A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

pH-responsive titratable inotropic performance of histidine-modified cardiac troponin I

Cardiac troponin I (cTnI) functions as the molecular switch of the thin filament. Studies have shown that a histidine button engineered into cTnI (cTnI A164H) specifically enhances inotropic function in the context of numerous pathophysiological challenges. To gain mechanistic insight into the basis of this finding, we analyzed histidine ionization states in vitro by studying the myofilament biophysics of amino acid substitutions that act as constitutive chemical mimetics of altered histidine ionization. We also assessed the role of histidine-modified cTnI in silico by means of molecular dynamics simulations. A functional in vitro analysis of myocytes at baseline (pH 7.4) indicated similar cellular contractile function and myofilament calcium sensitivity between myocytes expressing wild-type (WT) cTnI and cTnI A164H, whereas the A164R variant showed increased myofilament calcium sensitivity. Under acidic conditions, compared with WT myocytes, the myocytes expressing cTnI A164H maintained a contractile performance similar to that observed for the constitutively protonated cTnI A164R variant. Molecular dynamics simulations showed similar intermolecular atomic contacts between the WT and the deprotonated cTnI A164H variant. In contrast, simulations of protonated cTnI A164H showed various potential structural configurations, one of which included a salt bridge between His-164 of cTnI and Glu-19 of cTnC. This salt bridge was recapitulated in simulations of the cTnI A164R variant. These data suggest that differential histidine ionization may be necessary for cTnI A164H to act as a molecular sensor capable of modulating sarcomere performance in response to changes in the cytosolic milieu.     

Interaction between chondroitin-6-sulfate and poly-L-arginine in aqueous solution

The interactions between chondroitin-6-sulfate and poly-L -arginine in aqueous salt solution have been investigated by circular dichroism techniques. In the presence of chondroitin-6-sulfate, at neutral pH, poly-L -arginine adopts the α-helical conformation rather than “charged coil” form observed in the absence of mucopolysaccharide. This interaction is at a maximum when the ratio of arginine to disaccharide residues is 2:1. Elevation of the temperature leads to a sharp melting transition at 76.0 ± 1.0°C. This behavior is in marked contrast to that for poly-L -lysine-chondroitin-6-sulfate interactions, which are at a maximum at a 1:1 residue ratio and have a melting transition at 47.0 ± 1.0°C. These results indicate a stronger interaction for poly-L -arginine than for poly-L -lysine. The positive arginine side chains appear to interact with both the negative sulfate and carboxyl residues, while those of the lysines are involved only with the sulfates. Poly-L -ornithine at neutral pH shows no conformation directing interaction with chondroitin-6-sulfate, although a small proportion of α-helix is formed on dilution of the mixture with methanol. The extent of the interaction of cationic polypeptides with chondroitin-6-sulfate increases in the order poly-L -ornithine, poly-L -lysine, poly-L -arginine, i.e., in the order of increasing side-chain length.     

Coarse‐grained simulations of proton‐dependent conformational changes in lactose permease

During lactose/H⁺ symport, the Escherichia coli lactose permease (LacY) undergoes a series of global conformational transitions between inward‐facing (open to cytoplasmic side) and outward‐facing (open to periplasmic side) states. However, the exact local interactions and molecular mechanisms dictating those large‐scale structural changes are not well understood. All‐atom molecular dynamics simulations have been performed to investigate the molecular interactions involved in conformational transitions of LacY, but the simulations can only explore early or partial global structural changes because of the computational limits (< 100 ns). In this work, we implement a hybrid force field that couples the united‐atom protein models with the coarse‐grained MARTINI water/lipid, to investigate the proton‐dependent dynamics and conformational changes of LacY. The effects of the protonation states on two key glutamate residues (Glu325 and Glu269) have been studied. Our results on the salt‐bridge dynamics agreed with all‐atom simulations at early short time period, validating our simulations. From our microsecond simulations, we were able to observe the complete transition from inward‐facing to outward‐facing conformations of LacY. Our results showed that all helices have participated during the global conformational transitions and helical movements of LacY. The inter‐helical distances measured in our simulations were consistent with the double electron‐electron resonance experiments at both cytoplasmic and periplasmic sides. Our simulations indicated that the deprotonation of Glu325 induced the opening of the periplasmics side and partial closure of the cytoplasmic side of LacY, while protonation of the Glu269 caused a stable cross‐domain salt‐bridge (Glu130‐Arg344) and completely closed the cytoplasmic side. Proteins 2016; 84:1067–1074. © 2016 Wiley Periodicals, Inc.     

Role of Hydrophobic Core on the Thermal Stability of Proteins—Molecular Dynamics Simulations on a Single Point Mutant of Sso7d

Abstract

The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 μs long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.     

GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis Study

RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.Abbreviations used: nACh, nicotinic acetylcholine; AChBP, acetylcholine binding protein; GABA, gamma-aminobutyric acid; MD, molecular dynamics; RDL, resistant to dieldrin; RMSD, root mean-square displacement; RMSF, root mean-square fluctuation     

Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands β₅ and β₆, loop L₅ and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.     

Building a foundation for structure‐based cellulosome design for cellulosic ethanol: Insight into cohesin‐dockerin complexation from computer simulation

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high‐affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin‐dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin‐dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen‐bonding network in the recognition strips—the conserved loop regions previously proposed to be involved in binding—through electrostatic and salt‐bridge interactions between β‐strands 3 and 5 of the cohesin and α‐helix 3 of the dockerin. The mutation‐induced subtle disturbance in the local hydrogen‐bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin‐dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin‐dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin‐dockerin complex. The PMF shows a high‐free energy barrier against the dissociation and reveals a stepwise pattern involving both the central β‐sheet interface and its adjacent solvent‐exposed loop/turn regions clustered at both ends of the β‐barrel structure.     

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)⁺ with the concurrent reduction of O₂ to H₂O₂, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and ¹⁵N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.     

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.     

Structures and free-energy landscapes of the wild type and mutants of the Abeta(21-30) peptide are determined by an interplay between intrapeptide electrostatic and hydrophobic interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ_21-30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK_a values that compare well with the experimental estimates. The pK_a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in “dried” salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ_21-30 peptide into two basins, differentiated by the relative position of the DVG(23-25) and GSN(25-27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.     

A Second Look at Mini-Protein Stability: Analysis of FSD-1 Using Circular Dichroism, Differential Scanning Calorimetry, and Simulations

Mini-proteins that contain <50 amino acids often serve as model systems for studying protein folding because their small size makes long timescale simulations possible. However, not all mini-proteins are created equal. The stability and structure of FSD-1, a 28-residue mini-protein that adopted the ββα zinc-finger motif independent of zinc binding, was investigated using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The broad melting transition of FSD-1, similar to that of a helix-to-coil transition, was observed by using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The N-terminal β-hairpin was found to be flexible. The FSD-1 apparent melting temperature of 41°C may be a reflection of the melting of its α-helical segment instead of the entire protein. Thus, despite its attractiveness due to small size and purposefully designed helix, sheet, and turn structures, the status of FSD-1 as a model system for studying protein folding should be reconsidered.     

In silico study on the effect of surface lysines and arginines on the electrostatic interactions and protein stability

Charged amino acids are mostly exposed on a protein surface, thereby forming a network of interactions with the surrounding amino acids as well as with water. In particular, positively charged arginine and lysine have different side chain geometries and provide a different number of potential electrostatic interactions. This study reports a comparative analysis of the difference in the number of two representative electrostatic interactions, such as salt-bridges and hydrogen bonds, contributed by surface arginine and lysine, as well as their effect on protein stability using molecular modeling and dynamics simulation techniques. Two in silico variants, the R variant with all arginines and the K variant with all lysines on the protein surface, were modeled by mutating all the surface lysines to arginines and the surface arginines to lysines, respectively, for each of the 10 model proteins. A structural comparison of the respective two variants showed that the majority of R variants possessed more salt-bridges and hydrogen bond interactions than the K variants, indicating that arginine provides a higher probability of electrostatic interactions than lysine owing to its side chain geometry. Molecular dynamics simulations of these variants revealed the R variants to be more stable than the K variants at room temperature but this effect was not prominent under protein denaturating conditions, such as 353 and 333 K with 8 M urea. These results suggest that the arginine residues on a protein surface contribute to the protein stability slightly more than lysine by enhancing the electrostatic interactions.     

Mutations in alpha-helical solvent-exposed sites of eglin c have long-range effects: evidence from molecular dynamics simulations

Eglin c is a small protease inhibitor whose structural and thermodynamic properties have been well studied. Previous thermodynamic measurements on mutants at solvent-accessible positions in the protein's helix have shown the unexpected result that the data could be best fit by the inclusion of residue- and position-specific parameters to the model. To explore the origins of this surprising result, long molecular dynamics simulations in explicit solvent have been performed. These simulations indicate specific long-range interactions between the solvent-exposed residues in the eglin c alpha-helix and binding loop, an unexpected observation for such a small protein. The residues involved in the interaction are on opposite sides of the protein, about 25 A apart. Simulations of alanine substitutions at the solvent-exposed helix positions, arginine 22, glutamic acid 23, threonine 26, and leucine 27, show both small and large perturbations of eglin c dynamics. Two mutations exhibit large impacts on the long-range helix-loop interactions. Previous stability measurements (Yi et al., Biochemistry 2003;42:7594-7603) had indicated that an alanine substitution at position 27 was less stabilizing than at other solvent-exposed positions in the helix. The L27A mutation effects observed in these simulations suggest that the position-dependent loss of stability measured in wet bench experiments is derived from changes in dynamics that involve long-range interactions; thus, these simulations support the hypothesis that solvent-exposed positions in helices are not always equivalent.     

Comparative molecular dynamics simulation studies for determining factors contributing to the thermostability of chemotaxis protein “CheY”

Comparative molecular dynamics simulations of chemotaxis protein “CheY” from thermophilic origin Thermotoga maritima and its mesophilic counterpart Salmonella enterica have been performed for 10?ns each at 300 and 350?K, and 20?ns each at 400 and 450?K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein–solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall flexibility than its thermophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar direction of motions are observed for both proteins at 300?K. Overall, the mesophilic protein leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the first ever study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms living at different temperatures with less visible differences.     

Conformational dynamics of the EGFR kinase domain reveals structural features involved in activation

The epidermal growth factor receptor (EGFR) has been the focus of intensive studies because of its importance in cancer research. Thus, a broader understanding of the molecular mechanism of activation of the EGFR kinase will have profound significance for the development of novel therapeutics. Numerous crystal structures of EGFR kinase, including the structure of the activating‐kinase dimer, have provided snapshots of the specific pathway. Herein, we performed unrestrained‐, as well as targeted‐molecular dynamics simulations based on these data, to gain further insight into the conformational changes responsible for activation. Comparison of the monomer‐ versus activating‐EGFR‐dimer simulations indicates that the dimerization is stabilizing structural elements associated with the activated state and predicts new salt‐bridge interactions involving activation‐loop residues that may also be associated with that state. Targeted molecular dynamics simulations of the inactive‐to‐active EGFR transition, as well as the reverse pathway, confirm the formation of conserved structural features of functional importance for the activity or stabilization of either conformation. Interestingly, simulations of the L834R mutant, which is associated with cancer, suggest that the structural basis of the activation induced by that mutation might be the ability of the mutated R834 residue to consecutively form salt bridges with neighboring acidic residues and cause destabilization of a hydrophobic cluster in the inactive state. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

Molecular dynamics simulation of Kv channel voltage sensor helix in a lipid membrane with applied electric field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1-R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1-S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically ∼6.7 Å. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.     

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B‐factors of residue pairs in CalB wild type (CalB‐WT) followed by simple flexibility analysis of residues in CalB‐WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162‐K308, N169‐F304, Q156_‐L163, S50‐A273, and S239C‐D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C‐K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB‐WT. Remarkably, the temperature at which 50% of its activity remained after 60‐min incubation (T) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB‐WT (55 and 46.5°C, respectively). Additionally, the half‐life at 50°C of CalB A162C‐K308C was 4.5‐fold higher than that of CalB‐WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C‐K308C was elucidated at the molecular level by molecular dynamics (MD) simulation. Biotechnol. Bioeng. 2012; 109:867–876. © 2011 Wiley Periodicals, Inc.     

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol^?1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix–dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.