Calcium is one of the closely relevant metal ions that involves in enormous physicochemical activities in human body, including cell division and apoptosis, muscle contraction, neurotransmitter release, enzyme activation, and blood-clotting. Calcium fulfills its functions through the binding to different classes of calcium-binding proteins. To facilitate our understanding of the roles of calcium in biological systems, and to design novel metal-binding proteins with tailored binding capabilities, it is important to develop computation algorithms to predict calcium-binding sites in different classes of proteins. In literature, calcium-binding sites may be represented by either a spacial point, or the set of residues chelating calcium ion. A thorough statistical analysis of known calcium-binding proteins deposited in Protein Data Bank gives reference values of various parameters characterizing geometric and chemical features in calcium-binding sites including distances, angles, dihedral angles, the Hull property, coordination numbers, ligand types and formal charges. It also reveals clear differences between the well-known EF-hand calcium-binding motif and other calcium-binding motifs. Utilizing the above multiple geometric and chemical parameters in well-formed calcium binding sites, we developed MUG (MUltiple Geometries) program. MUG can re-identify the coordinates of the documented calcium ion and the set of ligand residues. Three previously published data sets were tested. They are comprised of, respectively, 19, 44 and 54 holo protein structures with 48, 92 and 91 documented calcium-binding sites. Defining a "correct hit" as a point within 3.5 angstrom to the documented calcium location, MUG has a sensitivity around 90% and selectivity around 80%. The set of ligand residues (calcium-binding pockets) were identified for 43, 66 and 63 documented calcium ion in these three data set respectively. In order to achieve true prediction, our program was then enhanced to predict calcium-binding pockets in apo (calcium-free) proteins. Our new program MUGSR accounts for the conformational changes involved in calcium-binding pockets before and after the binding of calcium ions. It is able to capture calcium binding pockets that may undergo local conformational changes or side chain torsional rotations, which is validated by referring back to the corresponding holo protein structure sharing more than 98% sequence similarity with the apo protein.

Kim, Straub, and Keyes introduced the statistical temperature molecular dynamics (STMD) algorithm to overcome broken ergodicity by sampling a non­-Boltzmann flat energy histogram as noted in Kim, Straub, and Keyes, Phys. Rev. Lett. 97: 050601 (2007). Canonical averages are calculated via reweighting to the desired temperature. While STMD is promising, its application has been almost entirely to simple or model systems. In this dissertation the implementation of STMD into the biosimulation package CHARMM is used to simulate the methionine enkephalin pentamer peptide with a methione terminal cap in a droplet of CHARMM TIP3P water molecules. Chain thermodynamics is analyzed from the novel perspective of the statistical temperature as a function of potential energy, $TS(U), automatically generated by STMD. Both the minimum in the slope of $TS(U), and the peak in the heat capacity as a function of temperature, calculated via reweighting, indicate a collapse transition at Tθ ≈ 253K. Distributions of dihedral angles are obtained as a function of temperature. Rotamer regions found in the literature are reproduced, along with unique regions not found previously, including with advanced algorithms, indicating the power of STMD enhanced sampling.

No / Most rotamer libraries are generated from subsets of the PDB and do not fully represent the conformational scope of protein side chains. Previous attempts to rectify this sparse coverage of conformational space have involved application of weighting and smoothing functions. We resolve these limitations by using physics-based molecular dynamics simulations to determine more accurate frequencies of rotameric states. This work forms part of our Dynameomics initiative and uses a set of 807 proteins selected to represent 97% of known autonomous protein folds, thereby eliminating the bias toward common topologies found within the PDB. Our Dynameomics derived rotamer libraries encompass 4.8 × 10(9) rotamers, sampled from at least 51,000 occurrences of each of 93,642 residues. Here, we provide a backbone-dependent rotamer library, based on secondary structure ϕ/ψ regions, and an update to our 2011 backbone-independent library that addresses the doubling of our dataset since its original publication. / NIH

The study of enzyme binding sites is an essential but rather demanding process of increased complexity since the amino acids lining these areas are not rigid. At the same time, the minimization of side effects and the specificity of new ligands is a great challenge in the structure-based drug design approach. Using glycogen phosphorylase - a validated target for the development of new antidiabetic agents - as a case study, this project focuses on the examination of side-chain conformations of amino acids that play a key role in the catalytic site of the enzyme. Specifically, different rotamers of each amino acid were collected to build a dataset of different conformations of the catalytic site. The rotamers were filtered by their probability of occurrence and subsequently, all rotamers that create steric clashes were rejected. Then, these conformations were clustered based on their similarity. Three different clustering algorithms and multiple numbers of clusters were tested using the silhouette scores evaluation for the clustering process. In order to measure the similarity, the Euclidean metric was used which due to the correspondence of the coordinates between the conformations was very similar to the cRMSD metric. Two-level clustering was applied to the dataset for more in-depth observations. According to the clustering results, specific aminoacids with major geometrical variations in their rotamers play the most important role in the separation of the clusters. Additionally, all rotamers of an amino acid can be grouped based on their structure, something that was confirmed using “Chimera” software as a visualization tool. To this end, the ultimate aim of this study is to examine whether the clustering of conformations produces clusters with points geometrically similar to each other, in order to identify near neighbors, i.e. conformations that are quite similar in structure but do not play a determinant role in the function and those that are quite diverse and could be further exploited.

The cytoskeletal protein vinculin plays a key role in the control of cell-cell or cell-matrix adhesions. It is involved in the assembly and disassembly of focal adhesions and affects their mechanical stability. While many facts highlight the importance and significance of vinculin for vital processes, its precise role in the regulation of cell adhesions is still only partially understood. Various EPR methods are used in this work in order to study the vinculin tail (Vt) domain in an aqueous buffer solution and its structural changes induced by F-actin and acidic phospholipids. EPR results in combination with a rotamer library approach (RLA), MD simulation and other computational methods allowed the construction of molecular models of Vt and dimeric Vt in the presence and absence of its binding partners. Furthermore, X-band orientation selective DEER measurements were applied on a Vt double mutant. It could be shown that the determination of the mutual orientation of protein bound spin labels is possible at X-band frequencies, if the orientation correlation of the spin label pair is strong. The method established here can be used to determine valuable information about proteins and nucleic acids, expanding the virtue of DEER spectroscopy as a tool for structure determination.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.

Die Elektronen-Spin-Resonanz-Spektroskopie (ESR) in Verbindung mit ortsspezifischer Spinmarkierung stellt eine hervorragende Möglichkeit dar, um die Struktur und Dynamik von Proteinen aufzuklären. In dieser Dissertation wurden mit Hilfe der Hochfeld-ESR-Spektroskopie (W-Band, 95 GHz, T=160 K) für dreizehn spinmarkierte Colicin A Proben die Polarität und die Protizität der Umgebung der Spinlabelbindestelle bestimmt. Wasserzugänglichkeiten und Wasserstoffbrückenbindungen zum Spinlabel wurden mittels Puls-ESR Methoden (3-Puls-D-ESEEM und Hahn-Echozerfall) bestimmt und die Ergebnisse mit den Polaritäts- und Protizitätswerten korreliert. Raumtemperaturspektren dieser Proben im X-Band (9.5 GHz), Q-Band (34 GHz) und W-Band (95 GHz) liefern Informationen über die Spinlabelbewegung. Mit Hilfe von Molekulardynamiksimulationen (MD) der spinmarkierten kanalbildenden Domäne von Colicin A konnten die Konformationen (Rotameranalyse) und die Dynamik der Spinlabelseitenketten in den unterschiedlichen Umgebungen charakterisiert werden. Der Vergleich der experimentellen mit den aus MD-Trajektorien berechneten ESR-Spektren liefert die Beiträge der unterschiedlichen Rotamerübergänge, die für die beobachteten Spektrenformen charakteristisch sind.