Protein Globularization During Folding. A Study by Synchrotron Small-angle X-ray Scattering

• Published in: Journal of molecular biology 1996-10, Vol.262 (4), p.559-574
• Main Authors: Semisotnov, Gennady V., Kihara, Hiroshi, Kotova, Nina V., Kimura, Kazumoto, Amemiya, Yoshiyuki, Wakabayashi, Katsuzo, Serdyuk, Igor N., Timchenko, Alexander A., Chiba, Kaori, Nikaido, Kiyokazu, Ikura, Teikichi, Kuwajima, Kunihiro
• Format: Article
• Language: English
• Subjects: Animals; Cattle; folding intermediates; globularization; Molecular Weight; nonspecific associates; Polyglutamic Acid - chemistry; Polylysine - chemistry; Protein Conformation; protein folding; Scattering, Radiation; synchrotron small-angle X-ray scattering; Synchrotrons
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0535

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and “molten globule”) and in non-globular states (unfolded protein as well as randomly coiled, partially α-helical and partially β-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein “molten globule”-like intermediates accumu lated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity 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 integrated 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 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.