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author:("alexon, Emil")
1.  On the Modeling of Polar Component of Solvation Energy using Smooth Gaussian-Based Dielectric Function 
Journal of theoretical & computational chemistry  2014;13(3):10.1142/S0219633614400021.
Traditional implicit methods for modeling electrostatics in biomolecules use a two-dielectric approach: a biomolecule is assigned low dielectric constant while the water phase is considered as a high dielectric constant medium. However, such an approach treats the biomolecule-water interface as a sharp dielectric border between two homogeneous dielectric media and does not account for inhomogeneous dielectric properties of the macromolecule as well. Recently we reported a new development, a smooth Gaussian-based dielectric function which treats the entire system, the solute and the water phase, as inhomogeneous dielectric medium (J Chem Theory Comput. 2013 Apr 9; 9(4): 2126-2136.). Here we examine various aspects of the modeling of polar solvation energy in such inhomogeneous systems in terms of the solute-water boundary and the inhomogeneity of the solute in the absence of water surrounding. The smooth Gaussian-based dielectric function is implemented in the DelPhi finite-difference program, and therefore the sensitivity of the results with respect to the grid parameters is investigated, and it is shown that the calculated polar solvation energy is almost grid independent. Furthermore, the results are compared with the standard two-media model and it is demonstrated that on average, the standard method overestimates the magnitude of the polar solvation energy by a factor 2.5. Lastly, the possibility of the solute to have local dielectric constant larger than of a bulk water is investigated in a benchmarking test against experimentally determined set of pKa's and it is speculated that side chain rearrangements could result in local dielectric constant larger than 80.
doi:10.1142/S0219633614400021
PMCID: PMC4092036  PMID: 25018579
dielectric constant; Poisson-Boltzmann equation; electrostatics; finite-difference method; protein flexibility
2.  A rational free energy-based approach to understanding and targeting disease-causing missense mutations 
Background and significance
Intellectual disability is a condition characterized by significant limitations in cognitive abilities and social/behavioral adaptive skills and is an important reason for pediatric, neurologic, and genetic referrals. Approximately 10% of protein-encoding genes on the X chromosome are implicated in intellectual disability, and the corresponding intellectual disability is termed X-linked ID (XLID). Although few mutations and a small number of families have been identified and XLID is rare, collectively the impact of XLID is significant because patients usually are unable to fully participate in society.
Objective
To reveal the molecular mechanisms of various intellectual disabilities and to suggest small molecules which by binding to the malfunctioning protein can reduce unwanted effects.
Methods
Using various in silico methods we reveal the molecular mechanism of XLID in cases involving proteins with known 3D structure. The 3D structures were used to predict the effect of disease-causing missense mutations on the folding free energy, conformational dynamics, hydrogen bond network and, if appropriate, protein-protein binding free energy.
Results
It is shown that the vast majority of XLID mutation sites are outside the active pocket and are accessible from the water phase, thus providing the opportunity to alter their effect by binding appropriate small molecules in the vicinity of the mutation site.
Conclusions
This observation is used to demonstrate, computationally and experimentally, that a particular condition, Snyder-Robinson syndrome caused by the G56S spermine synthase mutation, might be ameliorated by small molecule binding.
doi:10.1136/amiajnl-2012-001505
PMCID: PMC3721167  PMID: 23408511
In Silico Modeling; Experimenatal Measurements; Mental Disorders; Missense Mutation; Small Molecule Screening
3.  Computational and Experimental Approaches to Reveal the Effects of Single Nucleotide Polymorphisms with Respect to Disease Diagnostics 
DNA mutations are the cause of many human diseases and they are the reason for natural differences among individuals by affecting the structure, function, interactions, and other properties of DNA and expressed proteins. The ability to predict whether a given mutation is disease-causing or harmless is of great importance for the early detection of patients with a high risk of developing a particular disease and would pave the way for personalized medicine and diagnostics. Here we review existing methods and techniques to study and predict the effects of DNA mutations from three different perspectives: in silico, in vitro and in vivo. It is emphasized that the problem is complicated and successful detection of a pathogenic mutation frequently requires a combination of several methods and a knowledge of the biological phenomena associated with the corresponding macromolecules.
doi:10.3390/ijms15069670
PMCID: PMC4100115  PMID: 24886813
single nucleotide polymorphism (SNP); pathogenic mutation; missense mutations; disease diagnostics; protein stability; protein interactions
4.  A novel p.Leu(381)Phe mutation in Presenilin 1 is associated with very early onset and unusually fast progressing dementia, and lysosomal inclusions typically seen in Kufs disease 
Whole exome sequencing in a family with suspected dominant Kufs disease identified a novel Presenilin 1 mutation p.Leu(381)Phe in three brothers who, along with their father, developed progressive dementia and motor deficits in their early 30s. All affected relatives had unusually rapid disease progression (on average 3.6 years from disease onset to death). In silico analysis of mutation p.Leu(381)Phe predicted more detrimental effects when compared to the common Presenilin 1 mutation p.Glu(280)Ala. Electron microscopy study of peripheral fibroblast cells of the proband showed lysosomal inclusions typical for Kufs disease. However his brain autopsy demonstrated typical changes of Alzheimer disease.
doi:10.3233/JAD-131340
PMCID: PMC4013718  PMID: 24121961
5.  Using DelPhi capabilities to mimic protein’s conformational reorganization with amino acid specific dielectric constants 
Many molecular events are associated with small or large conformational changes occurring in the corresponding proteins. Modeling such changes is a challenge and requires significant amount of computing time. From point of view of electrostatics, these changes can be viewed as a reorganization of local charges and dipoles in response to the changes of the electrostatic field, if the cause is insertion or deletion of a charged amino acid. Here we report a large scale investigation of modeling the changes of the folding energy due to single mutations involving charged group. This allows the changes of the folding energy to be considered mostly electrostatics in origin and to be calculated with DelPhi assigning residue-specific value of the internal dielectric constant of protein. The predicted energy changes are benchmarked against experimentally measured changes of the folding energy on a set of 257 single mutations. The best fit between experimental values and predicted changes is used to find out the effective value of the internal dielectric constant for each type of amino acid. The predicted folding free energy changes with the optimal, amino acid specific, dielectric constants are within RMSD=0.86 kcal/mol from experimentally measured changes.
PMCID: PMC3966310  PMID: 24683422
DelPhi; protein electrostatics; dielectric constant; Poisson-Boltzmann equation; protein flexibility; energy calculations; single point mutations
6.  DelPhi Web Server: A comprehensive online suite for electrostatic calculations of biological macromolecules and their complexes 
Here we report a web server, the DelPhi web server, which utilizes DelPhi program to calculate electrostatic energies and the corresponding electrostatic potential and ionic distributions, and dielectric map. The server provides extra services to fix structural defects, as missing atoms in the structural file and allows for generation of missing hydrogen atoms. The hydrogen placement and the corresponding DelPhi calculations can be done with user selected force field parameters being either Charmm22, Amber98 or OPLS. Upon completion of the calculations, the user is given option to download fixed and protonated structural file, together with the parameter and Delphi output files for further analysis. Utilizing Jmol viewer, the user can see the corresponding structural file, to manipulate it and to change the presentation. In addition, if the potential map is requested to be calculated, the potential can be mapped onto the molecule surface. The DelPhi web server is available from http://compbio.clemson.edu/delphi_webserver.
PMCID: PMC3966485  PMID: 24683424
DelPhi; electrostatics; proteins; continuum models; electrostatic potential; Finite-Difference Poisson-Boltzmann solver
7.  In silico investigation of pH-dependence of prolactin and human growth hormone binding to human prolactin receptor 
Experimental data shows that the binding of human prolactin (hPRL) to human prolactin receptor (hPRLr-ECD) is strongly pH-dependent, while the binding of the same receptor to human growth hormone (hGH) is pH-independent. Here we carry in silico analysis of the molecular effects causing such a difference and reveal the role of individual amino acids. It is shown that the computational modeling correctly predicts experimentally determined pKa’s of histidine residues in an unbound state in the majority of the cases and the pH-dependence of the binding free energy. Structural analysis carried in conjunction with calculated pH-dependence of the binding revealed that the main reason for pH-dependence of the binding of hPRL-hPRLr-ECD is a number of salt- bridges across the interface of the complex, while no salt-bridges are formed in the hGH-hPRlr-ECD. Specifically, most of the salt-bridges involve histidine residues and this is the reason for the pH-dependence across a physiological range of pH. The analysis not only revealed the molecular mechanism of the pH-dependence of the hPRL-hPRLr-ECD, but also provided critical insight into the underlying physic-chemical mechanism.
PMCID: PMC3966486  PMID: 24683423
human prolactin; human prolactin receptor; human growth hormone; pKa calculations; pH-dependence; electrostatics
8.  BION web server: predicting non-specifically bound surface ions 
Bioinformatics  2013;29(6):805-806.
Motivation: Ions are essential component of the cell and frequently are found bound to various macromolecules, in particular to proteins. A binding of an ion to a protein greatly affects protein’s biophysical characteristics and needs to be taken into account in any modeling approach. However, ion’s bounded positions cannot be easily revealed experimentally, especially if they are loosely bound to macromolecular surface.
Results: Here, we report a web server, the BION web server, which addresses the demand for tools of predicting surface bound ions, for which specific interactions are not crucial; thus, they are difficult to predict. The BION is easy to use web server that requires only coordinate file to be inputted, and the user is provided with various, but easy to navigate, options. The coordinate file with predicted bound ions is displayed on the output and is available for download.
Availability: http://compbio.clemson.edu/bion_server/
Supplementary information: Supplementary data are available at Bioinformatics online.
Contact: ealexov@clemson.edu
doi:10.1093/bioinformatics/btt032
PMCID: PMC3597141  PMID: 23380591
9.  Progress in developing Poisson-Boltzmann equation solvers 
Molecular based mathematical biology  2013;1:10.2478/mlbmb-2013-0002.
This review outlines the recent progress made in developing more accurate and efficient solutions to model electrostatics in systems comprised of bio-macromolecules and nano-objects, the last one referring to objects that do not have biological function themselves but nowadays are frequently used in biophysical and medical approaches in conjunction with bio-macromolecules. The problem of modeling macromolecular electrostatics is reviewed from two different angles: as a mathematical task provided the specific definition of the system to be modeled and as a physical problem aiming to better capture the phenomena occurring in the real experiments. In addition, specific attention is paid to methods to extend the capabilities of the existing solvers to model large systems toward applications of calculations of the electrostatic potential and energies in molecular motors, mitochondria complex, photosynthetic machinery and systems involving large nano-objects.
doi:10.2478/mlbmb-2013-0002
PMCID: PMC3816640  PMID: 24199185
Continuum electrostatics; Poisson-Boltzmann equation; numerical techniques; dielectric constant; molecular surface
10.  An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity 
Human Molecular Genetics  2012;21(20):4497-4507.
Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca2+ release channels. These RyR proteins play a major role in the regulation of Ca2+ signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca2+ signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via ‘leaky mutations’ can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca2+ release through RyRs in neuronal cells and cardiac muscle cells.
doi:10.1093/hmg/dds292
PMCID: PMC3459470  PMID: 22814392
11.  Highly efficient and exact method for parallelization of grid-based algorithms and its implementation in DelPhi 
Journal of computational chemistry  2012;33(24):1960-1966.
The Gauss-Seidel method is a standard iterative numerical method widely used to solve a system of equations and, in general, is more efficient comparing to other iterative methods, such as the Jacobi method. However, standard implementation of the Gauss-Seidel method restricts its utilization in parallel computing due to its requirement of using updated neighboring values (i.e., in current iteration) as soon as they are available. Here we report an efficient and exact (not requiring assumptions) method to parallelize iterations and to reduce the computational time as a linear/nearly linear function of the number of CPUs. In contrast to other existing solutions, our method does not require any assumptions and is equally applicable for solving linear and nonlinear equations. This approach is implemented in the DelPhi program, which is a finite difference Poisson-Boltzmann equation solver to model electrostatics in molecular biology. This development makes the iterative procedure on obtaining the electrostatic potential distribution in the parallelized DelPhi several folds faster than that in the serial code. Further we demonstrate the advantages of the new parallelized DelPhi by computing the electrostatic potential and the corresponding energies of large supramolecular structures.
doi:10.1002/jcc.23033
PMCID: PMC3412928  PMID: 22674480
electrostatics; DelPhi; Poisson- Boltzmann equation; Gauss-Seidel iteration; parallel computing
12.  DelPhi web server v2: incorporating atomic-style geometrical figures into the computational protocol 
Bioinformatics  2012;28(12):1655-1657.
Summary: A new edition of the DelPhi web server, DelPhi web server v2, is released to include atomic presentation of geometrical figures. These geometrical objects can be used to model nano-size objects together with real biological macromolecules. The position and size of the object can be manipulated by the user in real time until desired results are achieved. The server fixes structural defects, adds hydrogen atoms and calculates electrostatic energies and the corresponding electrostatic potential and ionic distributions.
Availability and implementation: The web server follows a client–server architecture built on PHP and HTML and utilizes DelPhi software. The computation is carried out on supercomputer cluster and results are given back to the user via http protocol, including the ability to visualize the structure and corresponding electrostatic potential via Jmol implementation. The DelPhi web server is available from http://compbio.clemson.edu/delphi_webserver.
Contact: nsmith@clemson.edu, ealexov@clemson.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts200
PMCID: PMC3371833  PMID: 22531215
13.  Cancer Missense Mutations Alter Binding Properties of Proteins and Their Interaction Networks 
PLoS ONE  2013;8(6):e66273.
Many studies have shown that missense mutations might play an important role in carcinogenesis. However, the extent to which cancer mutations might affect biomolecular interactions remains unclear. Here, we map glioblastoma missense mutations on the human protein interactome, model the structures of affected protein complexes and decipher the effect of mutations on protein-protein, protein-nucleic acid and protein-ion binding interfaces. Although some missense mutations over-stabilize protein complexes, we found that the overall effect of mutations is destabilizing, mostly affecting the electrostatic component of binding energy. We also showed that mutations on interfaces resulted in more drastic changes of amino acid physico-chemical properties than mutations occurring outside the interfaces. Analysis of glioblastoma mutations on interfaces allowed us to stratify cancer-related interactions, identify potential driver genes, and propose two dozen additional cancer biomarkers, including those specific to functions of the nervous system. Such an analysis also offered insight into the molecular mechanism of the phenotypic outcomes of mutations, including effects on complex stability, activity, binding and turnover rate. As a result of mutated protein and gene network analysis, we observed that interactions of proteins with mutations mapped on interfaces had higher bottleneck properties compared to interactions with mutations elsewhere on the protein or unaffected interactions. Such observations suggest that genes with mutations directly affecting protein binding properties are preferably located in central network positions and may influence critical nodes and edges in signal transduction networks.
doi:10.1371/journal.pone.0066273
PMCID: PMC3682950  PMID: 23799087
14.  The Role of Protonation States in Ligand-Receptor Recognition and Binding 
Current pharmaceutical design  2013;19(23):4182-4190.
In this review we discuss the role of protonation states in receptor-ligand interactions, providing experimental evidences and computational predictions that complex formation may involve titratable groups with unusual pKa’s and that protonation states frequently change from unbound to bound states. These protonation changes result in proton uptake/release, which in turn causes the pH-dependence of the binding. Indeed, experimental data strongly suggests that almost any binding is pH-dependent and to be correctly modeled, the protonation states must be properly assigned prior to and after the binding. One may accurately predict the protonation states when provided with the structures of the unbound proteins and their complex; however, the modeling becomes much more complicated if the bound state has to be predicted in a docking protocol or if the structures of either bound or unbound receptor-ligand are not available. The major challenges that arise in these situations are the coupling between binding and protonation states, and the conformational changes induced by the binding and ionization states of titratable groups. In addition, any assessment of the protonation state, either before or after binding, must refer to the pH of binding, which is frequently unknown. Thus, even if the pKa’s of ionizable groups can be correctly assigned for both unbound and bound state, without knowing the experimental pH one cannot assign the corresponding protonation states, and consequently one cannot calculate the resulting proton uptake/release. It is pointed out, that while experimental pH may not be the physiological pH and binding may involve proton uptake/release, there is a tendency that the native receptor-ligand complexes have evolved toward specific either subcellular or tissue characteristic pH at which the proton uptake/release is either minimal or absent.
PMCID: PMC3625499  PMID: 23170880
protonation states; receptor-ligand interactions; pKa calculations; pH-dependence; electrostatics
15.  Between algorithm and model: different Molecular Surface definitions for the Poisson-Boltzmann based electrostatic characterization of biomolecules in solution 
The definition of a molecular surface which is physically sound and computationally efficient is a very interesting and long standing problem in the implicit solvent continuum modeling of biomolecular systems as well as in the molecular graphics field. In this work, two molecular surfaces are evaluated with respect to their suitability for electrostatic computation as alternatives to the widely used Connolly-Richards surface: the blobby surface, an implicit Gaussian atom centered surface, and the skin surface. As figures of merit, we considered surface differentiability and surface area continuity with respect to atom positions, and the agreement with explicit solvent simulations. Geometric analysis seems to privilege the skin to the blobby surface, and points to an unexpected relationship between the non connectedness of the surface, caused by interstices in the solute volume, and the surface area dependence on atomic centers. In order to assess the ability to reproduce explicit solvent results, specific software tools have been developed to enable the use of the skin surface in Poisson-Boltzmann calculations with the DelPhi solver. Results indicate that the skin and Connolly surfaces have a comparable performance from this last point of view.
doi:10.4208/cicp.050711.111111s
PMCID: PMC3601494  PMID: 23519863
Molecular Surface; Connolly Surface; Blobby Surface; Skin Surface; Poisson-Boltzmann
16.  Predicting folding free energy changes upon single point mutations 
Bioinformatics  2012;28(5):664-671.
Motivation: The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability due to naturally occurring mutations, missense mutations, are typically causing diseases. Single point mutations made in vitro are frequently used to assess the contribution of given amino acid to the stability of the protein. In both cases, it is desirable to predict the change of the folding free energy upon single point mutations in order to either provide insights of the molecular mechanism of the change or to design new experimental studies.
Results: We report an approach that predicts the free energy change upon single point mutation by utilizing the 3D structure of the wild-type protein. It is based on variation of the molecular mechanics Generalized Born (MMGB) method, scaled with optimized parameters (sMMGB) and utilizing specific model of unfolded state. The corresponding mutations are built in silico and the predictions are tested against large dataset of 1109 mutations with experimentally measured changes of the folding free energy. Benchmarking resulted in root mean square deviation = 1.78 kcal/mol and slope of the linear regression fit between the experimental data and the calculations was 1.04. The sMMGB is compared with other leading methods of predicting folding free energy changes upon single mutations and results discussed with respect to various parameters.
Availability: All the pdb files we used in this article can be downloaded from http://compbio.clemson.edu/downloadDir/mentaldisorders/sMMGB_pdb.rar
Contact: ealexov@clemson.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts005
PMCID: PMC3289912  PMID: 22238268
17.  Enhancing Human Spermine Synthase Activity by Engineered Mutations 
PLoS Computational Biology  2013;9(2):e1002924.
Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing.
Author Summary
Proteins are constantly subjected to evolutionary pressure to assure the organism's survival and reproduction. At the same time, the proteins' amino acid sequence undergoes mutations, some of which may cause diseases while others may be reflecting natural differences within the population (non-synonymous single nucleotide polymorphism, nsSNP). In this study we examine the human spermine synthase (HsSMS), for which currently there are no nsSNPs, while rare disease mutations are known to cause Snyder-Robinson syndrome. What is so special with this protein? Maybe the HsSMS is so well optimized for its function that any change of the wild type sequence should be degrading its performance. To check such a possibility, we engineered a mutant of HsSMS with enhanced stability, electrostatic and mechanical properties. The mutant was confirmed experimentally to be a better enzyme than the wild type. Thus, the HsSMS is not evolutionally optimized with respect to its enzymatic reaction, its amino acid sequence differs only in sick individuals and so far its sequence was found to be identical in all healthy individuals. Therefore, it can be speculated that the HsSMS function is precisely tuned toward the wild type characteristics such so any deviation is unwanted and is disease-causing.
doi:10.1371/journal.pcbi.1002924
PMCID: PMC3585406  PMID: 23468611
18.  Protein Nano-Object Integrator (ProNOI) for generating atomic style objects for molecular modeling 
Background
With the progress of nanotechnology, one frequently has to model biological macromolecules simultaneously with nano-objects. However, the atomic structures of the nano objects are typically not available or they are solid state entities. Because of that, the researchers have to investigate such nano systems by generating models of the nano objects in a manner that the existing software be able to carry the simulations. In addition, it should allow generating composite objects with complex shape by combining basic geometrical figures and embedding biological macromolecules within the system.
Results
Here we report the Protein Nano-Object Integrator (ProNOI) which allows for generating atomic-style geometrical objects with user desired shape and dimensions. Unlimited number of objects can be created and combined with biological macromolecules in Protein Data Bank (PDB) format file. Once the objects are generated, the users can use sliders to manipulate their shape, dimension and absolute position. In addition, the software offers the option to charge the objects with either specified surface or volumetric charge density and to model them with user-desired dielectric constants. According to the user preference, the biological macromolecule atoms can be assigned charges and radii according to four different force fields: Amber, Charmm, OPLS and PARSE. The biological macromolecules and the atomic-style objects are exported as a position, charge and radius (PQR) file, or if a default dielectric constant distribution is not selected, it is exported as a position, charge, radius and epsilon (PQRE) file. As illustration of the capabilities of the ProNOI, we created a composite object in a shape of a robot, aptly named the Clemson Robot, whose parts are charged with various volumetric charge densities and holds the barnase-barstar protein complex in its hand.
Conclusions
The Protein Nano-Object Integrator (ProNOI) is a convenient tool for generating atomic-style nano shapes in conjunction with biological macromolecule(s). Charges and radii on the macromolecule atoms and the atoms in the shapes are assigned according to the user’s preferences allowing various scenarios of modeling. The default output file is in PQR (PQRE) format which is readable by almost any software available in biophysical field. It can be downloaded from: http://compbio.clemson.edu/downloadDir/ProNO_integrator.tar.gz
doi:10.1186/1472-6807-12-31
PMCID: PMC3532097  PMID: 23217202
Biological macromolecules; Electrostatic calculations; Molecular modeling; Nano technology; DelPhi; Poisson-Boltzmann equation
19.  Developing hybrid approaches to predict pKa values of ionizable groups 
Proteins  2011;79(12):3389-3399.
Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure. On another hand, if the change of the charge state is accompanied by a structural reorganization of the target protein, then the relevant conformational changes have to be taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups, which ionization is expected to cause conformational changes, termed “problematic” residues, then applies a special protocol on them, while the rest of the pKa’s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for “problematic” groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the pKa of the “problematic” residues and then the results are averaged.
doi:10.1002/prot.23097
PMCID: PMC3220190  PMID: 21744395
pKa calculations; conformational changes; MD simulations; homology modeling; electrostatics
20.  PROGRESS IN THE PREDICTION OF pKa VALUES IN PROTEINS 
Proteins  2011;79(12):3260-3275.
The pKa-cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pKa values and protein electrostatics in general. The first round of the pKa-cooperative, which challenged computational labs to carry out blind predictions against pKas experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6–10, 2009). This paper serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here we briefly outline existing approaches for pKa calculations, emphasizing methods that were used by the participants in calculating the blind pKa values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pKa calculations.
doi:10.1002/prot.23189
PMCID: PMC3243943  PMID: 22002859
pKa; protein electrostatics; pH dependent properties of proteins; predicting pKa values in proteins
21.  A Missense Mutation in CLIC2 Associated with Intellectual Disability is Predicted by In Silico Modeling to Affect Protein Stability and Dynamics 
Proteins  2011;79(8):2444-2454.
Large-scale next generation resequencing of X chromosome genes identified a missense mutation in the CLIC2 gene on Xq28 in a male with X-linked intellectual disability (XLID) and not found in healthy individuals. At the same time, numerous nsSNPs (nonsynonomous SNP) have been reported in the CLIC2 gene in healthy individuals indicating that the CLIC2 protein can tolerate amino acid substitutions and be fully functional. To test the possibility that p.H101Q is a disease-causing mutation, we performed in silico simulations to calculate the effects of the p.H101Q mutation on CLIC2 stability, dynamics and ionization states while comparing the effects obtained for presumably harmless nsSNPs. It was found that p.H101Q, in contrast with other nsSNPs, (a) lessens the flexibility of the joint loop which is important for the normal function of CLIC2, (b) makes the overall 3D structure of CLIC2 more stable and thus reduces the possibility of the large conformational change expected to occur when CLIC2 moves from a soluble to membrane form and (c) removes the positively charged residue, H101, which may be important for the membrane association of CLIC2. The results of in silico modeling, in conjunction with the polymorphism analysis, suggest that p.H101Q may be a disease-causing mutation, the first one suggested in the CLIC family.
doi:10.1002/prot.23065
PMCID: PMC3132293  PMID: 21630357
CLIC2; missense mutations; mental disorder; energy calculations; pKa calculations; electrostatics; molecular dynamics simulations
22.  On the role of electrostatics on protein-protein interactions 
Physical biology  2011;8(3):035001.
The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity.
doi:10.1088/1478-3975/8/3/035001
PMCID: PMC3137121  PMID: 21572182
electrostatics; protein-protein interactions; pH; salt; nsSNPs; missense mutations
23.  DelPhi: a comprehensive suite for DelPhi software and associated resources 
BMC Biophysics  2012;5:9.
Background
Accurate modeling of electrostatic potential and corresponding energies becomes increasingly important for understanding properties of biological macromolecules and their complexes. However, this is not an easy task due to the irregular shape of biological entities and the presence of water and mobile ions.
Results
Here we report a comprehensive suite for the well-known Poisson-Boltzmann solver, DelPhi, enriched with additional features to facilitate DelPhi usage. The suite allows for easy download of both DelPhi executable files and source code along with a makefile for local installations. The users can obtain the DelPhi manual and parameter files required for the corresponding investigation. Non-experienced researchers can download examples containing all necessary data to carry out DelPhi runs on a set of selected examples illustrating various DelPhi features and demonstrating DelPhi’s accuracy against analytical solutions.
Conclusions
DelPhi suite offers not only the DelPhi executable and sources files, examples and parameter files, but also provides links to third party developed resources either utilizing DelPhi or providing plugins for DelPhi. In addition, the users and developers are offered a forum to share ideas, resolve issues, report bugs and seek help with respect to the DelPhi package. The resource is available free of charge for academic users from URL: http://compbio.clemson.edu/DelPhi.php.
doi:10.1186/2046-1682-5-9
PMCID: PMC3463482  PMID: 22583952
DelPhi; Poisson-Boltzmann equation; Implicit solvation model; Electrostatics; Biological macromolecules; Software
24.  Analyzing Effects of Naturally Occurring Missense Mutations 
Single-point mutation in genome, for example, single-nucleotide polymorphism (SNP) or rare genetic mutation, is the change of a single nucleotide for another in the genome sequence. Some of them will produce an amino acid substitution in the corresponding protein sequence (missense mutations); others will not. This paper focuses on genetic mutations resulting in a change in the amino acid sequence of the corresponding protein and how to assess their effects on protein wild-type characteristics. The existing methods and approaches for predicting the effects of mutation on protein stability, structure, and dynamics are outlined and discussed with respect to their underlying principles. Available resources, either as stand-alone applications or webservers, are pointed out as well. It is emphasized that understanding the molecular mechanisms behind these effects due to these missense mutations is of critical importance for detecting disease-causing mutations. The paper provides several examples of the application of 3D structure-based methods to model the effects of protein stability and protein-protein interactions caused by missense mutations as well.
doi:10.1155/2012/805827
PMCID: PMC3346971  PMID: 22577471
25.  Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction 
A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases.
doi:10.1504/IJCBDD.2010.038396
PMCID: PMC3319068  PMID: 21297231
Amino acid substitutions; homology modeling; folding energy; binding energy; protein stability; protein-protein interaction

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