STRUCTURAL CHARACTERIZATION OF THE PRESSURE-DENATURED STATE AND UNFOLDING REFOLDING KINETICS OF STAPHYLOCOCCAL NUCLEASE BY SYNCHROTRON SMALL-ANGLE X-RAY-SCATTERING AND FOURIER-TRANSFORM INFRARED-SPECTROSCOPY/

The pressure-induced unfolding of wild-type staphylococcal nuclease (S nase WT) was studied using synchrotron X-ray small-angle scattering (S AXS) and Fourier-transform infrared (FT-LR) spectroscopy, which monito r changes in the tertiary and secondary structural properties of the p rotein upon pressurization. The experimental results reveal that appli cation of high-pressure up to 3 kbar leads to an approximate twofold i ncrease of the radius of gyration R-g of the native protein (R-g appro ximate to 17 Angstrom) and a large broadening of the pair-distance-dis tribution function, indicating a transition from a globular to an elli psoidal or extended chain structure. Analysis of the FT-IR amide I' sp ectral components reveals that the pressure-induced denaturation proce ss sets in at 1.5 kbar at 25 degrees C and is accompanied by an increa se in disordered and turn structures while the content of beta-sheets and alpha-helices drastically decreases. The pressure-induced denature d state above 3 kbar retains nonetheless some degree of beta-like seco ndary structure and the molecule cannot be described as a fully extend ed random coil. Temperature-induced denaturation involves a further un folding of the protein molecule which is indicated by a larger R-g val ue and significantly lower fractional intensities of IR-bands associat ed with secondary-structure elements. In addition, we have carried out pressure-jump kinetics studies of the secondary-structural evolution and the degree of compactness in the folding/unfolding reactions of Sn ase. The effect of pressure on the kinetics arises from a larger posit ive activation volume for folding than for unfolding, and leads to a s ignificant slowing down of the folding rate with increasing pressure. Moreover, the system becomes two-state under pressure. These propertie s make it ideal for probing multiple order parameters in order to comp are the kinetics of changes in secondary structure by pressure-jump FT -IR and chain collapse by pressure-jump SAXS. After a pressure jump fr om 1 bar to 2.4 kbar at 20 degrees C, the radius of gyration Increases in a first-order manner from 17 Angstrom to 22.4 Angstrom over a time scale of approximately 30 minutes. The increase in R-g value is caused by the formation of an extended (ellipsoidal) structure as indicated by the corresponding pair-distance-distribution function. Pressure-jum p FT-IR studies reveal that the reversible first order changes in beta -sheet, alpha-helical and random structure occur on the same slow time scale as that observed for the scattering curves and for fluorescence. These studies indicate that the changes in secondary structure and ch ain compactness in the folding/unfolding reactions of Snase are probab ly dependent upon the same rate-limiting step as changes in tertiary s tructure. (C) 1998 Academic Press Limited.