Optimal oxidative folding of the novel antimicrobial cyclotide from Hedyotis biflora requires high alcohol concentrations

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.     

Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE

Many biologically active natural peptides are synthesized by nonribosomal peptide synthetases (NRPS). Product release is accomplished by dedicated thioesterase (TE) domains, some of which catalyze an intramolecular cyclization to form macrolactone or macrolactam cyclic peptides. The excised 28 kDa SrfTE domain, a member of the alpha/beta hydrolase enzyme family, exhibits a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and tolerates its folding to form a cyclic structure. A substrate analog confirms the substrate binding site and suggests a mechanism for substrate acylation/deacylation. Docking of the peptidyl carrier protein domain immediately preceding SrfTE positions the 4'-phosphopantheinyl prosthetic group that transfers the nascent acyl-peptide chain to SrfTE. The structure provides a basis for understanding the mechanism of acyl-PCP substrate recognition and for the cyclization reaction that results in release of the macrolactone cyclic heptapeptide.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.     

Low-molecular-mass surfactant protein type 1. The primary structure of a hydrophobic 8-kDa polypeptide with eight half-cystine residues

The low-molecular-mass surfactant protein fraction, soluble in chloroform/methanol, contains at least two separate polypeptide chains. The 8-kDa form (type I) was isolated, [14C]carboxymethylated after reduction, and submitted to structural analysis. Its highly hydrophobic nature complicated purification, proteolytic cleavages, and sequence analysis. Acid hydrolysis in 6 M HCl for 7 days was necessary for release of branched-chain residues in full yield. Pepsin was the only enzyme found to cleave the surfactant protein and was used to complement peptide generation by chemical cleavage with CNBr. The primary structure deduced consists of 79 residues with 8 half-cystine residues, and a total of 39% branched-chain hydrophobic residues. However, 11 residues are charged at physiological pH, and all properties of the primary structure are not entirely outstanding in relation to those of other proteins. Hydrophobic segments, coupled with a presumably tight folding from the presence of disulfide bridges, probably explain the unusual properties and the solubility in organic solvents.     

The folding of an enzyme. VI. The folding pathway of barnase: comparison with theoretical models.

The sequence of events in the refolding pathway of barnase has been analysed to search for general principles in protein folding. There appears to be a correlation between burying hydrophobic surface area and early folding events. All the regions that fold early interact extensively with the beta-sheet. These interactions involve predominantly hydrophobic interactions and the burial of very extensive hydrophobic areas in which multiple, close, hydrophobic-hydrophobic contacts are established around a central group of aliphatic residues. There is no burial of hydrophilic residues in these regions; those that are partly screened from the solvent make hydrogen bonds. All the regions or interactions that are made late in the folding pathway do not make extensive contacts with the beta-sheet. Their buried hydrophobic regions lack a central hydrophobic residue or residues around which other hydrophobic residues pack. Further, in some of these regions there is an extensive burial of hydrophilic residues. The results are consistent with one of the earlier events in protein folding being the local formation of native-like secondary structure elements driven by local hydrophobic surface burial. A possible candidate for an initiation site is a beta-hairpin between beta-strands 3 and 4 that is conserved in the microbial ribonuclease family. A comparison of structures in this family shows that those regions that can be superimposed, or have sequence homology, correspond to elements of structure that are formed and interact with each other early in the folding pathway, suggesting that some of these residues could be involved in directing the folding process. The data on barnase combined with results from other laboratories suggest the following tentative conclusions for the refolding of small monomeric proteins. (1) The refolding pathway is, at least in part, sequential and of compulsory order. (2) Secondary structure formation is driven by local hydrophobic surface burial and precedes the formation of most tertiary interactions. These elements are then stabilized and sometimes elongated by tertiary interactions. It is plausible that there are stop signals encoded in the linear sequence that prevent the elongation of isolated secondary structure elements in solution to a larger extent than is found in the folded protein. (3) Many tertiary interactions are not very constrained in the intermediate but become more and more defined as the hydrophobic cores consolidate, loop structures form and the configuration of surface residues takes place. The interactions between different elements of secondary structure are the last ones to be consolidated while the interactions within the secondary structure elements are consolidated earlier.(ABSTRACT TRUNCATED AT 400 WORDS)     

Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: a high resolution structural characterization of a peptide model in aqueous solution.

There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.     

Beta-alanine containing peptides: a novel molecular tool for the design of gamma-turns.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.     

NMR study of cyclic peptides with renin inhibitor activity

Several cyclic analogues of renin inhibitors, based on Glu-D-Phe-Lys motif have been investigated by NMR spectroscopy and molecular dynamics calculations (MD). The 15 membered macrocycle, resulting from Glu and Lys side-chain cyclization, exhibits conformational preference. The structural evidence from NMR shows the presence of hydrogen bond between Lys NH and Glu side-chain carbonyl, resulting in a 10 membered pseudo beta-turn-like structure. The structure of the cyclic moiety is similar in all the peptides, which takes at least two conformations around Calpha-Cbeta in Glu side chain. The restrained MD calculations further support such observations and show that the macrocycle is fairly rigid, with two conformations about the Glu Calpha-Cbeta bond. The linear peptide appendages, which are essential for activity in cyclic peptides, show an extended structure in the beta-region of Ramchandran plot. These calculations also demonstrate that for the most active peptide, two major conformers each exist about the Calpha-CO bond of the Lys, D-Trp and Leu residues. In this peptide, the cyclic moiety presents a negatively charged surface formed due to the carbonyl oxygens, which are thus available to form hydrogen bonds with the receptor. The linear fragment presents further binding sites with a surface which has the hydrophobic side chains of D-Trp, Leu and D-Met on one side and carbonyls on the other side.     

The importance of the leader sequence for directing lanthionine formation in lacticin 481

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

Molecular dynamics simulations of a highly charged peptide from an SH3 domain: possible sequence-function relationship

A seven-residue peptide that is highly conserved in SH3 domains despite being far from the active site has been shown by NMR to be stable in solution. This peptide, biologically important because it is a likely folding nucleus for SH3 domains, provides a challenging subject for molecular dynamics because it is highly charged. We present stable, 10-ns simulations of both the native-like diverging turn structure and a helical model. Free energies of these two conformations, estimated through MM-PBSA analysis using several force fields, suggest a comparable free energy (DeltaDeltaG < or =6 kcal/mol) for native and helix conformations. NOE intensities calculated from the native trajectory reproduce experimental data quite well, suggesting that the conformations sampled by the trajectory reasonably represent those observed in the NMR experiment. The molecular dynamics results, as well as sequence analysis of a diverse 690-member family of SH3 domain proteins, suggest that the presence of two elements is essential for formation of the diverging turn structure: a pair of residues with low helical propensity in the turn region and, as previously recognized, two hydrophobic residues to close the end of the diverging turn. Thus, these two sequence features may form the structural basis for the function of this peptide as a folding nucleus in this family of proteins.     

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.     

Effect of backbone cyclization on protein folding stability: chain entropies of both the unfolded and the folded states are restricted

Circular versions of a large number of proteins have been designed by connecting the N and C termini via peptide linkers. A motivation for these designs is the assumed enhancement in folding stability, because backbone cyclization reduces the chain entropy of the unfolded state. Here, it is recognized that backbone cyclization also reduces the chain entropy of a flexible peptide linker in the folded state. Specifically, the end-to-end distance of the linker is restricted to fluctuations around the average displacement between the N and C termini of the folded protein. The balance of the chain-entropy reductions in the folded and unfolded states is used to predict the change in the unfolding free energy, deltadeltaG(cycl), by backbone cyclization. Predicted values of deltadeltaG(cycl) are in quantitative agreement with results of a careful study on cyclizing the 34 residue PIN1 WW domain by linkers with two to seen residues. The experimental results of an optimal linker length l=4 and a maximum stabilization of 1.7 kcal/mol are reproduced. Calculations of deltadeltaG(cycl) for a broad selection of circular proteins suggest that the stabilizing effect of backbone cyclization is modest, reflecting entropy reductions in both the unfolded and the folded states.     

A 30-residue fragment of the carp granulin-1 protein folds into a stack of two beta-hairpins similar to that found in the native protein.

Upon air oxidation, a peptide corresponding to the 30-residue N-terminal subdomain of carp granulin-1 spontaneously formed the disulfide pairing observed in the native protein. Structural characterization using NMR showed the presence of a defined secondary structure within this peptide. The chemical shifts for most of the alphaCH protons of the peptide and the protein are very similar, and the observed NOE contacts of the peptide strongly resemble those in the protein. A structure calculation of the peptide using NOE distance constraints indicates that the peptide fragment adopts the same conformation as formed within the native protein. The 30-residue N-terminal peptide of carp granulin-1 is the first example of an independently folded stack of two beta-hairpins reinforced by two interhairpin disulfide bonds. Two key areas of the structure show a clustering of hydrophobic residues that may account for its exceptional conformational stability.     

Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity and flexibility

Antigenic site A of foot-and-mouth disease virus (serotype C) has been reproduced by means of cyclic versions of peptide A15, YTASARGDLAHLTTT, corresponding to residues 136-150 of envelope protein VP1. A structural basis for the design of the cyclic peptides is provided by crystallographic data from complexes between the Fab fragments of anti-site A monoclonal antibodies and A15, in which the bound peptide is folded into a quasi-cyclic pattern. Head-to-tail cyclizations of A15 do not provide peptides of superior antigenicity. Internal disulfide cyclization, however, leads to analogs which are recognized as one to two orders of magnitude better than linear A15 in both ELISA and biosensor experiments. CD and NMR studies show that the best antigen, CTASARGDLAHLTT-Ahx-C (disulfide), is very insensitive to environment-induced conformational change, suggesting that cyclization helps to stabilize a bioactive-like structure.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

Protein Structure Classification Based on Conserved Hydrophobic Residues

Protein folding is frequently guided by local residue interactions that form clusters in the protein core. The interactions between residue clusters serve as potential nucleation sites in the folding process. Evidence postulates that the residue interactions are governed by the hydrophobic propensities that the residues possess. An array of hydrophobicity scales has been developed to determine the hydrophobic propensities of residues under different environmental conditions. In this work, we propose a graph-theory-based data mining framework to extract and isolate protein structural features that sustain invariance in evolutionary-related proteins, through the integrated analysis of five well-known hydrophobicity scales over the 3D structure of proteins. We hypothesize that proteins of the same homology contain conserved hydrophobic residues and exhibit analogous residue interaction patterns in the folded state. The results obtained demonstrate that discriminatory residue interaction patterns shared among proteins of the same family can be employed for both the structural and the functional annotation of proteins. We obtained on the average 90 percent accuracy in protein classification with a significantly small feature vector compared to previous results in the area. This work presents an elaborate study, as well as validation evidence, to illustrate the efficacy of the method and the correctness of results reported.     

Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.     

Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.