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Molecular Dynamics Simulation of Calbindin D-9k in apo, Singly and Doubly Loaded States in Various Side-chains

Thapa, Mahendra B.
http://orcid.org/0000-0003-2442-1056
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470044265

Abstract Details

2016, PhD, University of Cincinnati, Arts and Sciences: Physics.
Calbindin D9k (CAB) is a single domain calcium-binding protein and is the smallest members of the calmodulin superfamily, possessing a pair of calcium-binding EF-hands, and structures for all four states have been determined and extensively characterized experimentally. Because of the tremendous advancement in hardware and software computer technologies in recent years, longer and more realistic molecular dynamics (MD) simulations of a protein are possible now in reasonable periods of time. These advances were exploited to generate multiple, all-atom MD simulations of CAB via the AMBER software package, and the resulting trajectories were employed to calculate backbone order parameters of the apo, the singly and the doubly loaded states of calcium in CAB. The results are in very good agreement with corresponding experimental NMR-based (Nuclear Magnetic Resonance spectroscopy) results, and are improved in comparison to those calculated over a decade ago; use of modified force fields played a key role in the observed improvements. The apo state is the most flexible, and the singly loaded and the doubly loaded states are similar, thus supporting positive cooperativity in line with the experimental results. Further, B-factor calculations of backbone atoms for these calcium-binding states of calbindin D9k also support such cooperativity. Although changes in side-chain motions are not necessarily correlated to changes in protein backbone mobility, past studies on the comparison of experimental and simulated methyl side-chain NMR relaxation parameters of CAB for the doubly-loaded state reported significant improvements in the quantitative representation of side-chain motion by MD simulation. In this project, the order parameters for various side chains in apo, singly loaded and doubly loaded states of CAB were calculated. The primary goal of this work was to determine whether or not the allosteric effect of calcium binding, as observed via the backbone order parameters, also extended to the amino acid side chains, and if so, to what extent. Such information could be useful in better understanding the physical basis of cooperative calcium binding in CAB. Most of the residues which provide ligands to bind calcium at the binding sites support positive cooperativity, as observed when Ca-Cß, Cß-C?, C-C bond and C-O bonds of COO groups of aspartic and glutamic acid residues, the C-N bond of the side-chain amide group in asparagine and glutamine residues, and the N-H bonds of amide (NH2) group order parameters were studied. There are only a few residues containing methyl groups that are involved in providing ligands to the calcium, and the studies of order parameters of C-C bond and C-H bond of these methyl groups did not exhibit the cooperativity effect upon calcium binding; the simulated C-C bond order parameter of the methyl group symmetry axis did correlate well with the experimental results for the fully loaded state of CAB (4ICB). Analysis of the MD trajectories using GSATools and MutInf, provided valuable insights into possible pathways for communicating allosteric effects between the two calcium-binding sites of CAB.
Mark Rance, Ph.D. (Committee Chair)
Eric Johnson, Ph.D. (Committee Member)
Thomas Beck, Ph.D. (Committee Member)
David Mast, Ph.D. (Committee Member)
Rostislav Serota, Ph.D. (Committee Member)
175 p.
Physics
Calbindin D-9k; Side-chain dynamics; Molecular Dynamics; 4ICB; AMBER; NMR

Recommended Citations

Citations

  • Thapa, M. B. (2016). Molecular Dynamics Simulation of Calbindin D-9k in apo, Singly and Doubly Loaded States in Various Side-chains [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470044265

    APA Style (7th edition)

  • Thapa, Mahendra. Molecular Dynamics Simulation of Calbindin D-9k in apo, Singly and Doubly Loaded States in Various Side-chains. 2016. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470044265.

    MLA Style (8th edition)

  • Thapa, Mahendra. "Molecular Dynamics Simulation of Calbindin D-9k in apo, Singly and Doubly Loaded States in Various Side-chains." Doctoral dissertation, University of Cincinnati, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470044265

    Chicago Manual of Style (17th edition)

ucin1470044265
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This open access ETD is published by University of Cincinnati and OhioLINK.