Secondary structure of globular proteins at the early and the final stages in protein folding

The ellipticities for an early transient intermediate in refolding observed by kinetic circular dichroism measurements at 220–225 nm for 14 different proteins are summarized, and the ellipticity values are compared with those for the final native proteins and also with the ellipticities expected from a physical theory of protein and polypeptide secondary structure. The results show that a substantial part of the protein secondary structure is in general formed in the earliest detectable intermediate in refolding and that the ellipticities in both the native and the intermediate states are consistent with the physical theory of protein secondary structure

An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

The refolding kinetics of the tryptophan synthase β2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’ decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’ state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains

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 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

Kinetic refolding of β-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy

β-Lactoglobulin (βLG) is a predominantly β-sheet protein with a markedly high helical propensity and forms non-native α-helical intermediate in the refolding process. We measured the refolding reaction of βLG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that βLG forms a compact, globular structure 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, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native α-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in βLG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial β-sheets and remarkable non-native α-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding