STUDY OF PROTEIN DYNAMICS UNDER HIGH HYDROSTATIC PRESSURE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ACERT has a long-standing collaboration with Wayne Hubbell, fueled by common interests in the study of molecular dynamics including protein dynamics. It has been shown recently by Wayne Hubbell that application of high hydrostatic pressure of up to 2 kbar populates higher energy conformers, and thereby enables one to detect dynamic exchange between the ground state and excited state, which is strongly believed to be functional. Hubbell has shown that these states can be probed by CW ESR and pulse saturation ESR. Since these states can undergo exchange on a microsecond time-scale, it would be logical to apply 2D ELDOR to study protein dynamics and conformational exchange in proteins under high pressure. This presents a great opportunity for 2D ELDOR to realize its potential in studying molecular dynamics. We will conduct the study initially on two spin-labeled proteins: Myoglobin and IFABP, which Hubbell's laboratory has already produced. It should be noted that the development of a 2D ELDOR high pressure method has broader implications, for example, by providing more tools to study by 2D-ELDOR protein folding, both in the steady state with or without denaturant and kinetically after a pressure jump. Furthermore, 2D-ELDOR at high pressure can be conducted at ACERT in a multi-frequency context, which would help to determine dynamic variables with greater confidence