Folding of apomyoglobin is characterized by formation of a compact intermediate that contains substantial helicity. To determine whether this intermediate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench-flow hydrogen-exchange pulse labeling techniques with electrospray ionization mass spectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway containing an obligatory intermediate with a significant hydrogen-bonded secondary structure content.

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local α-helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local β-sheet form as viewed from Gly10 and Phe22, and both β-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from α-helix to β-sheet, and from random coil to β-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local β-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic α-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel β-sheets at pH 7.5 and a mixture of antiparallel and parallel β-sheets at pH 3.3.

Following hints from X-ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547–10553; Yoshikawa S et al., 1998, Science 280:1723–1729), chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copperB-coordinated His240–Tyr244) cross-link at the O2-activating Heme Fea3–CuB center in the catalytic subunit I of the enzyme. The early evolutionary invention of this unusual structure may have prevented demaging [bull ]OH-radical release at e−-transfer to dioxygen and thus have enabled O2 respiration.

Outer membrane protein A (OmpA) of Escherichia coli is a β-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in β-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

A protein structure comparison method is described that allows the generation of large populations of high-scoring alternate alignments. This was achieved by incorporating a random element into an iterative double dynamic programming algorithm. The maximum scores from repeated comparisons of a pair of structures converged on a value that was taken as the global maximum. This lay 15% over the score obtained from the single fixed (unrandomized) calculation. The effect of the gap penalty was observed through the shift of the alignment populations, characterized by their alignment length and root-mean-square deviation (RMSD). The best (lowest RMSD) values found in these populations provided a base-line against which other methods were compared.

The biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post-translational linkage of biotin to biotin-dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine-rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a number of mononucleotide binding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a three-dimensional structure has been determined, the conserved sequence is found in a partially disordered surface loop. Mutations in the sequence have previously been isolated and characterized in vivo. In this work these single-site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purified and biochemically characterized with respect to binding of small molecule substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP binding, this glycine-rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl-5′-AMP. These results are of general significance for understanding structure-function relationships in biotin holoenzyme synthetases.

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditits elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.

Bovine β-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4–2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4–5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine α-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: α-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.

α-Synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of α-synuclein has not yet been established. We here present biochemical evidence that recombinant α-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, α-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and α-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of α-synuclein, which is believed to reorganize the molecular surface of α-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, α-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, α-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that α-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that α-synuclein could act as a molecular chaperone, and that the conformational change of the α-synuclein could explain the aggregation kinetics of α-synuclein, which may be related to the abolishment of the chaperonic-like activity.

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.

Escherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon-helix-helix (β-α-α) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N-terminal domain of NikR show that it has many features expected of a β-α-α protein including α-helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence-specific manner. Mutation of a residue predicted to be important for DNA-binding reduces operator affinity but does not affect the secondary structure or stability of the protein.

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (1) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

The structural and dynamical behavior of the 41–56 β-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300 K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364–12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 Å resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340–3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1′ substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.

This paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time-dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf-course and funnel-like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics.

A search for the occurrence of the rare π-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the π-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight π-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the π-helix has evolved to provide unique structural features within a variety of proteins.

The “calorimetric criterion” is one of the important experimental approaches for determining whether protein folding is an “all-or-none” two-state transition (i.e., whether intermediates are present at equilibrium). The calorimetric criterion states that the equivalence of the “measured” calorimetric enthalpy change and the effective two-state van't Hoff enthalpy change demonstrates that there is a two-state transition. This paper addresses the essential question of whether the calorimetric criterion is a necessary and sufficient condition for a two-state process and shows that it is necessary but not sufficient by means of specific examples. Analysis of simple models indicates that the heat capacity curve, regardless of whether it originates from a two-state process or not, can always be decomposed in such a way that the calorimetric criterion is satisfied. Exact results for a three-state model and a homopolymer tetramer demonstrate that the deviation from the calorimetric criterion is not simply related to the population of intermediate states. Analysis of a three-helix bundle protein model, which has a two-state folding from a random coil to ordered (molten) globule, shows that the calorimetric criterion may not be satisfied if the standard linear interpolation of baselines (weighted or unweighted) is employed. A specific example also suggests that the more recently introduced deconvolution method is not necessarily better than the simple calorimetric criterion for distinguishing a two-state transition from a three-state transition. Although the calorimetric criterion is not a sufficient condition for a two-state process, it is likely to continue to be of practical utility, particularly when its results are shown to be consistent with those from other experimental methods.

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 Å resolution. The Ile192–Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.