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College Park, Maryland      June 6 - 10 , 2004

MP1: SANS Study of High Pressure Unfolding of Staphylococcal Nuclease

A. Paliwal (Johns Hopkins University), D.P. Bossev (NIST Center for Neutron Research), ME Paulaitis (Johns Hopkins University)

Staphylococcal Nuclease (SN) is a 149 residues long, single domain globular protein whose folding characteristics conform to the two state model. Reversible folding of SN with pH, chemical denaturants and temperature as perturbing agents has been very well examined and characterized.1-2 A series of equilibrium and kinetics studies of pressure induced unfolding of SN by Royer et al.3-4 revealed that the folding reaction involves significant dehydration and collapse of the chain. The picture that emerges from these studies is that of a combination of increased solvation and decreased excluded volume in the unfolded state relative to the folded state. However, no direct efforts have been made towards quantifying this difference in the extent of hydration of the folded and the unfolded states from a large body of available experimental results. Recently, Hummer et al.5 proposed that proteins stabilized by hydrophobic driving forces at ambient pressure are destabilized at elevated pressures due to water penetration into the hydrophobic core (“protein unfolding”). They argue that pressure-induced protein unfolding corresponds to the transfer of water into the protein interior, rather than to the transfer of nonpolar residues into water. This hypothesis, if correct, would resolve Kauzmann’s paradox6 for the pressure-induced denaturation of proteins, which results from the fact that the volume change of transfer of hydrocarbons into water is positive at high pressures.

In this study, we present a novel method recently developed7 to extract the extent of hydration of SN as a function of pressure from our Small Angle Neutron Scattering (SANS) results at 25° C. The method, primarily makes use of the radius of gyration, Rg and the forward scattering intensity, I(0) values obtained directly from the scattering spectra. These hydration results are then discussed in context of the proposition put forward by Hummer et al. and the ‘apparent’ paradox first pointed out by Kauzmann.

Reference:

1) Shortle D. and Meeker A. K. (1986) Proteins: struct., funct. and genet. 1, 81.
2) Griko Y.; Privalov, P. L.; Sturtevant J.M and Venyaminov, S. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 3343.
3) Vidugiris, G.J.A; Markley, J.L. and Royer, C. A (1995) Biochemistry U.S 34(15), 4909.
4) Panick, G.; Vidugiris, G. J. A; Malessa, R; Rapp, G, Winter, R. and Royer, C. A (1999) Biochemistry U.S 38, 4157.
5) Hummer, G.; Garde, S; Paulaitis, M. E. and Pratt, L. R. Proc. Natl. Acad. Sci. U.S.A (1998) 95, 1552.
6) Kauzmann, W. Nature (1987) 325, 763.
7) Lesemann, M.; Nathan, H.; DiNoia, T.; Kirby, K.; McHugh, M. A.; van Zantan J.H. and Paulaitis, M.E. Ind. Eng. Chem. Res. (2003) 42(25), 6425.

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