Book of Abstracts: Albany 2007
June 19-23 2007
Conformational Dynamics of Acto-myosin Motor as Revealed by X-ray Protein Footprinting
Actin is a globular protein that polymerizes to form a filamentous network within a eukaryotic cell and provides mechanical and locomotive support to the cell. In muscle cells, actin filaments interact with myosin filaments to generate muscle movements. In the present study, radiolytic footprinting is used to derive structural and dynamic information of the actin-myosin complex. Specifically, hydroxyl radical mediated covalent modifications of amino acid side chain probes, which is dependent on the solvent accessibility of the side chains, is used to monitor the changes in the conformational environment of actin induced by myosin binding. Samples are subjected to synchrotron X-ray exposure (National Synchrotron Light Source, Brookhaven National Laboratory, New York) followed by enzymatic proteolysis and injection to an HPLC coupled mass spectrometer. Dose response curves are plotted to extract modification rates constants of peptide oxidation. The modification rate is calculated for various peptides of actin in the absence and presence of myosin subfragment 1. Modification rate values of probe residues are compared among actin monomer, actin filament, and actin filament-myosin complex, to define the regions of binding and conformational alterations of actin induced by myosin. A number of probe residues in subdomain 1 showed significant decreases in their rate of modification upon actin binding (some showed no modification at all) indicating a potential binding region. Peptides, not located in the putative binding surface, are also found to show differential modification; they identify the loci of major conformational changes accompanying myosin binding. This conformational dynamics of actin filament upon binding to myosin is discussed with regard to the acto-myosin?s muscle contractile activity. These results add an important step towards understanding the structural basis of muscle contraction.
J. K. Amisha Kamal1
1Center for Proteomics and Mass Spectrometry