Proteins in silico-modeling and sampling

Proteins are linear chain molecules made out of amino acids. Only when they fold to their native states, they become functional. This dissertation aims to model the solvent (environment) effect and to develop & implement enhanced sampling methods that enable a reliable study of the protein folding problem in silico. We have developed an enhanced solvation model based on the solution to the Poisson-Boltzmann equation in order to describe the solvent effect. Following the quantum mechanical Polarizable Continuum Model (PCM), we decomposed net solvation free energy into three physical terms– Polarization, Dispersion and Cavitation. All the terms were implemented, analyzed and parametrized individually to obtain a high level of accuracy. In order to describe the thermodynamics of proteins, their conformational space needs to be sampled thoroughly. Simulations of proteins are hampered by slow relaxation due to their rugged free-energy landscape, with the barriers between minima being higher than the thermal energy at physiological temperatures. In order to overcome this problem a number of approaches have been proposed of which replica exchange method (REM) is the most popular. In this dissertation we describe a new variant of canonical replica exchange method in the context of molecular dynamic simulation. The advantage of this new method is the easily tunable high acceptance rate for the replica exchange. We call our method Microcanonical Replica Exchange Molecular Dynamic (MREMD). We have described the theoretical frame work, comment on its actual implementation, and its application to Trp-cage mini-protein in implicit solvent. We have been able to correctly predict the folding thermodynamics of this protein using our approach

Emerging Biomedical Applications of the Vesicular Stomatitis Virus Glycoprotein

Nanoparticles (NPs) made of metals, polymers, micelles, and liposomes are increasingly being used in various biomedical applications. However, most of these NPs are hazardous for long- and short-term use and hence have restricted biomedical applications. Therefore, naturally derived, biocompatible, and biodegradable nanoconstructs are being explored for such applications. Inspired by the biology of viruses, researchers are exploring the viral proteins that hold considerable promise in biomedical applications. The viral proteins are highly stable and further amenable to suit specific biological applications. Among various viral proteins, vesicular stomatitis virus glycoprotein (VSV-G) has emerged as one of the most versatile platforms for biomedical applications. Starting with their first major use in lentivirus/retrovirus packaging systems, the VSV-G-based reagents have been tested for diverse biomedical use, many of which are at various stages of clinical trials. This manuscript discusses the recent advancements in the use of the VSV-Gbased reagents in medical, biological research, and clinical applications particularly highlighting emerging applications in biomedical imaging

Conformational Preferences of Triantennary and Tetraantennary Hybrid N-Glycans in Solution: Insights from 20 μS Long Atomistic Molecular Dynamic Simulations

In the current study, we have investigated the conformational dynamics of a triantennary and a tetraantennary hybrid N-glycan associated with HIV glycoprotein using 20 micro-second long all-atom molecular dynamics simulations. <br /

Energetics of Mutation-Induced Changes in Potency of Lersivirine against HIV-1 Reverse Transcriptase

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy for the treatment of HIV-1. A common problem with the first generation NNRTIs is the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular, K103N and Y181C, which lead to resistance to the entire class of inhibitor. Here we have evaluated the relative affinity of the newly designed NNRTI lersivirine (LRV) against drug-resistant mutations in HIV-1 RT using the molecular mechanics generalized Born surface area (MM-GBSA) method. Eight single and one double mutant variants of RT are considered. Our results are in good agreement with experimental results and yield insights into the mechanisms underlying mutation-induced changes in the potency of LRV against RT. The strongest (54-fold) increase in the dissociation constant is found for the mutant F227C, originating from reduced electrostatic and van der Waals interactions between LRV and RT as well as a higher energetic penalty from the desolvation of polar groups. For the mutants K103N and Y181C only a moderate (2-fold) increase in the dissociation constant is found, due to a balance of opposite changes in the polar solvation as well as the electrostatic and van der Waals interactions between LRV and RT. The dissociation constant is decreased for the Y188C and G190A (2-fold), the M184V (5-fold), and the Y188C/Y188C mutant (10-fold), due to stronger electrostatic interactions between LRV and RT. Our results thus suggest that LRV is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses, which is in agreement with experimental observations

Mutation-Induced Loop Opening and Energetics for Binding of Tamiflu to Influenza N8 Neuraminidase

Tamiflu, also known as oseltamivir (OTV), binds to influenza A neuraminidase (H5N1) with very high affinity (0.32 nM). However, this inhibitor binds to other neuraminidases as well. In the present work, a systematic computational study is performed to investigate the mechanism underlying the binding of oseltamivir to N8 neuraminidase (NA) in “open” and “closed” conformations of the 150-loop through molecular dynamics simulations and the popular and well established molecular mechanics Poisson–Boltzmann (MM-PBSA) free energy calculation method. Whereas the closed conformation is stable for wild type N8, it transforms into the open conformation for the mutants Y252H, H274Y, and R292K, indicating that bound to oseltamivir these mutants are preferentially in the open conformation. Our calculations show that the binding of wild type oseltamivir to the closed conformation of N8 neuraminidase is energetically favored compared to the binding to the open conformation. We observe water mediated binding of oseltamivir to the N8 neuraminidase in both conformations which is not seen in the case of binding of the same drug to the H5N1 neuraminidase. The decomposition of the binding free energy reveals the mechanisms underlying the binding and changes in affinity due to mutations. Considering the mutant N8 variants in the open conformation adopted during the simulations, we observe a significant loss in the size of the total binding free energy for the N8_Y252H–OTV, N8_H274Y–OTV, and N8_R292K–OTV complexes compared to N8_WT–OTV, mainly due to the decrease in the size of the intermolecular electrostatic energy. For R292K, an unfavorable shift in the van der Waals interactions also contributes to the drug resistance. The mutations cause a significant expansion in the active site cavity, increasing its solvent accessible surface compared to the crystal structures of both the open and closed conformations. Our study underscores the need to consider dynamics in rationalizing the structure–function relationships of various antiviral inhibitor–NA complexes

Theoretical mimicry of biomembranes

The study of membrane proteins requires a proper consideration of the specific environment provided by the biomembrane. The compositional complexity of this environment poses great challenges to all experimental and theoretical approaches. In this article a rather simple theoretical concept is discussed for its ability to mimic the biomembrane. The biomembrane is approximated by three mimicry solvents forming individual continuum layers of characteristic physical properties. Several specific structural problems are studied with a focus on the biological significance of such an approach. Our results support the general perception that the biomembrane is crucial for correct positioning and embedding of its constituents. The described model provides a semi-quantitative tool of potential interest to many problems in structural membrane biology. © 2009 Federation of European Biochemical Societies

Systematic study of the boundary composition in poisson boltzmann calculations

We describe a three-stage procedure to analyze the dependence of Poisson Boltzmann calculations on the shape, size and geometry of the boundary between solute and solvent. Our study is carried out within the boundary element formalism, but our results are also of interest to finite difference techniques of Poisson Boltzmann calculations. At first, we identify the critical size of the geometrical elements for discretizing the boundary, and thus the necessary resolution required to establish numerical convergence. In the following two steps we perform reference calculations on a set of dipeptides in different conformations using the Polarizable Continuum Model and a high-level Density Functional as well as a high-quality basis set. Afterwards, we propose a mechanism for defining appropriate boundary geometries. Finally, we compare the classic Poisson Boltzmann description with the Quantum Chemical description, and aim at finding appropriate fitting parameters to get a close match to the reference data. Surprisingly, when using default AMBER partial charges and the rigorous geometric parameters derived in the initial two stages, no scaling of the partial charges is necessary and the best fit against the reference set is obtained automatically

Importance of Polar Solvation and Configurational Entropy for Design of Antiretroviral Drugs Targeting HIV‑1 Protease

Both KNI-10033 and KNI-10075 are high affinity preclinical HIV-1 protease (PR) inhibitors with affinities in the picomolar range. In this work, the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method has been used to investigate the potency of these two HIV-1 PR inhibitors against the wild-type and mutated proteases assuming that potency correlates with the affinity of the drugs for the target protein. The decomposition of the binding free energy reveals the origin of binding affinities or mutation-induced affinity changes. Our calculations indicate that the mutation I50V causes drug resistance against both inhibitors. On the other hand, we predict that the mutant I84V causes drug resistance against KNI-10075 while KNI-10033 is more potent against the I84V mutant compared to wild-type protease. Drug resistance arises mainly from unfavorable shifts in van der Waals interactions and configurational entropy. The latter indicates that neglecting changes in configurational entropy in the computation of relative binding affinities as often done is not appropriate in general. For the bound complex PR_I50V–KNI-10075, an increased polar solvation free energy also contributes to the drug resistance. The importance of polar solvation free energies is revealed when interactions governing the binding of KNI-10033 or KNI-10075 to the wild-type protease are compared to the inhibitors darunavir or GRL-06579A. Although the contributions from intermolecular electrostatic and van der Waals interactions as well as the nonpolar component of the solvation free energy are more favorable for PR–KNI-10033 or PR–KNI-10075 compared to PR–DRV or PR–GRL-06579A, both KNI-10033 and KNI-10075 show a similar affinity as darunavir and a lower binding affinity relative to GRL-06579A. This is because of the polar solvation free energy which is less unfavorable for darunavir or GRL-06579A relative to KNI-10033 or KNI-10075. The importance of the polar solvation as revealed here highlights that structural inspection alone is not sufficient for identifying the key contributions to binding affinities and affinity changes for the design of drugs but that solvation effects must be taken into account. A detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs