Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

Abstract

Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others.

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

“…6), which is in accordance with the experimental observations in the literature (17)(18)(19)(20)23,24), whereas 121 out of 128 simulations showed four-phase unfolding, i.e., 1(AB) N , 2(CD) N 3(EF) N , and 4(G) N phases, in simulation N (Fig. S4).…”

“…According to the experimental studies (13,16,23,24), the magnitude and the appearance frequency of the force peaks of the F-D curves were affected by the interhelix interactions, whereas the peak positions were not. In what follows, we discuss possible roles of the class-II interaction.…”

“…Sapra et al (23) performed forced unfolding experiments on P50A, M56A, Y57A, P91A, and P186A mutants of bR. As a result, they observed no change in the peak positions in the F-D curves, although changes in interhelical packing were expected in these mutants.…”

“…These studies indicate that the force peak originates from intrinsic properties of each TM helix rather than the interhelix interactions. Furthermore, experimental studies (13,16,23,24) suggested that the magnitude and the appearance frequency of the force peaks of the F-D curves were affected by the interhelix interactions, whereas the peak positions were not. Kessler et al (31) performed unfolding and refolding experiments of bR by lifting the AFM tip up and down repeatedly, and observed that the peak positions of the F-D curves for the helix pairs ED and CB were always reproduced whereas the magnitudes of these peaks were sometimes reproduced and sometimes not.…”

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…As theoretically predicted (46,47) and experimentally verified using membrane proteins (35,36,(48)(49)(50)(51), increasing the loading rate increases the unfolding forces. We observed a linear relationship between the most probable unfolding force and the logarithm of the loading rate for every interaction.…”