GB1 Is Not a Two-State Folder: Identification and Characterization of an On-Pathway Intermediate

Abstract

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.

Characterization of the free-energy landscapes of proteins by NMR-guided metadynamics

“…A control unbiased molecular dynamics simulation of 200 ns starting from a structure corresponding to the intermediate free-energy minimum remained extremely stable, with an average rmsd of 2.4 Å from the equilibrated initial structure. These results are consistent with the observation of the presence of an intermediate state of GB1 (44,45), which shares 88% of the sequence identity of GB3. In particular, that work, which was based on the measurement of the kinetic folding constant as a function of the pH and de- (44,45).…”

“…These results are consistent with the observation of the presence of an intermediate state of GB1 (44,45), which shares 88% of the sequence identity of GB3. In particular, that work, which was based on the measurement of the kinetic folding constant as a function of the pH and de- (44,45). However, the structure of the intermediate of GB1 is likely to be more native-like than the one that we find here.…”

“…To better characterize the folding mechanism of GB3, we simulated by a kinetic Monte Carlo approach (46) the dynamics on the multidimensional freeenergy landscape reconstructed by our procedure (Methods). All the trajectories connecting the folded and unfolded states go through the intermediate state, confirming that it is an on-pathway intermediate, like the one observed for GB1 (45). The black dashed line in Fig.…”

Residue level quantification of protein stability in living cells

“…With this caveat in mind, we suggest two additional sources of deviation. First, although most evidence points to equilibrium two-state folding of GB1 (36)(37)(38), there is evidence of complex kinetic pathways, so we cannot rule out the possibility that intermediates may be populated at equilibrium in cells or in buffer. Second, despite our knowledge that k int values do not change under crowded conditions (61), deviations could arise because intrinsic rates are derived from model peptides, not the specific primary structure of GB1.…”

“…We chose the B1 domain of streptococcal protein G (GB1) (27) as our test protein because its structure, stability and folding kinetics have been extensively studied in dilute solution (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”