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On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations.

ABSTRACT: Single-molecule force spectroscopy has proven extremely beneficial in elucidating folding pathways for membrane proteins. Here, we simulate these measurements, conducting hundreds of unfolding trajectories using our fast Upside algorithm for slow enough speeds to reproduce key experimental features that may be missed using all-atom methods. The speed also enables us to determine the logarithmic dependence of pulling velocities on the rupture levels to better compare to experimental values. For simulations of atomic force microscope measurements in which force is applied vertically to the C-terminus of bacteriorhodopsin, we reproduce the major experimental features including even the back-and-forth unfolding of single helical turns. When pulling laterally on GlpG to mimic the experiment, we observe quite different behavior depending on the stiffness of the spring. With a soft spring, as used in the experimental studies with magnetic tweezers, the force remains nearly constant after the initial unfolding event, and a few pathways and a high degree of cooperativity are observed in both the experiment and simulation. With a stiff spring, however, the force drops to near zero after each major unfolding event, and numerous intermediates are observed along a wide variety of pathways. Hence, the mode of force application significantly alters the perception of the folding landscape, including the number of intermediates and the degree of folding cooperativity, important issues that should be considered when designing experiments and interpreting unfolding data.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6817726 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam-modified ultrashort cantilevers. However, unambiguously discriminating between such closely spaced states remains challenging, in part because any individual unfolding trajectory only occupies a subset of the total number of intermediates. Moreover, structural assignment of these intermediates via worm-like-chain analysis is hindered by brief dwell times compounded with thermal and instrumental noise. To overcome these issues, we moved the cantilever in a sawtooth pattern of 6-12 nm, offset by 0.25-1 nm per cycle, generating a "zigzag" force ramp of alternating positive and negative loading rates. We applied this protocol to the model membrane protein bacteriorhodopsin (bR). In contrast to conventional studies that extract bR's photoactive retinal along with the first transmembrane helix, we unfolded bR in the presence of its retinal. To do so, we introduced a previously developed enzymatic-cleavage site between helices E and F and pulled from the top of the E helix using a site-specific, covalent attachment. The resulting zigzag unfolding trajectories occupied 40% more states per trajectory and occupied those states for longer times than traditional constant-velocity records. In total, we identified 31 intermediates during the unfolding of five helices of EF-cleaved bR. These included a previously reported, mechanically robust intermediate located between helices C and B that, with our enhanced resolution, is now shown to be two distinct states separated by three amino acids. Interestingly, another intermediate directly interacted with the retinal, an interaction confirmed by removing the retinal.

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On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations.

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