KINETIC REFOLDING OF BETA-LACTOGLOBULIN - STUDIES BY SYNCHROTRON X-RAY-SCATTERING, AND CIRCULAR-DICHROISM, ABSORPTION AND FLUORESCENCE SPECTROSCOPY

beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein wit h a markedly high helical propensity and forms non-native alpha-helica l intermediate in the refolding process. We measured the refolding rea ction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burs t phase of measurements, i.e. the burst-phase intermediate. Time-resol ved stopped-flow X-ray scattering measurements using the integral inte nsity of scattering show that beta LG forms a compact, globular struct ure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensurin g that the burst-phase intermediate is compact. The presence of a maxi mum peak in a Kratky plot shows a globular shape attained within 100 m s of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structur e regains rapidly in the burst phase with concurrent non-native alpha- helix formation, and that the subsequent compaction process is accompa nied by annealing of non-native secondary structure and slow acquisiti on of tertiary structure. These findings strongly suggest that both co mpaction and secondary structure formation in protein folding are quit e rapid processes, taking place within a millisecond time-scale. The s tructure of the burst-phase intermediate in beta LG refolding was char acterized as having a compact size, a globular shape, a hydrophobic co re, substantial beta-sheets and remarkable non-native alpha-helical st ructure, but little tertiary structure. These results suggest that bot h local interactions and non-local hydrophobic interactions are domina nt forces early in protein folding. The interplay of local and non-loc al interactions throughout folding processes is important in understan ding the mechanisms of protein folding. (C) 1998 Academic Press Limite d.