Several diverse projects are being pursued. These are the major ones pursued during the past year. Calcium ATPase Conformational Transition through Self-Guided Langevin Dynamics Simulation The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) transport calcium ions from cytoplasm into the reticulum and relaxes the muscle cells. Many crystal structures of SERCA1 in various binding states have been determined, which provide insights into the mechanism of transport Ca2+ across the membrane. Molecular modeling and simulation studies are also devoted to the understanding of this important process. SERCA1a is an integral membrane protein. It comprises a single polypeptide chain of 994 amino acid residues. It is clear from the crystal structures that SERCA has a 10 helices trans-membrane domain (M), an actuator domain (A), a nucleotide binding domain (N), and a phosphorylation domain (P). The Ca2+ transport cycle starts with Ca2E1 through the Ca2+ dependent phosphorylation by ATP, leading to the formation of the Ca2E1P high-energy intermediate. Ca2E1P transits to Ca2E2P, which releases Ca2+ into the lumen of SR and leads to the E2P state. After dephosphorylation, E2P transits to E2 state and closes the luminal gate. Through thermo agitation, E2 transits to E1 by releasing protons into the cytoplasm. E1 has high Ca2+ affinity and binds with Ca2+ to form Ca2E1. To understand the transport mechanism, it is desirable to study the dynamic process during the conformation transition. Self-guided Langevin dynamics (SGLD) is a simulation method capable of studying events with large conformational change. SGLD simulations of SERCA at different binding states produce conformational transitions between conformational states. New conformations for E1.2Ca2+ and E2.P state have been identified and at E2 state the crystal structure is a preferred conformation. Atomic mechanism of the kinesin walking on microtubule Kinesin is a protein belonging to the class of Cytoskeletal motor proteins. Kinesin converts the energy of ATP hydrolysis into stepping movement along microtubules, which supports several vital cellular functions including mitosis, meiosis, and the transport of cellular cargo. Because kinesin is a fundamental protein, further research on the topic will provide important information as to how it functions. Combined with low resolution electron microscopic images, self-guided Langevin dynamics simulations are performed to study molecular motion and conformational change of kinesin motor domain in water and binding with microtubule. SGLD enable simulation to reach the time scale required for conformational change to understand the role of ATP binding and interaction with microtubules. 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. Cyclic nucleotide modulation of structure and dynamics of a hyperpolarization-activated cyclic nucleotide-gated ion channel Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels play a fundamental role in electric signaling in nerves, muscles, and synapses; however, their ligand gating mechanism is not well understood. There is little structural information on HCN2, as only its cytoplasmic domain in the holo form has been solved by X-ray crystallography. At the N-terminus, this structure contains the C-linker (helices A-F) and the cyclic nucleotide binding domain (CNBD), comprising of eight β-strands and helices A-C. cAMP modulates the HCN2 channel opening and it also promotes oligomerization. Through collaboration between experimental and computational methods, this study brings insight into the mechanism of cyclic adenosine monophosphate (cAMP) modulation of the HCN2 channel by exploring the monomer and tetramer dynamics in the apo and holo states. We performed all-atom, as well as coarse-grained (CG) molecular dynamics simulations. In the CNBD the distance between the C-helix and the β-roll in the binding pocket increases in the absence of cAMP. Our results corroborate recent transition metal ion Frster resonance energy transfer and double electron-electron resonance studies showing the outward movement of the C-helix in the absence of cAMP. The most striking dynamics observed in simulations of the apo monomer is the unfolding of the C-helix together with loss of contact between A and B helices in the C-linker, and the CNBD. In contrast, in the holo monomer, these contacts are retained, although the C-helix is unstable. In both the apo and the holo tetramer, the C-helix remains folded. We inferred that this is due to a reduced solvent accessible surface area, as the C-helix of a subunit is sheltered by the adjacent subunit. The folding/unfolding interplay of the C-helix translates into the vertical movement of the CNBD. As recently observed in MloK1, an analogous prokaryotic ion channel, this vertical displacement induces CNBD disinhibition, which promotes channel opening. This study is conducted in collaboration with Dr. R. Best (NIDDK) and Dr. W. Zagotta (University of Washington). Computational study of β-galactosidase With study is a collaborative project with the group of Dr. S. Subramaniam (NCI). Their cryo-electron (cryo-EM) microscopy work has recently produced a very high-resolution (2.2 ) for β-galactosidase, with promise to improve even further. Their technique is able to pin-point ions and water molecules interacting with the molecule. Our computational study focuses on the dynamic behavior of the resulting cryo-EM and that of previously-published X-ray structures. This comparative study provides insight into minuscule, albeit crucial, differences in structural aspects that could influence protein dynamics and function. Structure and dynamics of human islet amyloid polypeptide Islet amyloid polypeptide (IAPP, or amylin), 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 types and perform molecular dynamics simulations on the resulting structures. By analyzing the stability of each structure we infer the relative contributions of mutations on different structural elements, and establish which has a dominant modulating effect. This is a comprehensive study in collaboration with the group of Dr. N-V. Buchete (University College Dublin, Ireland).
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