This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We continue to develop and refine the methods of coherent infrared photon echo spectroscopy to examine structural fluctuations in peptides and proteins through their vibrational correlation functions. Heterodyned transient gratings and three pulse photon echo methods for determining vibrational frequency correlation functions are now being evaluated to optimize the signal to noise ratio and extend the time scales over which the structural relaxation can be examined. We are also developing methods to determine the correlations between the structure fluctuations at different spatial locations of a wide variety of secondary structures. We have developed 2D-IR and 3D-IR methods in the region of the N-H and O-H (and isotopic variants) stretch modes. Combined amide-I and amide-A four-wave experiments have been accomplished in which couplings between remote N-H and amide carbonyls have been determined. Infrared methods with appropriate band width and time resolution have been developed to measure (i) The overall protein rotation, based on intrinsic probes; (ii) the motion of the chromophore as a whole relative to the protein; (iii) the orientational motion of the particular IR active bond; (iv) both the order parameters and time constants for these motions; (v) correlations between infrared absorption and chromophore absorption; (vi) correlations between different IR transitions of the same species (e.g. amino acid); (vii) probing the amide-I substructure by dynamic hole-burning and (viii) infrared photon echo measurements. The Resource has proved the principle of a number of important experiments aimed at using molecular vibrational transitions to study molecular reorientation, in particular internal rotations. The basic idea is to excite a bond vibration with a polarized IR pulse and probe it with another polarized IR pulse at a later time. The ensemble average of the projection of the initial dipole onto itself at later time is the orientational correlation function of the bond. To further utilize these techniques, we are combining the 2D-IR technology with laser-based T-jump methods. We are exploring methods of generating the pulses that can be directly absorbed into the overtone bands of the solvent water or D2O. The temperature jump method will be applied to induce conformational changes associated with unfolding/folding in proteins and peptides, probed with our 2D IR technique. A study of the T-jump induced folding dynamics of the villian head piece using this combined 2D IR/T-jump approach is under way in our laboratories.
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