Coupled flexibility change in cytochrome P450cam substrate binding determined by neutron scattering, NMR, and molecular dynamics simulation.

Neutron scattering and nuclear magnetic resonance relaxation experiments are combined with molecular dynamics (MD) simulations in a novel, to our knowledge, approach to investigate the change in internal dynamics on substrate (camphor) binding to a protein (cytochrome P450cam). The MD simulations agree well with both the neutron scattering, which furnishes information on global flexibility, and the nuclear magnetic resonance data, which provides residue-specific order parameters. Decreased fluctuations are seen in the camphor-bound form using all three techniques, dominated by changes in specific regions of the protein. The combined experimental and simulation results permit a detailed description of the dynamical change, which involves modifications in the coupling between the dominant regions and concomitant substrate access channel closing, via specific salt-bridge, hydrogen-bonding, and hydrophobic interactions. The work demonstrates how the combination of complementary experimental spectroscopies with MD simulation can provide an in-depth description of functional dynamical protein changes.