Identification of conserved residue patterns in small beta-barrel proteins

Our abilities to predict three-dimensional conformation of a polypeptide, given its amino acid sequence, remain limited despite advances in structure analysis. Analysis of structures and sequences of protein families with similar secondary structural elements, but varying topologies, might help in addressing this problem. We have studied the small beta-barrel class of proteins characterized by four strands (n = 4) and a shear number of 8 (S = 8) to understand the principles of barrel formation. Multiple alignments of the various protein sequences were generated for the analysis. Positional entropy, as a measure of residue conservation, indicated conservation of non-polar residues at the core positions. The presence of a type II beta-turn among the various barrel proteins considered was another strikingly invariant feature. A conserved glycyl-aspartyl dipeptide at the beta-turn appeared to be important in guiding the protein sequence into the barrel fold. Molecular dynamics simulations of the type II beta-turn peptide suggested that aspartate is a key residue in the folding of the protein sequence into the barrel. Our study suggests that the conserved type II beta-turn and the non-polar residues in the barrel core are crucial for the folding of the protein's primary sequence into the beta-barrel conformation.     

Cotranslational, cosecretory protein folding and its renaturation from a denatured state

On the basis of the available experimental data on structure, biosynthesis and secretion of globular proteins it is concluded that an alpha-helix is a starting conformation at formation of the native structure of any globular protein (alpha-helical model for initiation of protein folding). The structural invariant (clusterization of hydrophobic side chains on the alpha-helix surface) in the amino acid sequences of globular proteins is found which is predicted by alpha-helical model for the initiation of protein folding. The model predicts the pyramidization of the atoms C and N of peptide groups during the formation of spatial structure of proteins and a number of other effects that can be put to the experimental test. In the work the mechanism for protein translocation across membrane lipid bilayer is also suggested.     

A folding inhibitor of the HIV-1 protease

Because the human immunodeficiency virus type 1 protease (HIV-1-PR) is an essential enzyme in the viral life cycle, its inhibition can control AIDS. The folding of single-domain proteins, like each of the monomers forming the HIV-1-PR homodimer, is controlled by local elementary structures (LES, folding units stabilized by strongly interacting, highly conserved, as a rule hydrophobic, amino acids). These LES have evolved over myriad generations to recognize and strongly attract each other, so as to make the protein fold fast and be stable in its native conformation. Consequently, peptides displaying a sequence identical to those segments of the monomers associated with LES are expected to act as competitive inhibitors and thus destabilize the native structure of the enzyme. These inhibitors are unlikely to lead to escape mutants as they bind to the protease monomers through highly conserved amino acids, which play an essential role in the folding process. The properties of one of the most promising inhibitors of the folding of the HIV-1-PR monomers found among these peptides are demonstrated with the help of spectrophotometric assays and circular dichroism spectroscopy.     

Structural malleability of plasticins: preorganized conformations in solution and relevance for antimicrobial activity

Plasticins (23 long-residue glycine-leucine-rich dermaseptin-related peptides produced by the skin of South American hylids) have very similar amino acid sequences, hydrophobicities, and amphipathicities, but differ in their membrane-damaging properties and structurations (i.e. destabilized helix states, beta-hairpin, beta-sheet, and disordered states) at anionic and zwitterionic membrane interfaces. Structural malleability of plasticins in aqueous solutions together with parameters that may govern their ability to fold within beta-hairpin like structures were analyzed through circular dichroism and FTIR spectroscopic studies completed by molecular dynamics simulations in polar mimetic media. The goal of this study was to probe to which extent pre-existent peptide conformations, i.e. intrinsic "conformational landscape", may be responsible for variability in bioactive conformation and antimicrobial/hemolytic mechanisms of action of these peptides in relation with their various membrane disturbing properties. All plasticins present a turn region that does not always result in folding into a beta-hairpin shaped conformation. Residue at position 8 plays a major role in initiating the folding, while position 12 is not critical. Conformational stability has no major impact on antimicrobial efficacy. However, preformed beta-hairpin in solution may act as a conformational lock that prevents switch to alpha-helical structure. This lock lowers the antimicrobial efficiency and explains subtle differences in potencies of the most active antimicrobial plasticins.     

Uncovering conserved patterns in bioactive peptides in Metazoa

Bioactive (neuro)peptides play critical roles in regulating most biological processes in animals. Peptides belonging to the same family are characterized by a typical sequence pattern that is conserved among the family's peptide members. Such a conserved pattern or motif usually corresponds to the functionally important part of the biologically active peptide. In this paper, all known bioactive (neuro)peptides annotated in Swiss-Prot and TrEMBL protein databases are collected, and the pattern searching program Pratt is used to search these unaligned peptide sequences for conserved patterns. The obtained patterns are then refined by combining the information on amino acids at important functional sites collected from the literature. All the identified patterns are further tested by scanning them against Swiss-Prot and TrEMBL protein databases. The diagnostic power of each pattern is validated by the fact that any annotated protein from Swiss-Prot and TrEMBL that contains one of the established patterns, is indeed a known (neuro)peptide precursor. We discovered 155 novel peptide patterns in addition to the 56 established ones in the PROSITE database. All the patterns cover 110 peptide families. Fifty-five of these families are not characterized by the PROSITE signatures, and 12 are also not identified by other existing motif databases, such as Pfam and SMART. Using the newly identified peptide signatures as a search tool, we predicted 95 hypothetical proteins as putative peptide precursors.     

Structural assessment of glycyl mutations in invariantly conserved motifs

Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L-disallowed as compared with the L-allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long-range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L-allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L-disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site depending upon the conformational preference of the glycyl residue.     

Identification of a Post-targeting Step Required for Efficient Cotranslational Translocation of Proteins across the Escherichia coli Inner Membrane

Recent studies have shown that cytoplasmic proteins are exported efficiently in Escherichia coli only if they are attached to signal peptides that are recognized by the signal recognition particle and are thereby targeted to the SecYEG complex cotranslationally. The evidence suggests that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by the signal recognition particle were exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position, and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.Since the “signal hypothesis” was proposed over 30 years ago (1), it has become clear that signal sequences are not simply generic hydrophobic peptides that earmark proteins for secretion. In bacteria, the features of a signal peptide determine the mechanism by which a given presecretory protein is targeted to the SecYEG translocation complex in the inner membrane (IM).² Whereas most or all signal peptides are recognized by the signal recognition particle (SRP) in mammalian cells, only a small fraction of Escherichia coli signal peptides are recognized by SRP. These signal peptides are typically extremely hydrophobic (2, 3), but SRP apparently can also recognize slightly less hydrophobic signal peptides that contain a highly basic N terminus (4). SRP recognizes signal peptides as they emerge from translating ribosomes and then targets ribosome-nascent chain complexes to the IM cotranslationally (5). The binding of SRP to its receptor (FtsY), which interacts with the SecYEG complex (6), leads to the release of the nascent chain in the immediate vicinity of the translocation machinery. By targeting nascent polypeptides to the SecYEG complex at an early stage of translation, SRP prevents its substrates from folding into a conformation that is incompatible with translocation through the narrow channel formed by the SecYEG complex (7). Because most signal peptides are not recognized by E. coli SRP, the majority of presecretory proteins are fully synthesized and targeted post-translationally to the IM. These proteins are maintained in a translocation-competent conformation by molecular chaperones such as SecB that keep them unfolded (or loosely folded) (8). Signal peptides themselves also appear to play a role in maintaining translocation competence (9, 10). After mediating the targeting reaction, signal peptides likely play a role in gating open the SecYEG complex to initiate translocation.Interestingly, although signal sequences are the most salient feature of presecretory proteins, they are neither completely necessary nor sufficient to mediate protein export in E. coli (11–13). A version of alkaline phosphatase that lacks a signal peptide is still exported, albeit very inefficiently (11). The export of the leaderless protein, unlike the export of wild-type alkaline phosphatase, is strictly dependent on SecB (11). Conversely, the attachment of signal peptides to cytoplasmic proteins often does not promote their export (14). In light of evidence that folding and export are competing events, these observations led to the proposal that exported proteins tend to fold slowly (or are prevented from folding by chaperones) and therefore remain translocation-competent even without a signal peptide, whereas cytoplasmic proteins fold rapidly into a conformation that is incompatible with export. Recent studies that used thioredoxin as a model protein have validated this hypothesis. Whereas the wild-type protein attached to a typical signal peptide remained trapped in the cytoplasm, four of five slow folding mutants were exported efficiently (15). Furthermore, attachment of a signal peptide that is recognized by SRP to thioredoxin led to efficient export (16). This idea was further confirmed by a report in which various DARPins (designed ankyrin Repeat proteins) were attached to different signal peptides. Most of the DARPins were exported efficiently when they were fused to signal peptides that mediate cotranslational targeting but remained in the cytoplasm when they were attached to signal peptides that are bypassed by SRP (17).Despite these observations, there are several lines of evidence suggesting that export efficiency is not simply dictated by the ability of a protein to reach the SecYEG complex before folding into a translocation-incompetent conformation. For reasons that are unclear, some DARPins are secreted inefficiently even when they are routed into the SRP pathway (17). In addition, numerous reports have indicated that the amino acid composition of the segment of post-translationally targeted presecretory proteins that lies just beyond the signal peptide cleavage site has a dramatic effect on export efficiency. Statistical analysis has shown that the first ∼5–15 residues of the mature region of most presecretory proteins produced by Gram-negative bacteria is neutral or has a net negative charge (18). Consistent with the observed sequence bias, the presence of multiple basic residues at the N terminus of the mature region often leads to accumulation of the secretory precursor, whereas conversion of the basic residues to acidic residues restores export (19–22). Because different combinations of proteins and signal peptides were used in these studies, the exact number and location of charged residues that impinge on the efficiency of export is unclear. In any case, the effect of the net charge in the region distal to the signal peptide on protein export has never been explained. Although basic residues might conceivably promote premature folding of presecretory proteins or block the cleavage of signal peptides by leader peptidase, it is also possible that they inhibit an uncharacterized post-targeting event. Even if effects on signal peptide cleavage could have been ruled out in the aforementioned studies, however, it would not have been possible to distinguish between effects on protein folding and effects on a hypothetical post-targeting step because only proteins that are targeted post-translationally were monitored.To gain further insight into the factors that govern the efficiency of protein export, we sought an explanation for the observation that the cotranslational targeting of at least some cytoplasmic proteins is insufficient to guarantee their translocation across the IM. We found that the export of several different endogenous E. coli cytoplasmic proteins required not only the attachment of a signal peptide that is recognized by SRP but also a net negative charge just past the signal peptide cleavage site. Taken together with previous results, our data show that the charge of the segment just beyond the signal peptide influences export efficiency irrespective of the mechanism by which a protein is targeted to the IM. Because proteins that are targeted cotranslationally reach the IM before they have a chance to fold, our results imply the existence of a post-targeting step (most likely the initiation of translocation) that is facilitated by acidic residues distal to the signal peptide and inhibited or delayed by basic residues. These results help to resolve a long-standing puzzle about the influence of the mature region of presecretory proteins on protein export and have significant implications for optimizing the export of cytosolic and heterologous proteins in E. coli.     

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.     

Ab initio structure prediction for small polypeptides and protein fragments using genetic algorithms

Ab initio folding simulations have been performed on three peptides, using a genetic algorithm-based search method which operates on a full atom representation. Conformations are evaluated with an empirical force field parameterized by a potential of mean force analysis of experimental structures. The dominant terms in the force field are local and nonlocal main chain electrostatics and the hydrophobic effect. Two of the simulated structures were for fragments of complete proteins (eosinophil-derived neurotoxin (EDN) and the subtilisin propeptide) that were identified as being likely initiation sites for folding. The experimental structure of one of these (EDN) was subsequently found to be consistent with that prediction (using local hydrophobic burial as the determinant for independent folding). The simulations of the structures of these two peptides were only partly successful. The most successful folding simulation was that of a 22-residue peptide corresponding to the membrane binding domain of blood coagulation factor VIII (Membind). Three simulations were performed on this peptide and the lowest energy conformation was found to be the most similar to the experimental structure. The conformation of this peptide was determined with a C_α rms deviation of 4.4 Å. Although these simulations were partly successful there are still many unresolved problems, which we expect to be able to address in the next structure prediction experiment. © 1995 Wiley-Liss, Inc.     

A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics.

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.     

Folding of the nascent peptide chain into a biologically active protein

The refolding of denatured proteins with complete sequences may not be fast enough to account for the in vivo folding of growing peptide chains during biosynthesis. As some peptide fragments have secondary structures not unlike those of the corresponding segments in the intact molecules and native disulfide bonds of some proteins can form cotranslationally, it is suggested that the folding of the nascent chain begins early during synthesis. However, further adjustments may be necessary during chain elongation and after posttranslational modifications of the completed peptide chain to generate the native conformation of a biologically active protein.     

PII structure in the model peptides for unfolded proteins: studies on ubiquitin fragments and several alanine-rich peptides containing QQQ, SSS, FFF, and VVV

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far-reaching implications of order in unfolded states for protein folding. Recently, studies on oligo-Ala, oligo-Lys, oligo-Asp, and oligo-Glu, as well as oligo-Pro, have indicated that the left-handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine-rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or beta conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models.     

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.     

AMPs and OMPs: Is the folding and bilayer insertion of β-stranded outer membrane proteins governed by the same biophysical principles as for α-helical antimicrobial peptides?

The folding and function of membrane proteins is controlled not only by specific but also by unspecific interactions with the constituent lipids. In this review, we focus on the influence of the spontaneous lipid curvature on the folding and insertion of peptides and proteins in membranes. Amphiphilic α-helical peptides, as represented by various antimicrobial sequences, are compared with β-barrel proteins, which are found in the outer membrane of Gram-negative bacteria. It has been shown that cationic amphiphilic peptides are always surface-bound in lipids with a negative spontaneous curvature like POPC, i.e. they are oriented parallel to the membrane plane. On the other hand, in lipids like DMPC with a positive curvature, these peptides can get tilted or completely inserted in a transmembrane state. Remarkably, the folding and spontaneous membrane insertion of β-barrel outer membrane proteins also proceeds more easily in lipids with a positive intrinsic curvature, while it is hampered by negative curvature. We therefore propose that a positive spontaneous curvature of the lipids promotes the ability of a surface-bound molecule to insert more deeply into the bilayer core, irrespective of the conformation, size, or shape of the peptide, protein, or folding intermediate. This article is part of a Special Issue entitled: Lipid-protein interactions.     

Complex protein targeting to dinoflagellate plastids

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.     

Binding-induced folding transitions in calpastatin subdomains A and C

Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.     

Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.     

The family feud: do proteins with similar structures fold via the same pathway?

Theoretical and experimental studies of protein folding have suggested that the topology of the native state may be the most important factor determining the folding pathway of a protein, independent of its specific amino acid sequence. To test this concept, many experimental studies have been carried out with the aim of comparing the folding pathways of proteins that possess similar tertiary structures, but divergent sequences. Many of these studies focus on quantitative comparisons of folding transition state structures, as determined by Phi(f) value analysis of folding kinetic data. In some of these studies, folding transition state structures are found to be highly conserved, whereas in others they are not. We conclude that folds displaying more conserved transition state structures may have the most restricted number of possible folding pathways and that folds displaying low transition state structural conservation possess many potential pathways for reaching the native state.     

Comparative X-ray crystallographic evidence for a β-bend conformation as the active structure for peptide T in T4 receptor recognition

A sequence similarity has been found between two segments of endothiapepsin (acid proteinase, 2APE), bovine pancreatic ribonuclease A, and peptide T, a segment of the gp120 protein of human inmmune deficiency virus (HIV), which has been implicated in blocking viral attachment to the T4 receptor. The two similar sequences of the acid proteinase enzyme are Leu-Ile-Asp-Ser-Ser-Ala-Tyr-Thr (residues 169–176) and Tyr-Thr-Gly-Ser-Leu-Asn-Tyr-Thr (residues 175–182). Since the X-ray crystallographic structures of the acid proteinase and ribonuclease are known, it has been possible to determine whether the three-dimensional structures of the segments are similar. Portions of both of the segments of acid proteinase are directly superimposable on the structure of the RNase A 19–26 segment. The fact that the three similar sequences from two completely unrelated proteins give rise to almost identical structures raises the possibility that these segments may be involved in nucleating the folding of these proteins. In addition, this provides further support for the concept that the octapeptide sequence of peptide T of HIV, which is also similar in sequence to the 19–26 sequence of RNase A, is also structurally similar to these residues, which adopt a -bend conformation. Furthermore, comparison of similarities and differences in the structure of these similar sequences provides an explanation for alterations in the biological activity of various truncated or substituted derivatives of peptide T and additional confirmation of the structural requirements for peptide T in T4-receptor recognition.