Predicting binding affinities for GPCR ligands using free-energy perturbation

Medicinal ChemistryDrug Delivery Technolog

Coupling Constant pH Molecular Dynamics with Accelerated Molecular Dynamics

An extension of the constant pH method originally implemented by Mongan et al. (J. Comput. Chem.2004, 25, 2038−2048) is proposed in this study. This adapted version of the method couples the constant pH methodology with the enhanced sampling technique of accelerated molecular dynamics, in an attempt to overcome the sampling issues encountered with current standard constant pH molecular dynamics methods. Although good results were reported by Mongan et al. on application of the standard method to the hen egg-white lysozyme (HEWL) system, residues which possess strong interactions with neighboring groups tend to converge slowly, resulting in the reported inconsistencies for predicted pKa values, as highlighted by the authors. The application of the coupled method described in this study to the HEWL system displays improvements over the standard version of the method, with the improved sampling leading to faster convergence and producing pKa values in closer agreement to those obtained experimentally for the more slowly converging residues

Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy

Thermodynamic Selection of Steric Zipper Patterns in the Amyloid Cross-β Spine

At the core of amyloid fibrils is the cross-β spine, a long tape of β-sheets formed by the constituent proteins. Recent high-resolution x-ray studies show that the unit of this filamentous structure is a β-sheet bilayer with side chains within the bilayer forming a tightly interdigitating “steric zipper” interface. However, for a given peptide, different bilayer patterns are possible, and no quantitative explanation exists regarding which pattern is selected or under what condition there can be more than one pattern observed, exhibiting molecular polymorphism. We address the structural selection mechanism by performing molecular dynamics simulations to calculate the free energy of incorporating a peptide monomer into a β-sheet bilayer. We test filaments formed by several types of peptides including GNNQQNY, NNQQ, VEALYL, KLVFFAE and STVIIE, and find that the patterns with the lowest binding free energy correspond to available atomistic structures with high accuracy. Molecular polymorphism, as exhibited by NNQQ, is likely because there are more than one most stable structures whose binding free energies differ by less than the thermal energy. Detailed analysis of individual energy terms reveals that these short peptides are not strained nor do they lose much conformational entropy upon incorporating into a β-sheet bilayer. The selection of a bilayer pattern is determined mainly by the van der Waals and hydrophobic forces as a quantitative measure of shape complementarity among side chains between the β-sheets. The requirement for self-complementary steric zipper formation supports that amyloid fibrils form more easily among similar or same sequences, and it also makes parallel β-sheets generally preferred over anti-parallel ones. But the presence of charged side chains appears to kinetically drive anti-parallel β-sheets to form at early stages of assembly, after which the bilayer formation is likely driven by energetics

First-Step Mutations for Adaptation at Elevated Temperature Increase Capsid Stability in a Virus

The relationship between mutation, protein stability and protein function plays a central role in molecular evolution. Mutations tend to be destabilizing, including those that would confer novel functions such as host-switching or antibiotic resistance. Elevated temperature may play an important role in preadapting a protein for such novel functions by selecting for stabilizing mutations. In this study, we test the stability change conferred by single mutations that arise in a G4-like bacteriophage adapting to elevated temperature. The vast majority of these mutations map to interfaces between viral coat proteins, suggesting they affect protein-protein interactions. We assess their effects by estimating thermodynamic stability using molecular dynamic simulations and measuring kinetic stability using experimental decay assays. The results indicate that most, though not all, of the observed mutations are stabilizing

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

ABSTRACT: BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. METHODS: Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. RESULTS: Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. CONCLUSIONS: Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression

Plasma and Liver Lipidomics Response to an Intervention of Rimonabant in ApoE*3Leiden.CETP Transgenic Mice

Background: Lipids are known to play crucial roles in the development of life-style related risk factors such as obesity, dyslipoproteinemia, hypertension and diabetes. The first selective cannabinoid-1 receptor blocker rimonabant, an anorectic anti-obesity drug, was frequently used in conjunction with diet and exercise for patients with a body mass index greater than 30 kg/m2 with associated risk factors such as type II diabetes and dyslipidaemia in the past. Less is known about the impact of this drug on the regulation of lipid metabolism in plasma and liver in the early stage of obesity. Methodology/Principal Findings: We designed a four-week parallel controlled intervention on apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE&z.ast;3Leiden.CETP) transgenic mice with mild overweight and hypercholesterolemia. A liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric approach was employed to investigate plasma and liver lipid responses to the rimonabant intervention. Rimonabant was found to induce a significant body weight loss (9.4%, p<0.05) and a significant plasma total cholesterol reduction (24%, p<0.05). Six plasma and three liver lipids in ApoE&z.ast;3Leiden.CETP transgenic mice were detected to most significantly respond to rimonabant treatment. Distinct lipid patterns between the mice were observed for both plasma and liver samples in rimonabant treatment vs. non-treated controls. This study successfully applied, for the first time, systems biology based lipidomics approaches to evaluate treatment effects of rimonabant in the early stage of obesity. Conclusion: The effects of rimonabant on lipid metabolism and body weight reduction in the early stage obesity were shown to be moderate in ApoE&z.ast;3Leiden.CETP mice on high-fat diet. © 2011 Hu et al

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

BACKGROUND: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. METHODOLOGY/PRINCIPAL FINDINGS: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. CONCLUSIONS/SIGNIFICANCE: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases

A Self-Organizing Algorithm for Modeling Protein Loops

Protein loops, the flexible short segments connecting two stable secondary structural units in proteins, play a critical role in protein structure and function. Constructing chemically sensible conformations of protein loops that seamlessly bridge the gap between the anchor points without introducing any steric collisions remains an open challenge. A variety of algorithms have been developed to tackle the loop closure problem, ranging from inverse kinematics to knowledge-based approaches that utilize pre-existing fragments extracted from known protein structures. However, many of these approaches focus on the generation of conformations that mainly satisfy the fixed end point condition, leaving the steric constraints to be resolved in subsequent post-processing steps. In the present work, we describe a simple solution that simultaneously satisfies not only the end point and steric conditions, but also chirality and planarity constraints. Starting from random initial atomic coordinates, each individual conformation is generated independently by using a simple alternating scheme of pairwise distance adjustments of randomly chosen atoms, followed by fast geometric matching of the conformationally rigid components of the constituent amino acids. The method is conceptually simple, numerically stable and computationally efficient. Very importantly, additional constraints, such as those derived from NMR experiments, hydrogen bonds or salt bridges, can be incorporated into the algorithm in a straightforward and inexpensive way, making the method ideal for solving more complex multi-loop problems. The remarkable performance and robustness of the algorithm are demonstrated on a set of protein loops of length 4, 8, and 12 that have been used in previous studies