PROTEIN GLOBULARIZATION DURING FOLDING - A STUDY BY SYNCHROTRON SMALL-ANGLE X-RAY-SCATTERING

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of p roteins and model polypeptides in globular states (native and ''molten globule'') and in non-globular states (unfolded protein as well as ra ndomly coiled, partially alpha-helical and partially beta-structural s ynthetic polypeptides) were analyzed in terms of Guinier and Kratky pl ots. Large differences in the SAXS pattern have been found between glo bular and non-globular conformations of the polypeptide chains, and th ey have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular m ass. The equilibrium and time-resolved unfolding curves of bovine carb onic anhydrase and yeast phosphoglycerate kinase were monitored by int egrated SAXS intensity, and were found to be coincident with the curve s measured by other physicochemical techniques, such as tryptophan flu orescence and peptide circular dichroism spectra. The intermolecular a ssociation of the protein ''molten globule''-like intermediates accumu lated during the guanidine hydrochloride-induced unfolding of bovine c arbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensit ive to the protein intermolecular association than the zero angle inte nsity and the radius of gyration. We propose the integrated SAXS inten sity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrate d SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 se conds) kinetic processes for yeast phosphoglycerate kinase have been r ecorded. The kinetic processes reflect both protein intramolecular glo bularization and its intermolecular association. (C) 1996 Academic Pre ss Limited