Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets

Cross‐strand disulfides bridge two cysteines in a registered pair of antiparallel β‐strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross‐strand disulfides. Seventy‐six cross‐strand disulfides were found of which 75 and 1 occurred at non‐hydrogen‐bonded (NHB) and hydrogen‐bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T _m . All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG ⁰ = ?3.3 to ?6.7 kcal/mol). The data demonstrate that introduction of cross‐strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

High torsional energy disulfides: relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent beta-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained beta-sheet, are required.     

Insights into the determinants of beta-sheet stability: 1H and 13C NMR conformational investigation of three-stranded antiparallel beta-sheet-forming peptides.

In a previous study we designed a 20-residue peptide able to adopt a significant population of a three-stranded antiparallel beta-sheet in aqueous solution (de Alba et al. [1999]Protein Sci.8, 854-865). In order to better understand the factors contributing to beta-sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and 13Calpha conformational shifts. The populations of the target beta-sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic beta-sheet propensities greatly disfavor beta-sheet folding and that, as already found in other beta-sheet models, specific cross-strand side chain-side chain interactions contribute to beta-sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross-strand side chain-side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor beta-sheet-forming residue, such as Gly.     

Conformational changes in redox pairs of protein structures

Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox‐active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox‐active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox‐active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox‐activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.     

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.     

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.     

Disulfide conformation and design at helix N‐termini

To understand structural and thermodynamic features of disulfides within an α‐helix, a non‐redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty‐five examples were found of intrahelical disulfides involving a CXXC motif between the N‐Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N‐Cap residue for disulfide bonded CXXC motifs had average (?,ψ) values of (?112 ± 25.2°, 106 ± 25.4°). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N‐Cap‐3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N‐Cap‐3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild‐type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N‐Cap and 3 of an α‐helix is likely to have redox activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Contributions of residue pairing to beta-sheet formation: conservation and covariation of amino acid residue pairs on antiparallel beta-strands

In an effort to better understand beta-sheet assembly, we have investigated the evolutionary behavior of neighboring residues on adjacent antiparallel beta-strands. Residue pairs were classified according to solvent exposure as well as by whether their backbone NH and C==O groups are hydrogen bonded. The conservation and covariation of 19,241 pairs in 219 sequence alignments was analyzed. Buried pairs were found to be the most conserved, while stronger covariation was detected in the solvent-exposed pairs. However, residues on neighboring strands showed a degree of conservation and covariation similar to that of well-separated residues on the same strand, suggesting that evolutionary pressure to maintain complementarity between pairs on neighboring strands is weak. Moreover, in spite of the preference of certain amino acid pairs to occupy neighboring positions on adjacent strands, such favored pairs are neither more strongly mutually conserved nor covary more strongly than pairs of the same type in non-interacting positions. Although the beta-sheet pairs did not show outstanding evolutionary coupling, in many protein families significant conservation and covariation patterns were detected for some of the residue pairs. Overall, the weak evolutionary conservation and covariation of the beta-sheet pairs indicates that sheet structure is unlikely to be dictated by specific side-chain interactions.     

Direct evidence for spontaneous branch migration in antiparallel DNA Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms flanking a branch point. In naturally occurring Holliday junctions, the sequence flanking the branch point contains 2-fold (homologous) symmetry. As a consequence of this symmetry, the junction can undergo a conformational isomerization known as branch migration, which relocates the site of branching. In the absence of proteins and in the presence of Mg(2+), the four arms are known to stack in pairs, forming two helical domains whose orientations are antiparallel. Nevertheless, the mechanistic models proposed for branch migration are all predicated on a parallel alignment of helical domains. Here, we have used antiparallel DNA double crossover molecules to demonstrate that branch migration can occur in antiparallel Holliday junctions. We have constructed a DNA double crossover molecule with three crossover points. Two adjacent branch points in this molecule are flanked by symmetric sequences. The symmetric crossover points are held immobile by the third crossover point, which is flanked by asymmetric sequences. Restriction of the helices that connect the immobile junction to the symmetric junctions releases this constraint. The restricted molecule undergoes branch migration, even though it is constrained to an antiparallel conformation.     

Structure of a thermostable disulfide-bridge mutant of phage T4 lysozyme shows that an engineered cross-link in a flexible region does not increase the rigidity of the folded protein

A disulfide bond introduced between amino acid positions 9 and 164 in phage T4 lysozyme has been shown to significantly increase the stability of the enzyme toward thermal denaturation [Matsumura, M., Becktel, W.J., Levitt, M., & Matthews, B. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6562-6566]. To elucidate the structural features of the engineered disulfide, the crystal structure of the disulfide mutant has been determined at 1.8-A resolution. Residue 9 lies in the N-terminal alpha-helix, while residue 164 is located at the extreme C terminus of T4 lysozyme, which is the most mobile part of the molecule. The refined structure shows that the formation of the disulfide bond is accompanied by relatively large (approximately 2.5 A) localized shifts in C-terminal main-chain atoms. Comparison of the geometry of the engineered disulfide with those of naturally observed disulfides in proteins shows that the engineered bridge adopts a left-handed spiral conformation with a typical set of dihedral angles and C alpha-C alpha distance. The geometry of the engineered disulfide suggests that it is slightly more strained than the disulfide of oxidized dithiothreitol but that the strain is within the range observed in naturally occurring disulfides. The wild-type and cross-linked lysozymes have very similar overall crystallographic temperature factors, indicating that the introduction of the disulfide bond does not impose rigidity on the folded protein structure. In particular, residues 162-164 retain high mobility in the mutant structure, consistent with the idea that stabilization of the protein is due to the effect of the disulfide cross-link on the unfolded rather than the folded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ₂, χ₃, χ₂′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ₂～ ? 60°, χ₃～90, χ₂′～ ? 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.     

Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis

A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with C alpha-C alpha distances of less than or equal to 6.5 A and C beta-C beta distances of less than or equal to 4.5 A are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S-S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at less than or equal to 2 A resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.     

Striking stabilization of Arc repressor by an engineered disulfide bond

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.     

ATP synthase b subunit dimerization domain: a right-handed coiled coil with offset helices

The dimerization domain of Escherichia coli ATP synthase b subunit forms an atypical parallel two-stranded coiled coil. Sequence analysis reveals an 11-residue abcdefghijk repeat characteristic of right-handed coiled coils, but no other naturally occurring parallel dimeric structure of this class has been identified. The arrangement of the helices was studied by their propensity to form interhelix disulfide linkages and analysis of the stability and shape of disulfide-linked dimers. Disulfides formed preferentially between cysteine residues in an a position of one helix and either of the adjacent h positions of the partner. Such heterodimers were far more stable to thermal denaturation than homodimers and, on the basis of gel-filtration chromatography studies, were similar in shape to both non-covalent dimers and dimers linked through flexible Gly(1-3)Cys C-terminal extensions. The results indicate a right-handed coiled-coil structure with intrinsic asymmetry, the two helices being offset rather than in register. A function for the right-handed coiled coil in rotational catalysis is proposed.     

Factors governing selective formation of specific disulfides in synthetic variants of alpha-conotoxin

alpha-Conotoxin GI is a snail toxin protein consisting of 13 amino acids cross-linked by 2 intramolecular disulfide bridges. This toxin is an antagonist of acetylcholine receptors. The native sequence has been synthesized, along with nine additional variants in which non-cysteine residues are replaced by alanine or the cysteine positions are altered. Each reduced peptide has been oxidized by reaction with oxygen or glutathione both in a folding buffer and in 6 M guanidine hydrochloride. Purified products of oxidation have been characterized with respect to molecular weights and the positions of disulfides. The four cysteines in conotoxin can form two intramolecular disulfides in three different combinations. Relative yields of each of the three isomers have been determined, thereby permitting evaluation of the roles of non-cysteine residues and cysteine placements in the folding of conotoxin. Cysteine positions dominate factors directing formation of the nativelike isomer in a manner that may be predicted from equilibrium constants for loop formation in model peptides containing two cysteines. Alanine substitutions at several positions which are conserved in naturally occurring conotoxins affect the discrimination between the two most favored disulfide arrangements. Substitutions at three nonconserved positions have no structural effect on isomer yields. It therefore is possible to vary these latter three positions in a manner which might help to generate a functional binding surface which is complementary to receptors in the specific prey of a particular species of snail, without affecting the toxin's folding.     

The Effect of Tensile Stress on the Conformational Free Energy Landscape of Disulfide Bonds

Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C–C–S–S dihedrals, and . Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force–clamp spectroscopy and computer simulation. The and angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so–called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C–C–S–S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two –carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides.     

Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

1H NMR studies of echistatin in solution. Sequential resonance assignments and secondary structure.

Two-dimensional 1H-NMR methods have been used to obtain complete proton resonance assignments for the 49-residue protein echistatin from the viper Echis carinatus. The protein in solution contains only a small amount of regular secondary structure with four very short beta-strands. These beta-strands form two short segments of antiparallel beta-sheet, as evidenced by the observed cross-strand NOE. The first two strands are connected with a tight reverse turn, whereas the remaining two strands are linked together by an 11-residue loop forming a so-called hairpin. The tripeptide unit Arg-Gly-Asp, responsible for the binding of echistatin to the fibrinogen receptor glycoprotein GPIIb/IIIa, is located at the tip of this very hydrophilic loop.     

The crystallographically determined structures of atypical strained disulfides engineered into subtilisin

The geometries of two disulfide bridges genetically engineered into subtilisin have been characterized by x-ray crystallography to determine the structural and energetic constraints involved in introducing disulfide bonds into proteins. Both disulfide bridges (Cys-24-Cys-87 and Cys-22-Cys-87) exhibit atypical sets of dihedral angles compared to those for other reported disulfide structures in proteins. The geometric trends for naturally occurring disulfides in protein crystal structures are examined. Comparison of the disulfide-containing mutant protein structures with the wild-type structure shows that, in both cases, disulfide incorporation is accommodated by relatively minor changes in local main-chain conformation. The Cys-22-Cys-87 disulfide has two high energy dihedral angles (X2 = 121 degrees, X2' = 143 degrees). Both disulfides produce short non-bonded contacts with the main-chain.