Advanced high-resolution NMR spectroscopy, including two-dimensional NMR techniques, combined with high pressure capability, represents a powerful new tool in the study of proteins. This contribution is organized in the following way. First, the specialized instrumentation needed for high-pressure NMR experiments is discussed, with specific emphasis on the design features and performance characteristics of a high-sensitivity, high-resolution, variable-temperature NMR probe operating at 500 MHz and at pressures of up to 500 MPa. An overview of several recent studies using 1D and 2D high-resolution, high-pressure NMR spectroscopy to investigate the pressure-induced reversible unfolding and pressure-assisted cold denaturation of lysozyme, ribonuclease A, and ubiquitin is presented. Specifically, the relationship between the residual secondary structure of pressure-assisted, cold-denatured states and the structure of early folding intermediates is discussed.

At high (> 3.5 kbar) pressures and low (< -10 degrees C) temperatures, hen egg-white lysozyme denatures readily and reversibly. Amide hydrogen exchange methods were used to investigate the structure of the pressure-assisted cold-denatured state of lysozyme. Protection factors were obtained for 52 backbone amide protons. The extent of protection of many of these protons is markedly different from that in lysozyme denatured by high temperature, high urea concentration, or chemical modification; specifically, the protection factors are higher and are strongly correlated with elements of secondary structure present in the native state. Furthermore, the pattern of protection factors is similar to that observed in lysozyme during refolding from highly denatured states, particularly during the early stages (< 3.5 ms) of refolding [Gladwin, S. T., & Evans, P. A. (1996) Folding Des. 1, 407]. Previous data on cold-denatured ribonuclease A were reevaluated and compared to known folding intermediates [Houry, W. A. & Scheraga, H. A. (1996) Biochemistry 35, 11734; Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197] to further test the supposition that the pressure-assisted cold-denatured states of proteins resemble the early folding stages.

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