Synthetic symmetrization in the crystallization and structure determination of CelA from Thermotoga maritima

Protein crystallization continues to be a major bottleneck in X‐ray crystallography. Previous studies suggest that symmetric proteins, such as homodimers, might crystallize more readily than monomeric proteins or asymmetric complexes. Proteins that are naturally monomeric can be made homodimeric artificially. Our approach is to create homodimeric proteins by introducing single cysteines into the protein of interest, which are then oxidized to form a disulfide bond between the two monomers. By introducing the single cysteine at different sequence positions, one can produce a variety of synthetically dimerized versions of a protein, with each construct expected to exhibit its own crystallization behavior. In earlier work, we demonstrated the potential utility of the approach using T4 lysozyme as a model system. Here we report the successful application of the method to Thermotoga maritima CelA, a thermophilic endoglucanase enzyme with low sequence identity to proteins with structures previously reported in the Protein Data Bank. This protein had resisted crystallization in its natural monomeric form, despite a broad survey of crystallization conditions. The synthetic dimerization of the CelA mutant D188C yielded well‐diffracting crystals with molecules in a packing arrangement that would not have occurred with native, monomeric CelA. A 2.4 Å crystal structure was determined by single anomalous dispersion using a seleno‐methionine derivatized protein. The results support the notion that synthetic symmetrization can be a useful approach for enlarging the search space for crystallizing monomeric proteins or asymmetric complexes.     

Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag compatible with X‐ray crystallography

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein‐coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized‐metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X‐ray crystallography.     

Structural templates for modeling homodimers

Oligomeric proteins are more abundant in nature than monomeric proteins, and involved in all biological processes. In the absence of an experimental structure, their subunits can be modeled from their sequence like monomeric proteins, but reliable procedures to build the oligomeric assembly are scarce. Template‐based methods, which start from known protein structures, are commonly applied to model subunits. We present a method to model homodimers that relies on a structural alignment of the subunits, and test it on a set of 511 target structures recently released by the Protein Data Bank, taking as templates the earlier released structures of 3108 homodimeric proteins (H‐set), and 2691 monomeric proteins that form dimer‐like assemblies in crystals (M‐set). The structural alignment identifies a H‐set template for 97% of the targets, and in half of the cases, it yields a correct model of the dimer geometry and residue–residue contacts in the target. It also identifies a M‐set template for most of the targets, and some of the crystal dimers are very similar to the target homodimers. The procedure efficiently detects homology at low levels of sequence identities, and points to erroneous quaternary structures in the Protein Data Bank. The high coverage of the target set suggests that the content of the Protein Data Bank already approaches the structural diversity of protein assemblies in nature, and that template‐based methods should become the choice method for modeling oligomeric as well as monomeric proteins.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Can the propensity of protein crystallization be increased by using systematic screening with metals?

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well‐ordered epitopes possessing complementary surface and capable of producing stable inter‐protein interactions generate a regular three‐dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal‐mediated oligomerization and crystallization together with our experience on metal‐mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non‐redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ～14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.     

A survey of hemoglobin quaternary structures

We perform an analysis of the quaternary structure and dimer/dimer interface in the crystal structures of 165 human hemoglobin tetramers; 112 are in the T, 17 the R, 14 the Y (or R2) state; 11 are high-affinity T state mutants, and 11 may either be intermediates between the states, or off the allosteric transition pathway. The tertiary structure is fixed within each state, in spite of the different ligands, mutations, and chemical modifications present in individual entries. The geometry of the tetramer assembly is essentially the same in all the R or the Y state entries; it is slightly different in high salt and low salt crystals of T state hemoglobins. The dimer/dimer interface differs in terms of size, chemical composition and polar interactions, between the states. It is loosely packed, like crystal packing contacts or the subunit interface of weakly associated homodimers, and unlike most oligomeric proteins, which have close-packed interfaces. The loose packing is most obvious in the liganded forms, where the tetramer is known to dissociate at low concentration. We identify cavities that contribute to the loose packing of the α1β2 and α2β1 contacts. Two pairs of cavities occur recurrently in both the T and the R state tetramers. They may contribute to the allosteric mechanism by facilitating the subunit movements and the tertiary structure changes that accompany the transition from T to R to Y.     

The high‐molecular‐weight kininogen Domain 5 is an intrinsically unstructured protein and its interaction with ferritin is metal mediated

High‐molecular‐weight kininogen domain 5 (HK5) is an angiogenic modulator that is capable of inhibiting endothelial cell proliferation, migration, adhesion, and tube formation. Ferritin can bind to a histidine–glycine–lysine‐rich region within HK5 and block its antiangiogenic effects. However, the molecular intricacies of this interaction are not well understood. Analysis of the structure of HK5 using circular dichroism and nuclear magnetic resonance [¹H, ¹⁵N]‐heteronuclear single quantum coherence determined that HK5 is an intrinsically unstructured protein, consistent with secondary structure predictions. Equilibrium binding studies using fluorescence anisotropy were used to study the interaction between ferritin and HK5. The interaction between the two proteins is mediated by metal ions such as Co²⁺, Cd²⁺, and Fe²⁺. This metal‐mediated interaction works independently of the loaded ferrihydrite core of ferritin and is demonstrated to be a surface interaction. Ferritin H and L bind to HK5 with similar affinity in the presence of metals. The ferritin interaction with HK5 is the first biological function shown to occur on the surface of ferritin using its surface‐bound metals.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Prediction of phenotypes of missense mutations in human proteins from biological assemblies

Single nucleotide polymorphisms (SNPs) are the most frequent variation in the human genome. Nonsynonymous SNPs that lead to missense mutations can be neutral or deleterious, and several computational methods have been presented that predict the phenotype of human missense mutations. These methods use sequence‐based and structure‐based features in various combinations, relying on different statistical distributions of these features for deleterious and neutral mutations. One structure‐based feature that has not been studied significantly is the accessible surface area within biologically relevant oligomeric assemblies. These assemblies are different from the crystallographic asymmetric unit for more than half of X‐ray crystal structures. We find that mutations in the core of proteins or in the interfaces in biological assemblies are significantly more likely to be disease‐associated than those on the surface of the biological assemblies. For structures with more than one protein in the biological assembly (whether the same sequence or different), we find the accessible surface area from biological assemblies provides a statistically significant improvement in prediction over the accessible surface area of monomers from protein crystal structures (P = 6e‐5). When adding this information to sequence‐based features such as the difference between wildtype and mutant position‐specific profile scores, the improvement from biological assemblies is statistically significant but much smaller (P = 0.018). Combining this information with sequence‐based features in a support vector machine leads to 82% accuracy on a balanced dataset of 50% disease‐associated mutations from SwissVar and 50% neutral mutations from human/primate sequence differences in orthologous proteins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

Coordination chemistry of copper proteins: how nature handles a toxic cargo for essential function

Biological copper is coordinated predominantly by just three ligand types: the side chains of histidine, cysteine, and methionine, with of course some exceptions. The arrangement of these components, however, is fascinating. The diversity provided by just these three ligands provides choices of nitrogen vs. sulfur, neutral vs. charged, hydrophilic vs. hydrophobic, susceptibility to oxidation, and degree of pH-sensitivity. In this review we examine how the total number of ligands, their spatial arrangement and solvent accessibility, the various combinations of imidazole, thiolate, and thioether donors, all work together to provide binding sites that either enable copper to carry out a function, or safely transport it in a way that prevents toxic reactivity. We separate copper proteins into two broad classes, those that utilize the metal as a cofactor, or those that traffic the metal. Enzymes and proteins that utilize copper as a cofactor use high affinity sites of high coordination numbers of 4-5 that prevent loss of the metal during redox cycling. Copper trafficking proteins, on the other hand, promote metal transfer either by having low affinity binding sites with moderate coordination number ~ 4, or by having lower coordinate binding sites of 2-3 ligands that bind with high affinity. Both strategies retain the metal but allow transfer under appropriate conditions. Analysis of studies from our own lab on model peptides, combined with those from other labs, raises an interesting hypothesis that various methionine/histidine/cysteine combinations provide organisms with dynamic, multifunctional domains on copper trafficking proteins that facilitate copper transfer under different extracellular, subcellular, and tissue-specific scenarios of pH, redox environment, and presence of other copper carriers or target proteins.     

Crystal structures of MBP fusion proteins

Although chaperone‐assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose‐binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter‐domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP‐mediated structural distortions are very rare.     

Comparative analyses of quaternary arrangements in homo‐oligomeric proteins in superfamilies: Functional implications

A comprehensive analysis of the quaternary features of distantly related homo‐oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo‐oligomeric proteins have the same quaternary state. Considering these homologous homo‐oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo‐oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as “Russian doll effect.” The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo‐oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo‐oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo‐oligomers. Proteins 2016; 84:1190–1202. © 2016 Wiley Periodicals, Inc.     

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn²⁺ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn²⁺ and Cd²⁺ chelator with capacity to bind 10 Zn²⁺ ions per C terminus. When AtHMA4 is expressed in a Zn²⁺-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn²⁺ and Cd²⁺ tolerance and lowered Zn²⁺ and Cd²⁺ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn²⁺ and Cd²⁺ chelator (sensor) and as a regulator of the efficiency of Zn²⁺ and Cd²⁺ export. The identification of a post-translational handle on Zn²⁺ and Cd²⁺ transport efficiency opens new perspectives for regulation of Zn²⁺ nutrition and tolerance in eukaryotes.     

The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

Metallohistins: a new class of plant metal-binding proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. A reversible shift in histidine C1 and C2 protons in NMR analysis occurred during titration of this protein with ZnCl₂ strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.     

Process intensification for the removal of poly‐histidine fusion tags from recombinant proteins by an exopeptidase

This study describes the use of a hexa‐histidine tagged exopeptidase for the cleavage of hexa‐histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On‐column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on‐column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post‐load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post‐load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa‐histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa‐histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on‐column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on‐column exopeptidase cleavage makes the integration of the poly‐histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly‐histidine tag removal can now be combined with the on‐column tag cleavage operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

We describe the use of racemic crystallography to determine the X‐ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L ‐plectasin and D ‐plectasin were prepared by total chemical synthesis; interestingly, L ‐plectasin showed the expected antimicrobial activity, while D ‐plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X‐ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L ‐plectasin molecule and one D ‐plectasin molecule forming the unit cell. Dimer‐like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.     

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.