The specific goal of the proposed research is to determine the dominant mechanism for stimulated protein motion using myoglobin and hemoglobin proteins as model systems. Following ligand dissociation, the ensuing response function of heme proteins is to evolve to its deoxy tertiary structure. This motion involves the correlated displacement of thousands of atomic degrees of freedom. Exactly how the protein system evolves and propagates the structural changes is central to a general understanding of functionally relevant protein motion and molecular cooperativity. The forces that develop for the atomic displacements arise from the potential energy gradients that develop with ligand dissociation. The length scale over which these forces are distributed, the multiplicity of pathways, and the energetics for the motion are the key issues. In the proposed studies, the photodissociation of CO will be used as an optical trigger to initiate the structural changes. The emphasis of the research is on directly monitoring the structural relaxation with various optical probes to the protein motion. CO was chosen as the primary ligand for these studies as it exhibits minimal recombination on the time scale of interest to complicate the structural relaxation dynamics. To address the issue of length scale for the acting forces on the induced motion, one needs to determine the structural relaxation dynamics using probes that are sensitive to different length scales of the protein. The global motion (long length scale motion) will be followed using picosecond/femtosecond phase grating spectroscopy, while motion local to the epicenter for the forces at the Fe-CO binding site will focus on time-resolved Raman probes of the proximal histidine motion. These studies will determine the degree of collective atomic displacements during the initial phase of the triggered protein response. A new technique based on femtosecond librational scattering and optical Kerr effect detection will provide a direct measurement of the low frequency collective modes coupled to the structural relaxation coordinate. The overall energetics or driving force for the different phases of the motion will be followed by phase grating spectroscopy modified to selectively study thermally induced density changes. This thermal phase grating method is at the fundamental limit with respect to time resolution for the determination of bioenergetics. It has sufficient time resolution and sensitivity to distinguish between collective mode and conformational substate models as the dominant phase for the initial protein structural changes. The combined use of phase grating spectroscopy, femtosecond librational scattering, and energetics give a comprehensive experimental approach for studying the mechanics of protein motion. These studies will be extended to the microsecond range so that a complete connection can be made from the initial femtosecond/picosecond dynamics that initiate the motion to the longer time scale structural relaxations relative to functionality.
| Deak, J; Richard, L; Pereira, M et al. (1994) Picosecond phase grating spectroscopy: applications to bioenergetics and protein dynamics. Methods Enzymol 232:322-60 |
| Richard, L; Genberg, L; Deak, J et al. (1992) Picosecond phase grating spectroscopy of hemoglobin and myoglobin: energetics and dynamics of global protein motion. Biochemistry 31:10703-15 |
| Genberg, L; Richard, L; McLendon, G et al. (1991) Direct observation of global protein motion in hemoglobin and myoglobin on picosecond time scales. Science 251:1051-4 |
| Miller, R J (1991) Vibrational energy relaxation and structural dynamics of heme proteins. Annu Rev Phys Chem 42:581-614 |
