The severity of osteogenesis imperfecta: a comparison to the relative free energy differences of collagen model peptides

Molecular dynamics simulations were carried out to calculate free energy differences between the folded and unfolded states of wild type and mutant collagen model peptides. The calculated stability of the collagen models was compared with the severity of osteogenesis imperfecta. Free energy differences of Gly → Xaa (Xaa: Ser, Cys, Glu, and Asp) mutations between the wild type and the mutants at position 15 of the model peptide were 3.8, 4.2, 5.6, and 8.8 kcal/mol, respectively. The corresponding free energy differences of a second Gly mutation at the same position in different chains were, on average, 1.3, 1.5, 2.9, and 5.4 kcal/mol, respectively. Free energy simulations were also performed to estimate the relative stability between an oxidized form and a reduced form of the mutants containing two Cys residues, which indicated that the mutant of the collagen-like peptide containing an intramolecular disulfide bond was more stable than the mutant containing one Cys residue but less stable than the wild type. The calculated free energy differences between an oxidized and a reduced form of the mutants containing two Cys residues are 0.8 and 2.6 kcal/mol for the disulfide bonds between Chains A and B and between Chains A and C, respectively.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

Free energy simulation to investigate the effect of amino acid sequence environment on the severity of osteogenesis imperfecta by glycine mutations in collagen

Molecular dynamics simulations were carried out to calculate the free energy change difference of two collagen-like peptide models for Gly --> Ser mutations causing two different osteogenesis imperfecta phenotypes. These simulations were performed to investigate the impact of local amino acid sequence environment adjacent to a mutation site on the stability of the collagen. The average free energy differences for a Gly --> Ser mutant relative to a wild type are 3.4 kcal/mol and 8.2 kcal/mol for a nonlethal site and a lethal site, respectively. The free energy change differences of mutant containing two Ser residues relative to the wild type at the nonlethal and lethal mutation sites are 4.6 and 9.8 kcal/mol, respectively. Although electrostatic interactions stabilize mutants containing one or two Ser residues at both mutation sites, van der Waals interactions are of sufficient magnitude to cause a net destabilization. The presence of Gln and Arg near the mutation site, which contain large and polar side chains, provide more destabilization than amino acids containing small and nonpolar side chains.     

Conformational Dynamics of a Ligand-Free Adenylate Kinase

Adenylate kinase (AdK) is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven) undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol) free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme.     

Binding free energy differences in a TCR-peptide-MHC complex induced by a peptide mutation: a simulation analysis

Recognition by the T-cell receptor (TCR) of immunogenic peptides presented by class I major histocompatibility complexes (MHCs) is the determining event in the specific cellular immune response against virus-infected cells or tumor cells. It is of great interest, therefore, to elucidate the molecular principles upon which the selectivity of a TCR is based. These principles can in turn be used to design therapeutic approaches, such as peptide-based immunotherapies of cancer. In this study, free energy simulation methods are used to analyze the binding free energy difference of a particular TCR (A6) for a wild-type peptide (Tax) and a mutant peptide (Tax P6A), both presented in HLA A2. The computed free energy difference is 2.9 kcal/mol, in good agreement with the experimental value. This makes possible the use of the simulation results for obtaining an understanding of the origin of the free energy difference which was not available from the experimental results. A free energy component analysis makes possible the decomposition of the free energy difference between the binding of the wild-type and mutant peptide into its components. Of particular interest is the fact that better solvation of the mutant peptide when bound to the MHC molecule is an important contribution to the greater affinity of the TCR for the latter. The results make possible identification of the residues of the TCR which are important for the selectivity. This provides an understanding of the molecular principles that govern the recognition. The possibility of using free energy simulations in designing peptide derivatives for cancer immunotherapy is briefly discussed.     

Prediction of the mutation-induced change in thermodynamic stabilities of membrane proteins from free energy simulations

Comparative protein structure modeling and free energy perturbation simulation have been applied in a consecutive manner to investigate the mutation-induced stabilization of membrane proteins (MPs) in aqueous solution without knowledge of their three-dimensional structures. The calculated difference in protein solvation free energy between the wild type and a mutant compares well with their relative thermodynamic stabilities in solution. For monomeric MPs, a mutant reveals a higher stability than the wild type if the calculated solvation free energy indicates a favorable change. On the contrary, for oligomeric MPs the stability of a mutant increases as the solvation free energy of a mutated monomer becomes less favorable, indicating that the oligomeric MP mutant would be stabilized in solution due to the reduced desolvation cost for oligomerization. The present computational strategy is expected to find its way as a useful tool for assessing the relative stability of a mutant MP with respect to its wild type in solution.     

A fast method for predicting amino acid mutations that lead to unfolding

Amino acid mutation(s) that cause(s) partial or total unfolding of a protein can lead to disease states and failure to produce mutants. It is therefore very useful to be able to predict which mutations can retain the conformation of a wild-type protein and which mutations will lead to local or global unfolding of the protein. We have developed a fast and reasonably accurate method based on a backbone-dependent side-chain rotamer library to predict the (folded or unfolded) conformation of a protein upon mutation. This method has been tested on proteins whose wild-type 3D structures are known and whose mutant conformations have been experimentally characterized to be folded or unfolded. Furthermore, for the cases studied here, the predicted partially folded or denatured mutant conformation correlate with a decrease in the stability of the mutant relative to the wild-type protein. The key advantage of our method is that it is very fast and predicts locally or globally unfolded states fairly accurately. Hence, it may prove to be useful in designing site-directed mutagenesis, X-ray crystallography and drug design experiments as well as in free energy simulations by helping to ascertain whether a mutation will alter or retain the wild-type conformation.     

Analysis of a water mediated protein-protein interactions within RNase T1

Buried and well-ordered solvent molecules are an integral part of each folded protein. For a few individual water molecules, the exchange kinetics with solvent have been described in great detail. So far, little is known about the energetics of this exchange process. Here, we present an experimental approach to investigate water-mediated intramolecular protein-protein interactions by use of double mutant cycles. As a first example, we analyzed the interdependence of the contribution of two side chains (Asn9 and Thr93) to the conformational stability of RNase T1. In the folded state, both side chains are involved in the "solvation of the same water molecule WAT1. The coupling of the contributions of Asn9 and Thr93 to the conformational stability of RNase T1 was measured by urea unfolding and differential scanning calorimetry. The structural integrity of each mutant was analyzed by X-ray crystallography. We find that the effects of the Asn9Ala and the Thr93Ala mutations on the conformational stability are additive in the corresponding double mutant. We conclude that the free energy of the WAT1 mediated intramolecular protein-protein interaction in the folded state is very similar to solvent mediated protein-protein interaction in the unfolded state.     

The crystal structure of a mutant human lysozyme C77/95A with increased secretion efficiency in yeast

The three-dimensional structure of a mutant human lysozyme, C77/95A, in which residues Cys77 and Cys95 were replaced by alanine, was determined at 1.8-A resolution by x-ray crystallography. The properties of this mutant protein have been well characterized with respect to its thermal stability and secretion efficiency in a yeast expression system. The overall three-dimensional structure of C77/95A was found to be essentially identical to that of the wild-type human lysozyme, although the coordinates were shifted by more than 0.5 A and the thermal factors of the main-chain atoms were increased in the vicinity of residue 77. The reduction in thermal stability of this mutant has been previously explained by an increase in entropy of the unfolded state. In addition, a packing defect (cavity) produced by the removal of the disulfide bond was detected in the three-dimensional structure of C77/95A. This cavity can also be a reason why the stability of the protein is reduced because the free energy of the folded state could be expected to increase. The increased secretion efficiency cannot be due mainly to the three-dimensional structure, but may possibly be related to some event in the pathway of protein secretion. One of the possibilities might involve molecular flexibilities in the secondary or tertiary structure for lack of one of the disulfide bonds.     

How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme

To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.     

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.     

Crystal structural analysis of protein-protein interactions drastically destabilized by a single mutation

The complex of barnase (bn) and barstar (bs), which has been widely studied as a model for quantitative analysis of protein-protein interactions, is significantly destabilized by a single mutation, namely, bs Asp39 --> Ala, which corresponds to a change of 7.7 kcal x mol(-1) in the free energy of binding. However, there has been no structural information available to explain such a drastic destabilization. In the present study, we determined the structure of the mutant complex at 1.58 A resolution by X-ray crystallography. The complex was similar to the wild-type complex in terms of overall and interface structures; however, the hydrogen bond network mediated by water molecules at the interface was significantly different. Several water molecules filled the cavity created by the mutation and consequently caused rearrangement of the hydrated water molecules at the interface. The water molecules were redistributed into a channel-like structure that penetrated into the complex. Furthermore, molecular dynamics simulations showed that the mutation increased the mobility of water molecules at the interface. Since such a drastic change in hydration was not observed in other mutant complexes of bn and bs, the significant destabilization of the interaction may be due to this channel-like structure of hydrated water molecules.     

Exploring the mechanism of zanamivir resistance in a neuraminidase mutant: a molecular dynamics study

It is critical to understand the molecular basis of the drug resistance of influenza viruses to efficiently treat this infectious disease. Recently, H1N1 strains of influenza A carrying a mutation of Q136K in neuraminidase were found. The new strain showed a strong Zanamivir neutralization effect. In this study, normal molecular dynamics simulations and metadynamics simulations were employed to explore the mechanism of Zanamivir resistance. The wild-type neuraminidase contained a 3(10) helix before the 150 loop, and there was interaction between the 150 and 430 loops. However, the helix and the interaction between the two loops were disturbed in the mutant protein due to interaction between K136 and nearby residues. Hydrogen-bond network analysis showed weakened interaction between the Zanamivir drug and E276/D151 on account of the electrostatic interaction between K136 and D151. Metadynamics simulations showed that the free energy landscape was different in the mutant than in the wild-type neuraminidase. Conformation with the global minimum of free energy for the mutant protein was different from the wild-type conformation. While the drug fit completely into the active site of the wild-type neuraminidase, it did not match the active site of the mutant variant. This study indicates that the altered hydrogen-bond network and the deformation of the 150 loop are the key factors in development of Zanamivir resistance. Furthermore, the Q136K mutation has a variable effect on conformation of different N1 variants, with conformation of the 1918 N1 variant being more profoundly affected than that of the other N1 variants studied in this paper. This observation warrants further experimental investigation.     

A molecular dynamics study of a miRNA:mRNA interaction

In this paper we present a methodology to evaluate the binding free energy of a miRNA:mRNA complex through molecular dynamics (MD)–thermodynamic integration (TI) simulations. We applied our method to the Caenorhabditis elegans let-7 miRNA:lin-41 mRNA complex—a validated miRNA:mRNA interaction—in order to estimate the energetic stability of the structure. To make the miRNA:mRNA simulation possible and realistic, the methodology introduces specific solutions to overcome some of the general challenges of nucleic acid simulations and binding free energy computations that have been discussed widely in many previous research reports. The main features of the proposed methodology are: (1) positioning of the restraints imposed on the simulations in order to guarantee complex stability; (2) optimal sampling of the phase space to achieve satisfactory accuracy in the binding energy value; (3) determination of a suitable trade-off between computational costs and accuracy of binding free energy computation by the assessment of the scalability characteristics of the parallel simulations required for the TI. The experiments carried out demonstrate that MD simulations are a viable strategy for the study of miRNA binding characteristics, opening the way to the development of new computational target prediction methods based on three-dimensional structure information.     

Rational design of a more stable yeast iso-1-cytochrome c.

Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c. We have used a coordinated approach, combining the known functional and structural properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with the goal of selectively increasing the stability of the protein. The two redox forms of the native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed by differential scanning calorimetry (DSC). The relative stability, expressed as the difference in the Gibb's free energy of denaturation at a given temperature between the native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins. In both oxidation states, the mutant proteins C102T, T69E/C102T, T96A/C102T, and T69E/T96A/C102T were more stable than the wild-type protein, respectively. The increased stability of the mutant proteins is proposed to be due to the removal of a rare surface cysteine and the stabilization of two distorted alpha-helices.     

Binding of an antiviral agent to a sensitive and a resistant human rhinovirus. Computer simulation studies with sampling of amino acid side-chain conformations. II. Calculation of free-energy differences by thermodynamic integration.

Thermodynamic-cycle perturbation theory and molecular dynamics simulations were used to calculate the difference in the free energy of binding of the antiviral compound WIN53338 to the wild-type human rhinovirus 14 and to a drug-resistant mutant of the virus in which valine 188 of the viral protein 1 is mutated to leucine. Because of the difficulty of achieving adequate sampling of all of the rotational isomers of amino acid side-chains in molecular dynamics simulations, an explicit treatment of the effects of the existence of multiple rotational isomers of residue 188 on the calculated free energies was used. The rotamers of residue 188 were first mapped by steric and energetic techniques as described in the accompanying article. Thermodynamic integration was then carried out during simulations of the virus, both with and without the antiviral compound bound, by mutating residue 188 while restraining its side-chain to one conformation. The contributions of the other rotamers of residue 188 to the free-energy changes for this mutation were then added to those calculated by thermodynamic integration as correction factors. Binding of WIN53338 to the wild-type virus was calculated to be favored over binding to the mutant virus by 1.7(+/- 3.0) kcal/mol. This is consistent with experimental data which, if differences in activity are assumed to be due to differences in binding, indicate that the binding affinity of WIN53338 for the wild-type virus is at least 0.15 to 1.7 kcal/mol greater than for the mutant virus. Thermodynamic integration was also performed in the conventional manner without restraints and was found to give less accurate results.     

Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.     

Solution structure and internal dynamics of NSCP, a compact calcium-binding protein

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of N(H)-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.