TCB Publications - Abstract

Thomas W. Lynch, Mark A. McLean, Dorina Kosztin, Klaus Schulten, and Stephen G. Sligar. High pressure gel mobility shift analysis and molecular dynamics: Investigating specific protein-nucleic acid recognition. In R. Hayashi, editor, Trends in High Pressure Bioscience and Biotechnology, pp. 87-94. Elsevier, 2002.

LYNC2002A In this report, we describe the application of electrophoresis for monitoring protein-DNA binding equilibria at high pressure utilizing the gel mobility shift assay. The first protein-DNA recognition complex studied using this methodology is the restriction endonuclease BamHI binding the cognate DNA recognition sequence. The application of hydrostatic pressure to the specific recognition complex of BamHI-DNA favors dissociation, which is apparent due to the increase in the equilibrium dissociation constant ($K_{d}$) at elevated pressures. From the dependence of $K_{d}$ on pressure, the volume change ($\Delta V$) of dissociation was determined. Molecular Dynamic (MD) simulations on the BamHI-DNA complex at both ambient and elevated pressures were performed to identify the structural origins of the observed experimental results. The simulation trajectories have identified important protein-DNA recognition elements that are disrupted with pressure. The trajectories also illustrate an increased hydration of the BamHI-DNA interface at elevated pressure. Both of these calculated pressure effects would favor dissociation of the complex. The combination of MD simulations and high pressure gel shift analysis proved useful in identifying thesse factors for maintaining BamHI-DNA complex stability.

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