The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded β-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21–32–GSGS-NS33–631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21–32) fused to the full-length NS3 (a.a. 3–631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined “knobs-into-holes” interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar “hot spot.”

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1′)anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 Å2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 ± 1.8 Å3 and 24.7 ± 2.2 Å3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in β-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.

A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (ΔGu), melting temperatures (Tm), and cooperativity. Metropolis-driven simulated annealing and low-temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gβ1, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side-chain conformations remarkably similar to that of native were predicted.

Please note the corrections to the above referenced paper that appeared in Volume 9, Number 11, 2000, pages 2109–2117. On page 2110 (line 22) the KA has been mislabeled as Kdiss (the KD). Also in the legend to Figure 2, KD should be replaced with the KA (on line 2 and in Equation 7 on page 2111).

Please note the corrections to the above referenced paper that appeared in Volume 9, Number 11, 2000, pages 2109–2117. On page 2110 (line 22) the KA has been mislabeled as Kdiss (the KD). Also in the legend to Figure 2, KD should be replaced with the KA (on line 2 and in Equation 7 on page 2111).

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2′deoxyATP (mant-2′deoxyATP), and 3′deoxyATP (mant-3′deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3′deoxyATP is approximately fivefold higher than that for 3′deoxyATP while derivatization of 2′deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2′deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.

Escherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine-lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD′ structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage-site orientation and substrate binding subsites in the UmuD(D′) protease. The structural alignment of SPase and UmuD′ predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases.

We have studied the conformation of β-lactoglobulin in aqueous solution at room temperature over the pH range ∼2.0–9.0 using vibrational Raman optical activity (ROA). The ROA spectra clearly show that the basic up and down β-barrel core is preserved over the entire pH range, in agreement with other studies. However, from the shift of a sharp positive ROA band at ∼1268 to ∼1294 cm−1 on going from pH values below that of the Tanford transition, which is centered at pH ∼7.5, to values above, the Tanford transition appears to be associated with changes in the local conformations of residues in loop sequences possibly corresponding to a migration into the α-helical region of the Ramachandran surface from a nearby region. These changes may be related to those detected in X-ray crystal structures which revealed that the Tanford transition is associated with conformational changes in loops which form a doorway to the interior of the protein. The results illustrate how the ability of ROA to detect loop and turn structure separately from secondary structure is useful for studying conformational plasticity in proteins.

Bacteriorhodopsin (bR) and halorhodopsin (hR) found in the cell membrane of halobacteria are the simplest known light energy-converting systems. They are small proteins and require only light and a lipid bilayer to generate an electrochemical ion gradient. In overall structure, chromophore, and photoreactions, they closely resemble the visual pigments of animals, but those are signal, not energy, transducers and no obvious sequence homologies originally indicated an evolutionary connection. Signal-transducing archaebacterial rhodopsins have also been detected, but they are closely related to the energy transducers. These pigments show the same seven-helix structural motif common to energy-transducing bacterial rhodopsins, visual pigments, and other G-protein-linked sensors. Nevertheless, their sensory function is not mediated by either ion transport or G-proteins, but resembles the chemosensory transduction system of eubacteria. This review, however, will trace only the development of a mechanistic model for the active transport function of bR up to 1995.

Hydrophobic substitutions at solvent-exposed positions in two α-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a tm 6 °C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 °C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1–158 of HIV-p24 (fragment 1–158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 °C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

The assumption that the intrinsic α-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by ∼0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (±0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (±0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a “tripod amphiphile,” that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.

Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize β-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β-hairpin formation, we have designed two 15-residue β-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for β-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β-hairpin folding.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.