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.     

Sequence determinants of thermodynamic stability in a WW domain—An all‐β‐sheet protein

The stabilities of 66 sequence variants of the human Pin1 WW domain have been determined by equilibrium thermal denaturation experiments. All 34 residues composing the hPin1 WW three‐stranded β‐sheet structure could be replaced one at a time with at least one different natural or non‐natural amino acid residue without leading to an unfolded protein. Alanine substitutions at only four positions within the hPin1 WW domain lead to a partially or completely unfolded protein—in the absence of a physiological ligand. The side chains of these four residues form a conserved, partially solvent‐inaccessible, continuous hydrophobic minicore comprising the N‐ and C‐termini. Ala mutations at five other residues, three of which constitute the ligand binding patch on the concave side of the β‐sheet, significantly destabilize the hPin1 WW domain without leading to an unfolded protein. The remaining mutations affect protein stability only slightly, suggesting that only a small subset of side chain interactions within the hPin1 WW domain are mandatory for acquiring and maintaining a stable, cooperatively folded β‐sheet structure.     

An integrated approach for thermal stabilization of a mesophilic adenylate kinase

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure‐guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs. Proteins 2014; 82:1947–1959. © 2014 Wiley Periodicals, Inc.     

Structural analysis of missense mutations in galactokinase 1 (GALK1) leading to galactosemia type‐2

Galactosemia type 2 is an autosomal recessive disorder characterized by the deficiency of galactokinase (GALK) enzyme due to missense mutations in GALK1 gene, which is associated with various manifestations such as hyper galactosemia and formation of cataracts. GALK enzyme catalyzes the adenosine triphosphate (ATP)–dependent phosphorylation of α‐d ‐galactose to galactose‐1‐phosphate. We searched 4 different literature databases (Google Scholar, PubMed, PubMed Central, and Science Direct) and 3 gene‐variant databases (Online Mendelian Inheritance in Man, Human Gene Mutation Database, and UniProt) to collect all the reported missense mutations associated with GALK deficiency. Our search strategy yielded 32 missense mutations. We used several computational tools (pathogenicity and stability, biophysical characterization, and physiochemical analyses) to prioritize the most significant mutations for further analyses. On the basis of the pathogenicity and stability predictions, 3 mutations (P28T, A198V, and L139P) were chosen to be tested further for physicochemical characterization, molecular docking, and simulation analyses. Molecular docking analysis revealed a decrease in interaction between the protein and ATP in all the 3 mutations, and molecular dynamic simulations of 50 ns showed a loss of stability and compactness in the mutant proteins. As the next step, comparative physicochemical changes of the native and the mutant proteins were carried out using essential dynamics. Overall, P28T and A198V were predicted to alter the structure and function of GALK protein when compared to the mutant L139P. This study demonstrates the power of computational analysis in variant classification and interpretation and provides a platform for developing targeted therapeutics.     

Structural,energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity‐creating mutations

The effects of cavity‐creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C‐terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β‐barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. © 2012 Wiley Periodicals, Inc.     

Destabilization of the dimer interface is a common consequence of diverse ALS‐associated mutations in metal free SOD1

Neurotoxic misfolding of Cu, Zn‐superoxide dismutase (SOD1) is implicated in causing amyotrophic lateral sclerosis, a devastating and incurable neurodegenerative disease. Disease‐linked mutations in SOD1 have been proposed to promote misfolding and aggregation by decreasing protein stability and increasing the proportion of less folded forms of the protein. Here we report direct measurement of the thermodynamic effects of chemically and structurally diverse mutations on the stability of the dimer interface for metal free (apo) SOD1 using isothermal titration calorimetry and size exclusion chromatography. Remarkably, all mutations studied, even ones distant from the dimer interface, decrease interface stability, and increase the population of monomeric SOD1. We interpret the thermodynamic data to mean that substantial structural perturbations accompany dimer dissociation, resulting in the formation of poorly packed and malleable dissociated monomers. These findings provide key information for understanding the mechanisms and energetics underlying normal maturation of SOD1, as well as toxic SOD1 misfolding pathways associated with disease. Furthermore, accurate prediction of protein–protein association remains very difficult, especially when large structural changes are involved in the process, and our findings provide a quantitative set of data for such cases, to improve modelling of protein association.     

pH‐selective mutagenesis of protein–protein interfaces: In silico design of therapeutic antibodies with prolonged half‐life

Understanding the effects of mutation on pH‐dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein–protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH‐dependent energy profiles demonstrate excellent agreement with experimentally measured pH‐dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH‐dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH‐selective binding. The method can be used to select mutations that change the pH‐dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH‐dependent binding behavior of IgG to FcRn with the aim of improving the half‐life of therapeutic antibodies in the target organism. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of cancer‐linked BRCA1‐BRCT missense variants and their interaction with phosphoprotein targets

The breast cancer tumor suppressor protein BRCA1 is involved in DNA repair and cell cycle control. Mutations at the two C‐terminal tandem (BRCT) repeats of BRCA1 detected in breast tumor patients were identified either to lower the stability of the BRCT domain and/or to disrupt the interaction of BRCT with phoshpopeptides. The aim of this study was to analyze five BRCT pathogenic mutations for their effect on structural integrity and protein stability. For this purpose, the five cancer‐associated BRCT mutants: V1696L, M1775K, M1783T, V1809F, and P1812A were cloned in suitable prokaryotic protein production vectors, and the recombinant proteins were purified in soluble and stable form for further biophysical studies. The biophysical analysis of the secondary structure and the thermodynamic stability of the wild‐type, wt, and the five mutants of the BRCT domain were performed by Circular Dichroism Spectroscopy (CD) and Differential Scanning Microcalorimetry (DSC), respectively. The binding capacity of the wt and mutant BRCT with (pBACH1/BRIP1) and pCtIP were measured by Isothermal Titration Calorimetry (ITC). The experimental results demonstrated that the five mutations of the BRCT domain: (i) affected the thermal unfolding temperature as well as the unfolding enthalpy of the domain, to a varying degree depending upon the induced destabilization and (ii) altered and/or abolished their affinity to synthetic pBACH1/BRIP1 and pCtIP phosphopeptides by affecting the structural integrity of the BRCT active sites. The presented experimental results are one step towards the elucidation of the effect of various missense mutations on the structure and function of BRCA1‐BRCT. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

Exploiting the Link between Protein Rigidity and Thermostability for Data‐Driven Protein Engineering

Understanding and exploiting the relationship between microscopic structure and macroscopic stability is important for developing strategies to improve protein stability at high temperatures. The thermostability of proteins has been repeatedly linked to an enhanced structural rigidity of the folded native state. In the current study, the rigidity of protein structures from mesophilic and thermophilic organisms along a thermal unfolding trajectory is directly probed. In order to perform this, protein structures were modeled as constraint networks, and the rigidity in these networks was quantified using the Floppy Inclusion and Rigid Substructure Topography (FIRST) method. During the thermal unfolding, a phase transition was observed that defines the rigidity percolation threshold and corresponds to the folded‐unfolded transition in protein folding. Using concepts from percolation theory and network science, a higher phase transition temperature was observed for ca. two‐thirds of the proteins from thermophilic organisms compared to their mesophilic counterparts, when applied to a data set of 20 pairs of homologues. From both the analysis of the microstructure of the constraint networks and monitoring the macroscopic behavior during the thermal unfolding, direct evidence was found for the “corresponding states” concept, which states that mesophilic and thermophilic enzymes are in corresponding states of similar flexibility at their respective optimal temperature. Finally, the current approach facilitated the identification of structural features from which a destabilization of the structure originates upon thermal unfolding. These predictions show a good agreement with the experimental data. Therefore, the information might be exploited in data‐driven protein engineering by pointing to residues that should be varied to obtain a protein with higher thermostability.     

Structure‐Based Site‐Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites

Polyethylene glycol modification (PEGylation) can enhance the pharmacokinetic properties of therapeutic proteins by the attachment of polyethylene glycol (PEG) to the surface of a protein to shield the protein surface from proteolytic degradation and limit aggregation. However, current PEGylation strategies often reduce biological activity, potentially as a result of steric hindrance of PEG. Overall, there are no structure‐based guidelines for selection of conjugate sites that retain optimal biological activity with improved pharmacokinetic properties. In this study, site‐specific PEGylation based on the FGF2‐FGFR1‐heparin complex structure is performed. The effects of the conjugate sites on protein function are investigated by measuring the receptor/heparin binding affinities of the modified proteins and performing assays to measure cell‐based bio‐activity and in vivo stability. Comprehensive analysis of these data demonstrates that PEGylation of FGF2 that avoids the binding sites for fibroblast growth factor receptor 1 (FGFR1) and heparin provides optimal pharmacokinetic enhancement with minimal losses to biological activity. Animal experiments demonstrate that PEGylated FGF2 exhibits greater efficacy in protecting against traumatic brain injury‐induced brain damage and neurological functions than the non‐modified FGF2. This rational structure‐based PEGylation strategy for protein modification is expected to have a major impact in the area of protein‐based therapeutics.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Implications of aromatic–aromatic interactions: From protein structures to peptide models

With increasing structural information on proteins, the opportunity to understand physical forces governing protein folding is also expanding. One of the significant non‐covalent forces between the protein side chains is aromatic–aromatic interactions. Aromatic interactions have been widely exploited and thoroughly investigated in the context of folding, stability, molecular recognition, and self‐assembly processes. Through this review, we discuss the contribution of aromatic interactions to the activity and stability of thermophilic, mesophilic, and psychrophilic proteins. Being hydrophobic, aromatic amino acids tend to reside in the protein hydrophobic interior or transmembrane segments of proteins. In such positions, it can play a diverse role in soluble and membrane proteins, and in α‐helix and β‐sheet stabilization. We also highlight here some excellent investigations made using peptide models and several approaches involving aryl–aryl interactions, as an increasingly popular strategy in protein and peptide engineering. A recent survey described the existence of aromatic clusters (trimer, tetramer, pentamer, and higher order assemblies), revealing the self‐associating property of aryl groups, even in folded protein structures. The application of this self‐assembly of aromatics in the generation of modern bionanomaterials is also discussed.     

Nonspecific yet decisive: Ubiquitination can affect the native‐state dynamics of the modified protein

Ubiquitination is one of the most common post‐translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site‐specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48‐linked tetra‐ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half‐life through proteasomal degradation.     

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild‐type Rop is a homodimeric four‐helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys‐free variant of Rop, including solving the 2.3 Å X‐ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild‐type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine‐free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.     

Computer-aided NMR assay for detecting natively folded structural domains

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.     

DE-loop mutations affect ��2 microglobulin stability, oligomerization, and the low-pH unfolded form

β2 microglobulin (β2m) is the light chain of class‐I major histocompatibility complex (MHC‐I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of β2m aggregation, several β2m mutants have been previously studied. Among these, three single‐residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect β2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild‐type (w.t.) β2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray‐ionization mass spectrometry (ESI‐MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE‐loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, β2m DE‐loop mutations display long‐range effects, affecting stability and structural properties of the native protein and its low‐pH intermediate. The evidence presented here hints to a crucial role played by the DE‐loop in determining the overall properties of native and partially folded β2m.     

Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.     

Mining mammalian genomes for folding competent proteins using Tat‐dependent genetic selection in Escherichia coli

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin‐arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N‐terminal Tat export signal and a C‐terminal selectable marker, namely β‐lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.