Absence of a stable intermediate on the folding pathway of protein A

Abstract

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 elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the I16W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable 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 intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of ikT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.

Folding a protein in a computer: An atomic description of the folding/unfolding of protein A

“…These evolving ensembles exhibit a folding mechanism where secondary structure formation is coupled with solvent exclusion and core formation. We find that helix III forms early, consistent with experiments (19) and most calculations (12,15,17). The results of Bockzo and Brooks (7) differ from ours on this specific detail.…”

“…For our studies, a natural choice is the 10-55 helical fragment B of protein A from Staphylococcus aureus, using the replica exchange molecular dynamics (REMD) algorithm described by Sugita and Okamoto (5). Protein A folds into a simple three-helix bundle (6) whose folding has been widely studied by using minimalist and all-atom simulations (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) as well as experiments (18,19). All of these different studies provided some information about the folding mechanism and the nature of the transition-state ensemble (TSE).…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…Strong protection, > 40%, was observed in experiments and predicted by the simulation for peptides spanning the region from residues 48-159, (βα) 2-5 β 6 . The simulation overestimated the protection at the Nterminus for residues 9-47, α 0 (βα) 1 and near the C-terminus for residues 146-179 (βα) 6 . The most apparent discrepancy exists at the N-terminus where the simulation predicts very strong protection in peptide 1-8 and the HX-MS results show just the absence of protection.…”

“…An extensive mutational analysis of the equilibrium intermediate for αTS 54,55 demonstrated that a tightly-packed Nterminal region, (βα) [1][2][3][4] , is not tightly coupled to a molten globule-like C-terminal region containing (βα) [5][6][7][8] . This differential behavior was attributed to misfolding at the boundaries of the (βα) [1][2][3][4] region that precluded propagation to the C-terminal region in the I1 intermediate. The possibility of misfolding at the periphery of an N-terminal core is supported by the results of another mutational analysis of a pair of long-range H-bonds in the N-terminal region between aspartic acid side chains at the C-termini of two helices and main chain amide hydrogens near the N-termini of their preceding β-strands.…”

“…1,2 By contrast, larger proteins often acquire substantial far-UV ellipticity in the sub-millisecond time range, far faster than the appearance of the native state. [3][4][5] The significance of this secondary structure to the folding mechanism has been a subject of controversy for several years.…”