Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.

The following interproton distances are reported for the decapeptide tyrocidine A in solution: (a) r(phi) distances between NH(i) and H alpha (i), (b) r(psi) distances between NH (i + 1) and H alpha (i), (c) r(phi psi) distances between NH(i + 1) and NH(i), (d) NH in equilibrium NH transannular distances, (e) H alpha in equilibrium H alpha transannular distances, (f) r x 1 distances between H alpha and H beta protons, (g) NH(i) in equilibrium H beta (i) distances, (h) NH (i + 1) in equilibrium H beta (i) distances, (i) carboxamide-backbone protons and carboxamide-side chain proton distances, (j) side chain proton-side chain proton distances. The procedures for distance calculations were: NOE ratios and calibration distances, sigma ratios and calibration distances, and correlation times and sigma parameters. The cross-relaxation parameters were obtained from the product, say, of NOE 1 leads to 2 and the monoselective relaxation rate of proton 2; the NOEs were measured by NOE difference spectroscopy. The data are consistent with a type I beta-turn/ type II' beta-turn/ approximately antiparallel beta-pleated sheet conformation of tyrocidine A in solution and the NOEs, cross-relaxation parameters, and interproton distances serve as distinguishing criteria for beta-turn and beta-pleated sheet conformations. It should be borne in mind that measurement of only r phi and r psi distances for a decapeptide only defines the ( phi, psi)-space in terms of 4(10) possible conformations; the distances b-j served to reduce the degeneracy in possible (phi, psi)-space to one tyrocidine A conformation. The latter conformation is consistent with that derived from scalar coupling constants, hydrogen bonding studies, and proton-chromophore distance measurement, and closely resembles the conformation of gramicidin S.     

Refined structure of elongation factor EF-Tu from Escherichia coli.

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.     

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.     

Optimal relationship between average conformational entropy and average energy of interactions between residues for fast protein folding

Based on the known experimental data and using the theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue, that is an entropy capacity, for fast protein folding. Statistical analysis of conformational entropy and the number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, +/-/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts and average conformational entropy per residue. These class-specific features determine the folding rates: a proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones.     

Conformational analysis of a cyclic thymopoietin-analogue by 1H n.m.r. spectroscopy and restrained molecular dynamics simulations

The internuclear distances of the cyclic thymopoietin derivative c[D-Val-Tyr-Arg-Lys-Glu] have been determined using two-dimensional nuclear Overhauser n.m.r. spectroscopy. These distances are used as constraints for a restrained Molecular Dynamics (MD) simulation. The two starting structures used for the calculations consist of a beta and gamma turn for model 1 and two gamma turns for model 2. The rms difference in atomic positions of the two conformations is 0.242 nm. They converge during the restrained MD simulation to the same final structure. The positional rms difference of the time averaged (5-14 ps) conformations is 0.011 nm. The hydrogen bond pattern is similar to that of model 1, but in addition we find three more gamma turns. The vicinal NH-C alpha H couplings agree well with those calculated from the time averaged structures.     

Three high molecular weight protease inhibitors of rat plasma. Isolation, characterization, and acute phase changes

Rat blood plasma contains three high molecular weight thiol ester-containing proteinase inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2-macroglobulin (alpha 2M). Rat serums have been analyzed using a two-dimensional gel electrophoretic technique which optimizes recovery of high molecular weight proteins. alpha 1M, and (alpha beta)4-tetramer in native solution, separated in the second sodium dodecyl sulfate-containing electrophoretic dimension as a disulfide-linked (alpha beta)2-dimer with an approximate Mr of 360 kDa. alpha 1I3 separated in the gels as a single 190-kDa polypeptide. It is also a monomer in native solution by ultracentrifugation criteria. Native rat alpha 2M is a tetramer, but it separates in the gels as a disulfide-linked dimer with an Mr of approximately 360 kDa. The kinetics of changes in concentration of these proteins during the induction of polyarthritis was also measured by quantitative immunoelectrophoresis. In rats with adjuvant-induced polyarthritis, the concentration of alpha 1I3 dramatically decreases and alpha 2M appears and continues to increase in a biphasic manner for 2 weeks. The alpha 1M concentration remains relatively constant. All three macroglobulins were purified utilizing modern rapid chromatographic techniques, and parallel comparisons of their native physicochemical properties were carried out. The N-terminal sequence of the alpha-chain of rat alpha 1M was also shown to share sequence homology with that of alpha 2M. In agreement, Esnard et al. (Esnard, F., Gutman, N., El Moujahed, A., and Gauthier, F. (1985) FEBS Lett. 182, 125-129) recently reported that alpha 1I3 also contains a thiol ester bond, as do alpha 1M and alpha 2M, since it reacts covalently with [14C]methylamine and is cleaved autolytically at 80 degrees C. We have examined negatively stained preparations of native, trypsin-treated, and methylamine-treated human alpha 2M, rat alpha 2M, and rat alpha 1M in the electron microscope. Trypsin appears to convert globular ring-shaped native molecules to rectangular box-like structures, in agreement with the conclusions of a recent report on human alpha 2M (Tapon-Bretaudiere, J., Bros, A., Couture-Tosi, E., and Delain, E. (1985) EMBO J. 4, 85-89).     

Direct folding simulation of alpha-helices and beta-hairpins based on a single all-atom force field with an implicit solvation model

Recently, we have shown that a modified energy model based on the param99 force field with the generalized Born (GB) solvation model produces reliable free energy landscapes of mini-proteins with a betabetaalpha motif (BBA5, 1FSD, and 1PSV), with the native structures of the mini-proteins located in their lowest free energy minimum states. One of the main features in the modified energy model is a significant improvement for more balanced treatments of alpha and beta strands in proteins. In this study, using the replica exchange molecular dynamics (REMD) simulation method with this new force field, we have carried out extensive ab initio folding studies of several well-known peptides with alpha or beta strands (C-peptide, EK-peptide, le0q, and gbl). Starting from fully extended conformations as the initial conditions, all of the native-like structures of the target peptides were successfully identified by REMD, with reasonable representations of free energy surfaces. The present simulation results with the modified energy model are consistent with experiments, demonstrating an extended applicability of the energy model to folding studies of a variety of alpha-helices, beta-strands, and alpha/beta proteins.     

Predicting protein structure from long-range contacts

Short-range and long-range contacts are important in forming protein structure. The proteins can be grouped into four different structural classes according to the content and topology of alpha-helices and beta-strands, and there are all-alpha, all-beta, alpha/beta and alpha+beta proteins. However, there is much difference in statistical property for those classes of proteins. In this paper, we will discuss protein structure in the view of the relative number of long-range (short-range) contacts for each residue. We find the percentage of residues having a large number of long-range contacts in protein is small in all-alpha class of proteins, and large in all-beta class of proteins. However, the percentage of residues is almost the same in alpha/beta and alpha+beta classes of proteins. We calculate the percentage of residues having the number of long-range contacts greater than or equal to (>/=) N(L)=5, and 7 for 428 proteins. The average percentage is 13.3%, 54.8%, 41.4% and 37.0% for all-alpha, all-beta, alpha/beta and alpha+beta classes of proteins with N(L)=5, respectively. With N(L) increasing, the percentage decreases, especially for all-alpha class of proteins. In the meantime, the percentage of residues having the number of short-range contacts greater than or equal to N(S) (>/=N(S)) in protein samples is large for all-alpha class of proteins, and small for all-beta class of proteins, especially for large N(S). We also investigate the ability of amino residues in forming a large number of long-range and short-range contacts. Cys, Val, Ile, Tyr, Trp and Phe can form a large number of long-range contacts easily, and Glu, Lys, Asp, Gln, Arg and Asn can form a large number of long-range contacts, but with difficulty. We also discuss the relative ability in forming short-range contacts for 20 amino residues. Comparison with Fauchere-Pliska hydrophobicity scale and the percentage of residues having large number of long-range contacts is also made. This investigation can provide some insights into the protein structure.     

Importance of long-range interactions in protein folding

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts. The residues occurring in the range of 4-10 residues apart contribute more towards long-range contacts in all-alpha proteins while the range is 11-20 in all-beta proteins. The hydrophobic residues Cys, Ile and Val prefer the 11-20 range and all other residues prefer the 4-10 range. The residues in all-beta proteins have an average of 3-8 long-range contacts whereas the residues in other classes have 1-4 long-range contracts. Furthermore, the preference of residue pairs to the folding and stability will be discussed.     

Globular protein stability: aspects of interest in protein turnover

The conformational stability of globular proteins is remarkably low. Under physiological conditions, the native globular conformation is only from 5 to 15 kcal/mole more stable than unfolded conformations. In addition, small changes in the structure of a protein such as removing one terminal residue or cleaving a single peptide bond frequently lead to a substantial decrease in the stability. Likewise, single substitutions in the amino acid sequence can increase or decrease the stability by several kilocalories per mole. The low conformational stability of globular proteins and the sensitivity to small changes in structure suggest a possible role for conformational stability in the intracellular degradation of proteins. Several lines of evidence from in vivo studies of protein degradation are consistent with this idea.     

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

More compact protein globules exhibit slower folding rates

We have demonstrated that, among proteins of the same size, alpha/beta proteins have on the average a greater number of contacts per residue due to their more compact (more "spherical") structure, rather than due to tighter packing. We have examined the relationship between the average number of contacts per residue and folding rates in globular proteins according to general protein structural class (all-alpha, all-beta, alpha/beta, alpha+beta). Our analysis demonstrates that alpha/beta proteins have both the greatest number of contacts and the slowest folding rates in comparison to proteins from the other structural classes. Because alpha/beta proteins are also known to be the oldest proteins, it can be suggested that proteins have evolved to pack more quickly and into looser structures.     

Peptide-plane flipping in proteins

A peptide-plane flip is a large-scale rotation of the peptide plane that takes the phi,psi angles at residues i and i + 1 to different structural regions in the Ramachandran plot with a comparatively small effect on the relative orientation of their side chains. This phenomenon, which is expected to play an important role during the early stages of protein folding, has been investigated using 76 proteins for which two high-resolution X-ray conformations are available. Peptide-plane flips are identified by looking for those cases where changes in /psi(i)/ + /phi(i + 1)/ are large (>200 degrees), but changes in /psi(i) + phi(i + 1)/ are comparatively small (<50 degrees). Of a total of 23 cases, the most common peptide-plane flip was identified to be the type I to type II beta-turn interconversion. Although individually rarer, there are many other types of flips that are collectively more common. Given the four main accessible regions alpha(R), alpha(L), beta and epsilon, identified from the phi,psi distribution corresponding to non-hydrogen-bonded peptide planes, 32 main types of peptide-plane flip are identified. Only 8 of these are "passive," in that they require only relatively minor adjustments in the orientation of adjacent peptide planes. Of these, only the type I to type II beta-turn interconversion, denoted, beta(i) + alpha(L)(i + 1) <--> alpha(R)(i) + alpha(R)(i + 1), and the rarer alpha(R)(i) + alpha(L)(i + 1) <--> beta(i) + alpha(R)(i + 1), do not involve the epsilon region. "Active" peptide-plane flips affect the orientation of adjacent peptide planes. The flip, alpha(L)(i) + alpha(L)(i + 1) <--> beta(i) + beta(i + 1), of which one example was found, shows how concerted peptide-plane flips can convert the alpha(L) structure to the beta structure without affecting the relative orientations of the side chains.     

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence and nucleic acid binding properties of bovine leukemia virus nucleocapsid protein

Peptidyl-prolyl cis/trans isomerization, observed in the native state of an increasing number of proteins, is of considerable biological significance. The first evidence for an asymmetric transmission along the polypeptide chain of the structural effects of prolyl isomerization is now derived from the statistics of the C(alpha)/C(alpha)-atom distance distributions in the crystal structures of 848 non-homologous proteins. More detailed information on how isomerization affects segments adjacent to proline is obtained from crystal structures of proteins, that are more than 95% homologous, and that exhibit two different states of isomerization at a particular prolyl bond. The resulting 64 cases, which represent 3.8% of the database used, form pairs of coordinates which were analyzed for the existence of isomer-specific intramolecular nonbonded C(alpha)/C(alpha)-atom distances around the critical proline, and for the positional preferences for particular amino acids in the isomeric sequence segment. The probability that a native protein exhibits both prolyl isomers in the crystalline state increases in particular with a Pro at the third position N-terminal to the isomeric bond (-3 position), and with Ser, Gly and Asp at the position preceding the isomeric bond (-1 position). Structural alignment of matched pairs of isomeric proteins generates three classes with respect to position-specific distribution of C(alpha)-atom displacements around an isomeric proline imide bond. In the majority of cases the distribution of these intermolecular isomer-specific C(alpha)-atom distances shows a symmetric behavior for the N-terminal and C-terminal segment flanking the proline residue, and the magnitude did not exceed 1.3+/-0.6 A including the C(alpha) atoms in proximity to the prolyl bond. However, in the remaining 12 protein pairs the structural changes are unidirectional relative to the isomerizing bond whereby the magnitude of the isomer-specific effect exceeds 3.0+/-2.0 A even at positions remote to proline. Interestingly, the magnitude of the intramolecular isomer-specific C(alpha) atom displacements reveals a lever-arm amplification of the isomerization-mediated structural changes in a protein backbone. The observed backbone effects provide a structural basis for isomer-specific reactions of proline-containing polypeptides, and thus may play a role in biological recognition and regulation.     

Prediction of protein folding from amino acid sequence over discrete conformation spaces

Predicting the three-dimensional structure of a protein given only its amino acid sequence is a long-standing goal in computational chemistry. In the thermodynamic approach, one needs a potential function of conformation that resembles the free energy of the real protein to the extent that the global minimum of the potential is attained by the native conformation and no other. In practice, this has never been achieved with certainty because even with greatly simplified representations of the polypeptide chain, there are an astronomical number of local minima to examine. If one chooses instead a protein representation with only a large but manageable number of discrete conformations, then the global preference of the potential for the native can be directly verified. Representing a protein as a walk on a two-dimensional square lattice makes it easy to see that simple functions of the interresidue contacts are sufficient to globally favor a given "native" conformation, as long as it is a compact, globular structure. Explicit representation of the solvent is not required. Another more realistic way to confine the conformational search to a finite set is to draw alternative conformations from fragments of larger proteins having known crystal structure. Then it is possible to construct a simple function of interresidue contacts in three dimensions such that only 8 proteins are required to determine the adjustable parameters, and the native conformations of 37 other proteins are correctly preferred over all alternative conformations. The deduced function favors short-range backbone-backbone contacts regardless of residue type and long-range hydrophobic associations. Interactions over long distances, such as electrostatics, are not required.     

Energy of stabilization of the right-handed beta alpha beta crossover in proteins

An explanation in terms of conformational energies is provided for the observed nearly exclusive preference of the beta alpha beta structure for forming a right-handed, rather than a left-handed, crossover connection. Conformational energy computations have been carried out on a model beta alpha beta structure, consisting of two six-residue Val beta-strands and of a 12-residue Ala alpha-helix, connected by two flexible four-residue Ala links to the strands. The energy of the most favorable right-handed crossover is 15.51 kcal/mol lower than that of the corresponding left-handed cross-over. The right-handed crossover is a strain-free structure. Its energy of stabilization arises largely from the interactions of the two beta-strands with one another and with the alpha-helix. On the other hand, the left-handed crossover is either disrupted after energy minimization or it remains conformationally strained, as indicated by an energetically unfavorable left twisting of the beta-sheet and by the presence of high-energy local residue conformations. In the energetically most favorable right-handed crossover, the right twisting of the beta-sheet and its manner of interacting with the alpha-helix are identical with those computed earlier for isolated beta-sheets and for packed alpha/beta structures. This result supports a proposed principle that it is possible to account for the main features of frequently occurring structural arrangements in globular proteins in terms of the properties of their component structural elements.     

Distance-constraint approach to protein folding. II. Prediction of three-dimensional structure of bovine pancreatic trypsin inhibitor

The possibility of predicting the three-dimensional structure of bovine pancreatic trypsin inhibitor (BPTI) is examined using a distance-constraint approach. The mean distances between the amino acid residues in globular proteins, calculated in a previous paper, are utilized as distance constraints. In this study, as in previous work of others, root-mean-square deviations of the predicted conformations from the native one of less than 4 Å could not be obtained if the only input information consisted of the mean distances between amino acid residues (which involve information about the hydrophobicity and hydrophilicity of each amino acid residue) and the locations of disuifide bonds, -helices, and -structures. An examination is made of the kinds of structural features of BPTI that appear in the conformations predicted without explicit inclusion of information about such structural features, and of the kinds of information required in a given set of distance constraints for successful folding of BPTI. For example, structures that resemble incipient-forming -helices, bends, and -structures are observed in the conformations predicted when only the mean distances between the amino acid residues and the locations of the disulfide bonds (without information about the locations of -helices, bends, and -structures) are used. One type of additional required information is knowledge of spatially distant pairs involving the active site and the terminal residues. Furthermore, examination of the missing information indicates an important role for the strong nonbonded interaction between sulfur atoms and the side chains of aromatic amino acid residues in BPTI. When such information is introduced into the set of distance constraints, in terms of the exact distances of five pairs, (which implicitly include sulfur/aromatic interactions), the root-mean-square deviations of the predicted conformations decrease to 2.2–3.2 Å. Several methods for comparing conformations are also discussed; in particular, comparisons between conformations of short segments are carried out by a differential-geometry procedure.     

Independent and synergistic effects of disulfide bond formation, beta 2-microglobulin, and peptides on class I MHC folding and assembly in an in vitro translation system.

We have examined the post-translational processing, intrachain disulfide bond formation, folding, and assembly of MHC class I H chains with beta 2-microglobulin after coupled in vitro translation of homogeneous mRNA and transport of nascent chains into canine microsomal vesicles. The formation of native alpha 3 domain conformation was dependent on conditions that optimized intrachain disulfide bond formation, and efficient folding of the alpha 1 alpha 2 domain required exposure to antigenic peptide. beta 2-microglobulin and peptide acted synergistically in forming native alpha 1 alpha 2 domain structure, and a small proportion of molecules with native alpha 1 alpha 2, but non-native alpha 3 structure were detected, indicating that alpha 3 domain folding is not an absolute prerequisite for the formation of native alpha 1 alpha 2 domain structure.