mesleh, Nevzorov and Opella2003), as the wild-type protein yielded poor data quality (i.e., broad lines) in SSNMR (Tan et al. Reference Tan, Jelinek, Opella, Malik, Terry and Perham1999) or lack of structural convergence in the case of cryo-EM (Wang et al. Reference Wang, Yu, Overman, Tsuboi, Thomas and Egelman2006). For the study of DNA within fd, the choice of labeling scheme was key (Morag et al. Reference Morag, Abramov and Goldbourt2014). By including unlabeled aromatic amino acids in the expression media, the nucleotide resonances were not obscured by protein peaks. Broadband and efficient CORD ¹³C–¹³C correlation experiments (Hou et al. Reference Hou, Yan, Sun, Han, Byeon, Ahn, Concel, Samoson, Gronenborn and Polenova2011a, Reference Hou, Yan, Trebosc, Amoureux and Polenova2013a), described above in Section 2.2.1, were found to be ideal for the observation of DNA resonances, particularly for quaternary carbons with no directly attached protons. Nucleotide-specific assignments were achieved using a nucleotide walk, as shown in Fig. 25. In the CORD spectra acquired with long mixing times, sugar-to-capsid and base-to-capsid DNA–protein interactions were also observed. PHHC proton-mediated ³¹P–¹³C correlation spectra detected capsid-to-phosphate backbone interactions. Altogether, 56 capsid–ssDNA interactions could be assigned. These protein–DNA interactions occur primarily between positively charged Lys side chains near the C-terminus and the DNA phosphate backbone. Fd was found to have a similar sugar conformation to Pf1 (Sergeyev et al. Reference Sergeyev, Day, Goldbourt and Mcdermott2011) but greater tendency for base stacking and a different protein–DNA interface.

Fig. 25. Spin system assignments of DNA in fd bacteriophage. Nucleotide walks are shown for (a) dG – blue and dC – red, and (b) dA – green, and dT – pink. (c) Assignment grid indicating all observed DNA correlations. (d) Expansion of ¹³C–¹³C CORD spectrum. Capsid-to-sugar correlations are labeled green, while intra-nucleotide resonances are labeled black. (e) Model of protein–DNA interactions in fd. Adapted with permission from Morag et al. (Reference Morag, Abramov and Goldbourt2014). Copyright 2014 American Chemical Society.

Due to challenges with respect to spectral overlap and sample preparation, MAS NMR of nucleic acids has been much more limited than protein NMR. Abramov and Goldbourt were able to characterize the 40 kbp double-stranded DNA packaged within bacteriophage T7, a 50 MDa icosahedral phage (Abramov & Goldbourt, Reference Abramov and Goldbourt2014). Using ¹³C–¹³C DARR and ¹⁵N–¹³C TEDOR correlations, all carbons and most nitrogens of the nucleic acid could be detected. The DNA packaged inside the phage was determined to be B-form with expected Watson–Crick base pairing. This study, along with the studies of nucleic acids in fd and Pf1, adds to the growing database of nucleic acid chemical shifts (particularly for large, native systems) and the relationship of chemical shifts to structure.

Yu and Schaefer used ¹⁵N–³¹P REDOR experiments to characterize DNA packaging in bacteriophage T4 with U-¹⁵N and [ε-¹⁵N]Lys-labeled T4 (Yu & Schaefer, Reference Yu and Schaefer2008). The dependence of REDOR dephasing on the ¹⁵N–³¹P distance enabled the determination that the DNA packaged inside T4 is B-form by determining the distance between the phosphate backbone and nitrogen atoms of the nucleic acid bases. T4 is packed into the phage capsid with over 1000 molecules of three lysine-rich proteins (Karam, Reference Karam1994). The very close phosphate–lysine distances (as close as 3·5 Å) indicate that side-chain amine groups have an important role in charge balance in the phage. Further charge balance of DNA inside the phage comes from polyamines and ammonium cations.

Bacteriophages including Pf1, fd, and M13 have also been studied in membrane-associated states, as well as by static SSNMR techniques by Opella and others. These topics are beyond the scope of the current review. We direct readers to the following reviews and key publications: (Bechinger, Reference Bechinger1997; Cross et al. Reference Cross, Tsang and Opella1983; Glaubitz et al. Reference Glaubitz, Grobner and Watts2000; Marassi & Opella, Reference Marassi and Opella2003; Opella et al. Reference Opella, Zeri and Park2008; Park et al. Reference Park, Marassi, Black and Opella2010; Shon et al. Reference Shon, Kim, Colnago and Opella1991; Tan et al. Reference Tan, Jelinek, Opella, Malik, Terry and Perham1999; Thiriot et al. Reference Thiriot, Nevzorov, Zagyanskiy, Wu and Opella2004, Reference Thiriot, Nevzorov and Opella2005; Zeri et al. Reference Zeri, Mesleh, Nevzorov and Opella2003).