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. Pulsed dipolar ESR spectroscopy has high capacity for the determination of the structure of biomacromolecules/complexes by performing site-directed distance measurements between spin-labels that are covalently attached to the object of interest. To gain sufficient information for the structure that is studied, several constrains of both short and long distances are needed. However, it is a challenge to measure long inter-spin distances due to the necessity of considerably longer evolution times to be used. This leads to a poor signal-to-noise ratio and not reliable interpretation of the results. The sensitivity of pulsed dipolar ESR experiments can be improved by the increase of the phase memory time Tm. At sufficient low temperatures and concentrations the main relaxation mechanism for the electron spins is related to proton spin diffusion. Therefore, Tm can be extended by appropriate choice of solvent or further by using deuterated solvents as a matrix. The later approach was successfully applied to measure distances up to 7 nm in biological objects in solution. Further complications appear in studies of proteins in membrane-bound state due to the high proton content in the lipids used to prepare the mimetic membranes. Thus to date the longest distances, measured in membrane context, do not exceed 4.5 nm. Herein, for the first time we conducted distances measurements in systems with 70 or high percentage substitution of deuterium for hydrogen in context of matrix, protein molecule and phospholipids.
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