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1.  ProBLM Web Server: Protein and Membrane Placement and Orientation Package 
The 3D structures of membrane proteins are typically determined without the presence of a lipid bilayer. For the purpose of studying the role of membranes on the wild type characteristics of the corresponding protein, determining the position and orientation of transmembrane proteins within a membrane environment is highly desirable. Here we report a geometry-based approach to automatically insert a membrane protein with a known 3D structure into pregenerated lipid bilayer membranes with various dimensions and lipid compositions or into a pseudomembrane. The pseudomembrane is built using the Protein Nano-Object Integrator which generates a parallelepiped of user-specified dimensions made up of pseudoatoms. The pseudomembrane allows for modeling the desolvation effects while avoiding plausible errors associated with wrongly assigned protein-lipid contacts. The method is implemented into a web server, the ProBLM server, which is freely available to the biophysical community. The web server allows the user to upload a protein coordinate file and any missing residues or heavy atoms are regenerated. ProBLM then creates a combined protein-membrane complex from the given membrane protein and bilayer lipid membrane or pseudomembrane. The user is given an option to manually refine the model by manipulating the position and orientation of the protein with respect to the membrane.
doi:10.1155/2014/838259
PMCID: PMC4122144  PMID: 25126110
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.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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

Results 1-10 (10)