Several diverse projects are being pursued. These are the major ones pursued during the past year. Analysis of the glomerular phosphoproteome Diseases of the kidney filtration barrier are a leading cause of endstage renal failure. Most disorders affect the podocytes, polarized cells that are connected by a unique cell junctional complex, the slit diaphragm. Podocytes require tightly controlled signaling to maintain their integrity, viability and function. Here we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins including podocyte-specific gene products identified in an unbiased tandem mass spectrometry-based approach. We discovered 2,449 phosphorylated proteins corresponding to 4,171 identified high-confident phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. Among the 146 phosphorylation sites found on proteins abundantly expressed in podocytes, several sites resided close to residues known to be mutated in human genetic forms of proteinuria. One such site discovered on the slit diaphragm protein Podocin, threonine-234 (T234), resides at the interface of Podocin dimers with a distance between both T234 residues of less than 10 Angstrom. We show that phosphorylation critically regulates dimer formation and that this may represent a general principle for the assembly of the large family of PHB-domain containing proteins. Proteins are dynamic in nature and can have many conformational states, including active, inactive, and intermediate states. These conformational states can represent an array of structural features that distinguish the ability of the protein to bind other molecules. Elucidating the transitions between the conformational states of protein complexes is critical for effective drug discovery, design, and delivery methods, as it would allow direct correlation between conformational-activity and structure-activity relationships. In particular, the NF-B inducing kinase (NIK) and the inhibitor of B kinase-B (IKK), two protein kinases associated with inflammatory responses, were investigated. Molecular dynamics simulations were employed to consider how conformational changes and protein-protein interactions within multimeric assemblies are influenced by changes in the interacting subunits as well as the environment. Protein kinases complexed with small molecule activators or inhibitors were examined in the active, inactive, and mutant states to correlate structure-property and structure-function relationships as a function of intracellular ionic strength. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions and the binding of small molecules are sensitive to changes in the ionic strength. Ligand binding pockets either compress or expand, affecting both local and distal intermolecular interactions. Binding free energies of inhibitors to HIV protease Binding of drugs to proteins can result in change of protonation states of ionizable residues of the drugs, or in the active sites of proteins. In order to calculate accurate binding free energies, this change in protonation state needs to be accounted for. We apply the recently developed method for binding free energie calculations based on our EDS-HREM constant pH method to calculate binding free energies of inhibitors to HIV protease. Preliminary results indicate that change in protonation states upon binding makes a non-negligible contribution, and results in more accurate binding free energies. Protonation states in the selectivity filter of voltage activated Na channel We are using EDS-HREM constant pH simulations to determine protonation state of the selectivity filter of voltage activated Na channel. The selectivity filter consists of four glutamate residues. Previous MD simulations of this channel were performed with all four glutamic acid residues charged. However, our preliminary constant pH simulations indicate that the most likely state with Na bound in the selectivity filter is that where one of the four glutamates in protonated, i.e., uncharged. In the absence of Na, an additional glutamic acid residues may get protonated. This simulation indicates a greater need for careful consideration of protonation states in this important channel. Further simulations in the presence of external voltage will be performed to establish its effect on protonation states. Computational study of -galactosidase Careful analysis provided improvement in cryo-electron (cryo-EM) microscopy, with resolutions of 2.2 for -galactosidase and detection of water densities. Our in-depth analysis of previously-published X-ray structures and water dynamics in molecular dynamics simulations brings insight into differences in experimental placement of functional water with important biological functions in oligomerization and binding. This study is an on-going collaboration with Dr. S. Subramaniam in the National Cancer Institute. Structure and dynamics of human islet amyloid polypeptide Islet amyloid polypeptide (IAPP), is a 37-long peptide that is the main constituent of amyloid aggregates of type-II diabetes. Certain species acquire the disease, whereas others dont. Human (hIAPP) and cat IAPP have been shown to aggregate, but rat and pig do not. Starting from the solid-state NMR structure for hIAPP, we perform single-point mutations towards the other species and employ molecular dynamics simulations on the resulting structures. By analyzing the dynamics of each structure, we infer the relative contributions of mutations on different structural elements, and establish which has a dominant modulating effect. Results from this study will hint towards possible novel therapeutics for the treatment of type-II diabetes. This is a comprehensive study in collaboration with the group of Dr. N.-V. Buchete (University College Dublin, Ireland). Blind free energy calculations on CBClip-guest complexes We participated in the SAMPL5 blind challenge for the absolute binding free energy calculations of CBClip-guest complexes. For the calculations, the initial conformations of CBClip-guest complexes were obtained using docking and molecular dynamics simulations. Free energy calculations were performed using thermodynamic integration (TI) with soft-core potentials and Bennetts Acceptance Ratio (BAR) method based on a serial insertion scheme. We compared the results obtained with TI simulations with soft-core potentials and Hamiltonian replica exchange simulations with the serial insertion method combined with the BAR method. The results show that the difference between the two methods can be mainly attributed to the van der Waals free energies, suggesting that either
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