Preferred conformations of a linear RGD tripeptide.

Conformational study of RGD tripeptides in the nonhydrated and hydrated states was carried out using an empirical potential function ECEPP/3 and the hydration shell model in order to investigate preferred conformations and factors responsible for their stability. RGD tripeptides in the nonhydrated and hydrated states can be interpreted as existing as an ensemble of feasible conformations rather than as a single dominant conformation from the analysis of distributions of backbone conformations, hydrogen bonds and beta-turns. The different distributions of conformations for the neutral and zwitterionic RGD tripeptides in both states may indicate that the conformation of the RGD tripeptide is liable to depend on solvent polarity and pH values. beta-Turn populations for the neutral tripeptide in both states are reasonably consistent with NMR measurements on linear RGD-containing peptides. The degradation of RGD tripeptide seems to be attributed mainly to the hydrogen bonds between the Asp side-chain and the backbone of Asp residue or C-terminal NHMe group, rather than to the flexible backbones of Gly and Asp residues.     

Conformation dynamics of cyclic disulfides and selenosulfides in CXXC(U) (X = Gly,Ala) tetrapeptide redox motifs

Thioredoxin fold proteins often contain a Cys‐(Xxx)_n‐Cys(Sec) or CX_nC(U) motif, where the active cysteine (C) or selenocysteine (U) is bridged by X residues, which vary with protein function. The effect of the X residues on the conformation space of the oxidized disulfide and selenosulfide forms of the CXXC(U) motif has been investigated using molecular dynamics (MD) and density functional theory. Multi‐microsecond‐length MD simulations of the CGGC, CGAC, and CAGC cyclic peptides show that CGGC rings readily exchange between several conformations over the course of the simulation, but steric interactions with the methyl group of Ala limit the conformation space available to the cyclic peptide, especially for CGAC. The potential for the motif to be reduced, as measured by the energy of the lowest unoccupied molecular orbitals, is dependent upon the ring conformation. These results suggest that control of available conformations by the bridging residues and the protein tertiary structure may be important for defining the function of the CXXC motif. Theoretical ⁷⁷Se chemical shifts of the selenosulfide moiety are dependent upon the conformation and/or intramolecular Se···O interactions with the backbone carbonyl group of the C‐terminal U residue.     

Modeling an active conformation for linear peptides and design of a competitive inhibitor for HMG-CoA reductase

This study presents an approach that can be used to search for lead peptide candidates, including unconstrained structures in a recognized sequence. This approach was performed using the design of a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In a previous design for constrained peptides, a head-to-tail cyclic structure of peptide was used as a model of linear analog in searches for lead peptides with a structure close to an active conformation. Analysis of the conformational space occupied by the peptides suggests that an analogical approach can be applied for finding a lead peptide with an unconstrained structure in a recognized sequence via modeling a cycle using fixed residues of the peptide backbone. Using the space obtained by an analysis of the bioactive conformations of statins, eight cyclic peptides were selected for a peptide library based on the YVAE sequence as a recognized motif. For each cycle, the four models were assessed according to the design criterion ("V" parameter) applied for constrained peptides. Three cyclic peptides (FGYVAE, FPYVAE, and FFYVAE) were selected as lead cycles from the library. The linear FGYVAE peptide (IC(50) = 0.4 microM) showed a 1200-fold increase the inhibitory activity compared to the first isolated LPYP peptide (IC(50) = 484 microM) from soybean. Experimental analysis of the modeled peptide structures confirms the appropriateness of the proposed approach for the modeling of active conformations of peptides.     

Mimicry of beta II'-turns of proteins in cyclic pentapeptides with one and without D-amino acids.

The solution structure of eight cyclic pentapeptides has been determined by two-dimensional 1H-NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C-terminal cholecystokinin fragment CCK-4 enlarged with Asp1 resulting in the sequence (Asp-Trp-Met-Asp-Phe), one L-amino acid after the other was substituted by its D-analog. In addition, two peptides, including an all-L-amino-acid-containing cyclic pentapeptide, cyclo(Asp-Phe-Lys-Ala-Thr) and cyclo(Asp-Phe-Lys-Ala-D-Thr) were investigated. All D-amino-acid-containing peptides show beta II'-turn conformations with the D-amino acid in the i + 1 position, excepting the D-aspartic-acid-containing peptides. These two peptides are characterized by the lack of beta-turns at pH values less than 4, suggesting that D-aspartic acid in the full-protonized state avoids the formation of beta-turns in these compounds. At pH values greater than 5, a conformational change into the beta II'-turn conformation was also observed for these peptides. Conformations without beta-turns are expected for cyclic all-L pentapeptides, but both cyclo(Asp-Phe-Lys-Ala-Thr) and the D-Thr analog cyclo(Asp-Phe-Lys-Ala-D-Thr) exhibit beta II'-turn conformations around Thr-Asp and D-Thr-Asp. Thus cyclic all-L pentapeptides and those with one D-amino acid are able to form similar structures preferably with a beta II'-turn. The beta-turn formation in cyclic pentapeptides containing a D-aspartic acid is dependent on the ionization state. The relevance of the work to the design of beta'-turn mimetics is discussed.     

On the role of the cis-proline residue in the active site of DsbA

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.     

Molecular dynamics study of disulfide bond influence on properties of an RGD peptide.

Three 1 ns length molecular dynamics simulations of an RGD peptide (Ac-Pen-Arg-Gly-Asp-Cys-NH2, with Pen denoting penicillamine) have been performed in aqueous solution, one for the disulfide bridged, and two for the unbridged form. The trajectories were analyzed to identify conformations explored by the two forms and to calculate several properties: NMR vicinal coupling constants, order parameters, dipole moments and diffusion coefficients, in an effort to describe the physical role of the disulfide bond. The cyclic peptide was able to explore several distinct backbone conformations centered around a turn-extended-turn structure. However, its flexibility was limited and it appeared to be 'locked in' into a a family of structures characterized by a high dipole moment and a well-defined conformation of the pharmacophore, which has been previously identified as biologically active. Excellent agreement between the simulated and observed NMR vicinal coupling constants indicates that realistic structures were sampled in the cyclic peptide simulation. The linear form of the peptide was much more flexible than the cyclic one. In the two independent 1 ns simulations of the linear form the explored conformations could be roughly grouped into two classes, of cyclic-like and extended type. Within each simulation the peptide switched between the two classes of structures several times. Exact matches between conformations in the two linear peptide simulations were not found; several conformational regions with backbone rms deviations below 1A were identified, suggesting that representative structures of the linear form have also been identified. In the linear peptide simulations the RGD pharmacophore is able to adopt a wide range of conformations, including the one preferred by the cyclic form. The lower biological activity of the linear peptide compared to the cyclic one may be correlated with the lower population of this structure in the absence of the disulfide bond.     

Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.     

Empirical rules predicting conformation of cyclic tetrapeptides from primary structure

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: A conformation allowing an intramolecular hydrogen bond (IMHB) of gamma-turn is preferred, and an ester bond always adopts a trans form. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. The carbonyl group acylating a D proline or an N-methyl-D-amino acid residue is oriented to the lower side of the ring, forming a cis bond. The LDDL configurational sequence adopts a cis-trans-cis-trans backbone with Ci symmetry. A glycine residue behaves as a D residue in an L-peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo(-L-Pro-L-Leu-D-Tyr(Me)-L-Ile-)(1) and cyclo (-L-Pro-D-Leu-D-Tyr(Me)-L-Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a gamma-turn IMHB, and 2 has a cis-trans-cis-trans backbone with Ci symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (LD) and cis (DD) secondary amide bonds.     

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation

Bis (cysteinyl) octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10^?3 M concentration was found to be very similar, but opposite to that of the K_ox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of β-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (≈ 90%). Similarly, operating under denaturing conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of β-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. © 1994 John Wiley & Sons, Inc. © 1994 John Wiley & Sons, Inc.     

A turning point in the knowledge of the structure-function-activity relations of elastin]

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.     

Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

The utility of side-chain cyclization in determining the receptor-bound conformation of peptides: cyclic tripeptides and angiotensin II.

The effect of side-chain cyclization on accessible backbone conformations of tripeptides, X-Ala-Y (X and/or Y = Cys, Hcy (Hcy: homocysteine), cis 4-mercaptoproline (MPc), and trans 4-mercaptoproline (MPt)), was elucidated using two variants of systematic conformational search. In addition to cyclization through a disulfide bond, the thioether (-S-CH2-) and amide (-CO-NH-) side-chain analogues of Cys-Ala-Cys and Hcy-Ala-Hcy were evaluated. The number of valid backbone conformations and the allowed phi, psi space were evaluated for each compound, and the ability of the cyclic tripeptides to accommodate beta-turn conformations was examined in order to assess the value of cyclization in limiting conformational freedom. Based on the number of conformations, cyclization was highly effective in reducing the backbone degree of freedom: in order of decreasing number of conformations, Ala-Ala-Ala 1 > Hcy-Ala-Hcy 2 > Cys-Ala-Hcy 3 approximately equal to Hcy-Ala-Cys 4 > MPc-Ala-Hcy 5, 7 > Cys-Ala-Cys 6 > MPc-Ala-Cys 8 > Hcy-Ala-MPt 9 > Cys-Ala-MPt 10 approximately equal to MPc-Ala-MPt 11. Although Hcy-Ala-Hcy 2 had the greatest number of conformations of the cyclic peptides studied, it was still greatly constrained relative to its linear analogue 1. The bicyclic ring system introduced by MP was even more effective in constraining the cycle, having greater impact at position 3 than at position 1. Under the conditions of the study, cyclization of MP-containing analogues could be effected only with the cis isomer (MPc) at position 1 and/or the trans isomer (MPt) at position 3. Sterically allowed conformations of Ala2 for the cyclic tripeptides 2-4 were generally similar to those of the linear tripeptide 1, while those of Cys-Ala-Cys 6 and MPc-Ala-Hcy 7 were restricted to a smaller region of phi 2, psi 2 space: the right- and left-handed alpha-helical conformation and the beta-conformation. This trend was even more pronounced for Hcy-Ala-MPt 9, Cys-Ala-MPt 10, and MPc-Ala-MPt 11, in which Ala2 was severely restricted to a very small region of phi, psi space: the left-handed alpha-helical conformation for 9-11, plus the beta conformation for 9. This suggests that MP at the 3-position is incompatible with a right-handed alpha-helical conformation at position 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developing potent backbone cyclic peptides bearing the shared epitope sequence as rheumatoid arthritis drug-leads

Rheumatoid arthritis (RA) is a common human leukocyte antigen-associated disease. Most RA patients have a five-residue sequence motif called the shared epitope (SE) in the DRβ-chain of the HLA-DRB1 protein. The SE was found to activate nitric oxide (NO) production, suggesting a possible mechanism for RA development. The native conformation of the SE is presumed to be an α-helix, thus using cyclic peptides to stabilize this conformation may produce a potent SE mimetic which will have drug-like properties. We present the development of a backbone cyclic SE mimetic that activates NO production in the low nM range. Circular dichroism analysis revealed a conformational change from for the parent linear peptides to the cyclic analogs. The most active cyclic analog is completely stable towards trypsin/chymotrypsin degradation while the linear 15-mer analogs completely degraded within 30 min. The outcome of this study is a potent cyclic peptide with drug-like properties that can be used as a template for drug development.     

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V. hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.     

Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and Biophysical properties

Mammalian defensins (alpha as well as beta forms) have a beta-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine beta-defensin-2 (BNBD-2): VRNHVTC(1)RINRGFC(2)VPIRC(3)PGRTRQIGTC(4)FGPRIKC(5)C(6)RSW (positions of disulfide bonds are C(1)[bond]C(5), C(2)[bond]C(4), and C(3)[bond]C(6)). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of beta-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the beta-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.     

Unfolding the fold of cyclic cysteine-rich peptides

We propose a method to extensively characterize the native state ensemble of cyclic cysteine-rich peptides. The method uses minimal information, namely, amino acid sequence and cyclization, as a topological feature that characterizes the native state. The method does not assume a specific disulfide bond pairing for cysteines and allows the possibility of unpaired cysteines. A detailed view of the conformational space relevant for the native state is obtained through a hierarchic multi-resolution exploration. A crucial feature of the exploration is a geometric approach that efficiently generates a large number of distinct cyclic conformations independently of one another. A spatial and energetic analysis of the generated conformations associates a free-energy landscape to the explored conformational space. Application to three long cyclic peptides of different folds shows that the conformational ensembles and cysteine arrangements associated with free energy minima are fully consistent with available experimental data. The results provide a detailed analysis of the native state features of cyclic peptides that can be further tested in experiment.     

Hydrogen-bonded turns in proteins: the case for a recount

Beta-turns are sites at which proteins change their overall chain direction, and they occur with high frequency in globular proteins. The Protein Data Bank has many instances of conformations that resemble beta-turns but lack the characteristic N-H(i) --> O=C(i - 3) hydrogen bond of an authentic beta-turn. Here, we identify potential hydrogen-bonded beta-turns in the coil library, a Web-accessible database utility comprised of all residues not in repetitive secondary structure, neither alpha-helix nor beta-sheet (http://www.roselab.jhu.edu/coil). In particular, candidate turns were identified as four-residue segments satisfying highly relaxed geometric criteria but lacking a strictly defined hydrogen bond. Such candidates were then subjected to a minimization protocol to determine whether slight changes in torsion angles are sufficient to shift the conformation into reference-quality geometry without deviating significantly from the original structure. This approach of applying constrained minimization to known structures reveals a substantial population of previously unidentified, stringently defined, hydrogen-bonded beta-turns. In particular, 33% of coil library residues were classified as beta-turns prior to minimization. After minimization, 45% of such residues could be classified as beta-turns, with another 8% in 3(10) helixes (which closely resemble type III beta-turns). Of the remaining coil library residues, 37% have backbone dihedral angles in left-handed polyproline II structure.     

Cross‐strand disulfides in the hydrogen bonding site of antiparallel β‐sheet (aCSDhs): Forbidden disulfides that are highly strained,easily broken

Some disulfide bonds perform important structural roles in proteins, but another group has functional roles via redox reactions. Forbidden disulfides are stressed disulfides found in recognizable protein contexts, which currently constitute more than 10% of all disulfides in the PDB. They likely have functional redox roles and constitute a major subset of all redox‐active disulfides. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. Previously we characterized the most abundant forbidden disulfide in the Protein Data Bank, the aCSDn: a canonical motif in which disulfide‐bonded cysteine residues are positioned directly opposite each other on adjacent anti‐parallel β‐strands such that the backbone hydrogen‐bonded moieties are directed away from each other. Here we perform a similar analysis for the aCSDh, a less common motif in which the opposed cysteine residues are backbone hydrogen bonded. Oxidation of two Cys in this context places significant strain on the protein system, with the β‐chains tilting toward each other to allow disulfide formation. Only left‐handed aCSDh conformations are compatible with the inherent right‐handed twist of β‐sheets. aCSDhs tend to be more highly strained than aCSDns, particularly when both hydrogen bonds are formed. We discuss characterized roles of aCSDh motifs in proteins of the dataset, which include catalytic disulfides in ribonucleotide reductase and ahpC peroxidase as well as a redox‐active disulfide in P1 lysozyme, involved in a major conformation change. The dataset also includes many binding proteins.     

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.     

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. coli.