Proteins: Structure, Function, Bioinformatics
Crystal structure of Plasmodium
vivax FK506 binding protein 25 reveals conformation changes responsible for its non-canonical activity
The malarial parasites currently remain one of the most dreadful parasites, which show increasing trend of drug resistance to the currently available antimalarial drugs. Thus, the need to identify and characterize new protein targets in these parasites can aid to design novel therapeutic strategies to combat malaria. Recently, the conserved FK506 binding protein family members with molecular weight of 35kDa from Plasmodium falciparum and P. vivax (referred to as PfFKBP35 and PvFKBP35, respectively) were identified for drug targeting. Further data mining revealed a 25kDa FKBP (FKBP25) family member present in the parasites. FKBP25 belongs to a unique class of FKBP, since it is a nuclear FKBP with multiple protein-binding partners. Apart from immune regulation, it is also known for its chaperoning role in various cellular processes such as transcription regulation and trafficking. Here we present the biochemical characterization and 1.9Å crystal structure of an N-terminal truncated FKBP25 from Plasmodium vivax (PvFKBP2572-209). The protein reveals the non-canonical nature with unique structural changes observed in the loops flanking the active site, concealing the binding pocket. Further, a potential calmodulin-binding domain, which is absent in human FKBP25, is observed in this protein. Though the functional implication of Plasmodium FKBP25 in malaria still remains elusive, we speculate that the notable conformational changes in its structure might serve as an overture in understanding its molecular mechanism. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
The targets of CAPRI rounds 20–27
Eight CAPRI prediction rounds with a total of 15 targets were held in the years 2010–2012. Only five of the targets were protein assemblies comparable with those of earlier CAPRI rounds. In one target, the solvent positions at the interface had to be predicted; another was a protein–polysaccharide complex. The remainders were designed complexes issued from protein engineering experiments, and the prediction concerned either their structure or the binding affinity of the designed ligand. Affinity prediction was a new experiment in CAPRI, and a challenge for its participants. It pushed the community into developing novel procedures and score functions that will improve the performance of docking methods, help designing binders, and yield better structure-based estimates of the binding free energy of natural assemblies. Proteins 2013; 81:2075–2081. © 2013 Wiley Periodicals, Inc.
Ab initio protein folding simulations using atomic burials as informational intermediates between sequence and structure
The three-dimensional structure of proteins is determined by their linear amino acid sequences but decipherment of the underlying protein folding code has remained elusive. Recent studies have suggested that burials, as expressed by atomic distances to the molecular center, are sufficiently informative for structural determination while potentially obtainable from sequences. Here we provide direct evidence for this distinctive role of burials in the folding code, demonstrating that burial propensities estimated from local sequence can indeed be used to fold globular proteins in ab initio simulations. We have used a statistical scheme based on a Hidden Markov Model (HMM) to classify all heavy atoms of a protein into a small number of burial atomic types depending on sequence context. Molecular dynamics simulations were then performed with a potential that forces all atoms of each type towards their predicted burial level, while simple geometric constraints were imposed on covalent structure and hydrogen bond formation. The correct folded conformation was obtained and distinguished in simulations that started from extended chains for a selection of structures comprising all three folding classes and high burial prediction quality. These results demonstrate that atomic burials can act as informational intermediates between sequence and structure, providing a new conceptual framework for improving structural prediction and understanding the fundamentals of protein folding. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: Implications for 3-D modelling
With the preponderance of multi-domain proteins in eukaryotic genomes it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multi-domain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the 3D structures of individual domains of two-domain proteins to train Naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (˜85%) and specific (˜95%) to the domain-domain interfaces. This method is specific to multi-domain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest towards rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins
Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier-proteins (PCPs) or acyl-carrier-proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or stand-alone proteins, termed type I and type II, respectively. There have been many biochemical studies of the type I PKS and NRPS CPs, and of type II ACPs. However, recently a number of type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general, but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous NMR analysis of a prototypical type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both type I and type II PCPs. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
The C-terminal domain of the transcriptional regulator BldD from Streptomyces
coelicolor A3(2) constitutes a novel fold of winged-helix domains
BldD regulates transcription of key developmental genes in Streptomyces coelicolor. While the N-terminal domain is responsible for both dimerization and DNA binding, the structural and functional roles of the C-terminal domain (CTD) remain largely unexplored. Here, the solution structure of the BldD-CTD shows a novel winged-helix domain fold not compatible with DNA binding, due to the negatively charged surface and presence of an additional helix. Meanwhile, a small elongated groove with conserved hydrophobic patches surrounded by charged residues suggests that the BldD-CTD could be involved in protein-protein interactions that provide transcriptional regulation. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
Murine norovirus protein NS1/2 aspartate to glutamate mutation sufficient for persistence reorients sidechain of surface exposed tryptophan within a novel structured domain
Compact viral genomes such as those found in noroviruses, which cause significant enteric disease in humans, often encode only a few proteins, but affect a wide range of processes in their hosts and ensure efficient propagation of the virus. Both human and mouse noroviruses persistently replicate and are shed in stool, a highly effective strategy for spreading between hosts. For mouse norovirus (MNV), the presence of a glutamate rather than an aspartate at position 94 of the NS1/2 protein was previously shown to be essential for persistent replication and shedding. Here, we analyze these critical sequences of NS1/2 at the structural level. Using solution NMR methods we determined folded NS1/2 domain structures from a nonpersistent murine norovirus strain CW3, a persistent strain CR6, and a persistent mutant strain CW3D94E. We found an unstructured PEST-like domain followed by a novel folded domain in the N-terminus of NS1/2. All three forms of the domain are stable and monomeric in solution. Residue 94, critical for determining persistence, is located in a reverse turn following an α-helix in the folded domain. The longer sidechain of glutamate, but not aspartate, allows interaction with the indole group of the nearby tryptophan, reshaping the surface of the domain. The discrimination between glutamyl and aspartyl residue is imposed by the stable tertiary conformation. These structural requirements correlate with the in vivo function of NS1/2 in persistence, a key element of norovirus biology and infection. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells
Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals. Proteins 2013. © 2013 Wiley Periodicals, Inc.
The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics
The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs-Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔCp) of folding. If increased or enhanced non-covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow–Klentaq homologous pair, the folding enthalpy (ΔHfold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (ΔGfold) originates from a significantly reduced entropic penalty of folding (ΔSfold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic–thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar “reduced ΔCp mechanism” for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced-entropic-penalty model. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein
PII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site-directed mutagenesis on them in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson-Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ∼5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Antiapoptotic Bcl-2 homolog CED-9 in Caenorhabditis elegans: Dynamics of BH3 and CED-4 binding regions and comparison with mammalian antiapoptotic Bcl-2 proteins
Proteins belonging to Bcl-2 family regulate intrinsic cell death pathway. Although mammalian antiapoptotic Bcl-2 members interact with multiple proapoptotic proteins, the Caenorhabditis elegans Bcl-2 homolog CED-9 is known to have only two proapoptotic partners. The BH3-motif of proapoptotic proteins bind to the hydrophobic groove of prosurvival proteins formed by the Bcl-2 helical fold. CED-9 is also known to interact with CED-4, a homolog of the human cell death activator Apaf1. We have performed molecular dynamics simulations of CED-9 in two forms and compared the results with those of mammalian counterparts Bcl-XL, Bcl-w, and Bcl-2. Our studies demonstrate that the region forming the hydrophobic cleft is more flexible compared with the CED-4-binding region, and this is generally true for all antiapoptotic Bcl-2 proteins studied. CED-9 is the most stable protein during simulations and its hydrophobic pocket is relatively rigid explaining the absence of functional redundancy in CED-9. The BH3-binding region of Bcl-2 is less flexible among the mammalian proteins and this lends support to the studies that Bcl-2 binds to less number of BH3 peptides with high affinity. The C-terminal helix of CED-9 lost its helical character because of a large number of charged residues. We speculate that this region probably plays a role in intracellular localization of CED-9. The BH4-motif accessibility in CED-9 and Bcl-w is controlled by the loop connecting the first two helices. Although CED-9 adopts the same Bcl-2 fold, our studies highlight important differences in the dynamic behavior of CED-9 and mammalian antiapoptotic homologs. Proteins 2013. © 2013 Wiley Periodicals, Inc.
We report the first assessment of blind predictions of water positions at protein–protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions—20 groups submitted a total of 195 models—were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models—a very good docking performance per se—only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein–water interactions and their role in stabilizing protein complexes.Proteins 2013. © 2013 Wiley Periodicals, Inc.
Analyses of the folding properties of ferredoxin-like fold proteins by means of a coarse-grained Gō model: Relationship between the free energy profiles and folding cores
The folding mechanisms of proteins with multi-state transitions, the role of the intermediate states, and the precise mechanism how each transition occurs are significant on-going research issues. In this study, we investigate ferredoxin-like fold proteins which have a simple topology and multi-state transitions. We analyze the folding processes by means of a coarse-grained Gō model. We are able to reproduce the differences in the folding mechanisms between U1A, which has a high-free-energy intermediate state, and ADA2h and S6, which fold into the native structure through two-state transitions. The folding pathways of U1A, ADA2h, S6, and the S6 circular permutant, S6_p54-55, are reproduced and compared with experimental observations. We show that the ferredoxin-like fold contains two common regions consisting folding cores as predicted in other studies and that U1A produces an intermediate state due to the distinct cooperative folding of each core. However, because one of the cores of S6 loses its cooperativity and the two cores of ADA2h are tightly coupled, these proteins fold into the native structure through a two-state mechanism. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Conformational changes in DNA-binding proteins: Relationships with precomplex features and contributions to specificity and stability
Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA-binding proteins by superimposing DNA-bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA-binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Prediction and validation of the unexplored RNA-binding protein atlas of the human proteome
Detecting protein-RNA interactions is challenging both experimentally and computationally because RNAs are large in number, diverse in cellular location and function, and flexible in structure. As a result, many RNA-binding proteins (RBPs) remain to be identified. Here, a template-based, function-prediction technique SPOT-Seq for RBPs is applied to human proteome and its result is validated by a recent proteomic experimental discovery of 860 mRNA-binding proteins (mRBPs). The coverage (or sensitivity) is 42.6% for 1217 known RBPs annotated in the Gene Ontology and 43.6% for 860 newly discovered human mRBPs. Consistent sensitivity indicates the robust performance of SPOT-Seq for predicting RBPs. More importantly, SPOT-Seq detects 2418 novel RBPs in human proteome, 291 of which were validated by the newly discovered mRBP set. Among 291 validated novel RBPs, 61 are not homologous to any known RBPs. Successful validation of predicted novel RBPs permits us to further analysis of their phenotypic roles in disease pathways. The dataset of 2418 predicted novel RBPs along with confidence levels and complex structures is available at http://sparks-lab.org (in publications) for experimental confirmations and hypothesis generation. Proteins 2013. © 2013 Wiley Periodicals, Inc.
The addition of 2,2,2-trifluoroethanol prevents the aggregation of guanidinium around protein and impairs its denaturation ability: A molecular dynamics simulation study
TFE, 2,2,2-trifluoroethanol, and guanidinium (Gdm+) are two typical osmolytes. A great number of experimental and theoretical studies have shown that TFE and Gdm+ can accumulate on protein surface and thus exert their effects on protein structure in their respective solutions. Their accumulation manners are, however, different: the hydrophobic property of TFE makes its accumulation more preferential around hydrophobic side chains than other types whereas Gdm+ prefers to stack strongly against the planar side chains but only weakly binds to the hydrophobic groups. The present molecular dynamics simulation study shows a novel test to investigate the combined effects of TFE and Gdm+ on protein structure in mixed guanidinium/TFE solution. The results indicate that the accumulation of TFE is more competitive than Gdm+ in either GdmSCN/TFE or GdmCl/TFE solution. The preceding accumulation of TFE around protein surface limits the approach of Gdm+ and water to protein. As a result, the hydrogen bonding between Gdm+ and water to protein is highly forbidden and the secondary structure stability of protein is strongly enhanced. In contrast, without the presence of TFE, the protein structure is largely denatured in similarly concentrated GdmSCN or GdmCl solution. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Two 8-µs all-atom molecular dynamics simulations have been performed on the two highly homologous G protein-coupled receptor (GPCR) subtypes, β1- and β2-adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β-adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long-time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I-/V-/L-rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol-binding site for the class-A GPCRs. Proteins 2013. © 2013 Wiley Periodicals, Inc.
For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions. Proteins 2013. © 2013 Wiley Periodicals, Inc.
In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2013. © 2013 Wiley Periodicals, Inc.