Andrzej Sienkiewicz (1), Slaven Garaj (3), Charles P. Scholes (2) , and Laszlo Forro (3)
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(1) Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland;
(2) Chemistry Department, SUNY at Albany, 1400 Washington Ave., Albany NY 12222, USA;
(3) Institut de Genie Atomique, Departement de Physique, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland.
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Electron Spin Resonance (ESR) spectroscopy, a technique for studying paramagnetic, ferromagnetic and antiferromagnetic spin systems, is a widely used tool in many fields of contemporary science, ranging from physics and chemistry to biology and medicine. A rationale for combining high-hydrostatic pressures with the ESR technique is also widely recognized, since the principal quantities associated with the spin dynamics, such as the ESR line shape and its intensity, spectroscopic g- and A- factors, line width DHpp, spin relaxation times T1 and T2 are sensitive to variable pressure. There is growing evidence that similarly to high-hydrostatic pressure Nuclear Magnetic Resonance (NMR), biological applications of high-hydrostatic pressure EPR can be crucial for studying the structure, dynamics and conformation changes of protein molecules. Over the last decade, it has been shown that cw-EPR spectroscopy in combination with Site-Directed Spin-Labeling (SDSL) is a powerful tool to probe the local molecular dynamics of large organic molecules as well as to follow such challenging biophysical processes as protein folding and unfolding. The possibility of monitoring spin probe dynamics at a number of different locations provides a unique view of protein folding/unfolding in regions often not amenable to other methods (like UV-visible absorption spectroscopy, tryptophan fluorescence quenching, far UV Circular Dichroism (CD) or NMR spectroscopy). Principles of attaching nitroxide spin-labels to strategic sites of the polypeptide backbone as well as technical details of newly developed sensitive EPR probe heads for performing cw and Stopped-Flow EPR experiments will be presented. Perspectives of following protein unfolding under high-hydrostatic pressure will be discussed. Preliminary results of monitoring molecular dynamics of nitroxide spin probes in aqueous solutions under hydrostatic pressures up to 0.9 Gpa will be shown. We will also present technical details of the miniature Dielectric Resonator (DR)-based probe h AC), thus enabling one to perform sensitive high-pressure EPR measurements of crystalline and polycrystalline materials as well as of biological samples (aqueous solutions).

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