ABSENCE OF A STABLE INTERMEDIATE ON THE FOLDING PATHWAY OF PROTEIN-A

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elem entary steps in the folding of larger proteins. Earlier studies sugges ted that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-termin al helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichr oism and NMR hydrogen-deuterium exchange pulse labeling. Folding of th e entire molecule is essentially complete within the 6 ms dead time of the quench-now apparatus, indicating that the intermediate, if formed , progresses rapidly to the final folded state. Site-directed mutagene sis of the isoleucine residue at position 16 was used to generate a va riant protein containing tryptophan (the I16W mutant). The formation o f the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unf avorable steric interactions of the bulky tryptophan side chain in the folded state. The I16W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermedi ate could be detected by either kinetic or equilibrium measurements. S tudies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, i ts stability appears to be marginal (of the order of 1/2kT) and it cou ld not be an obligatory intermediate on a defined folding pathway. The se results explicitly demonstrate that the protein A B domain folds ex tremely rapidly by an apparent two-state mechanism without formation o f stable partly folded intermediates. Similar mechanisms may also be i nvolved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.