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

Abstract

Developments in atomic force microscopy have opened up a new path toward single-molecular phenomena; in particular, during the process of pulling a membrane protein out of a lipid bilayer. However, the characteristic features of the force-distance (F-D) curve of a bacteriorhodopsin in purple membrane, for instance, have not yet been fully elucidated in terms of physicochemical principles. To address the issue, we performed a computer simulation of bacteriorhodopsin with, to our knowledge, a novel coarse-grained (C-G) model. Peptide planes are represented as rigid spheres, while the surrounding environment consisting of water solvents and lipid bilayers is represented as an implicit continuum. Force-field parameters were determined on the basis of auxiliary simulations and experimental values of transfer free energy of each amino acid from water to membrane. According to Popot's two-stage model, we separated molecular interactions involving membrane proteins into two parts: I) affinity of each amino acid to the membrane and intrahelical hydrogen bonding between main chain peptide bonds; and II) interhelix interactions. Then, only part I was incorporated into the C-G model because we assumed that the part plays a dominant role in the forced unfolding process. As a result, the C-G simulation has successfully reproduced the key features, including peak positions, of the experimental F-D curves in the literature, indicating that the peak positions are essentially determined by the residue-lipid and intrahelix interactions. Furthermore, we investigated the relationships between the energy barrier formation on the forced unfolding pathways and the force peaks of the F-D curves.

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

“…One all-atom MD study (17) identified a number of key residues that resist mechanical unfolding in the intermediate states probed by the experiment (11), although its pulling speed (1-50 m/s) was too fast to observe WLC behavior for the unfolded segments. A 2016 CG study used the same pulling rate as ours ($10 6 nm/s) and found WLC behavior (20). Although many features are similar between this and our studies, we observed more intermediates (Table S2).…”

“…These methods have been proven beneficial in detecting sparsely populated intermediates and elucidating unfolding pathways of soluble (2)(3)(4)(5) and membrane proteins (6)(7)(8)(9)(10)(11)(12)(13)(14). Simulations and theory have aided the experimental SMFS studies by revealing the complexity of the folding process (15)(16)(17)(18)(19)(20)(21). These measurements are challenging from the computational standpoint, in part because of the demanding computation resources required to simulate the experimental timescales (17).…”

“…These measurements are challenging from the computational standpoint, in part because of the demanding computation resources required to simulate the experimental timescales (17). Coarse-grained models enable more extensive sampling and allow for slower, more realistic pulling velocities and lower forces (20) that better match the experimental studies in providing increased likelihood of observing transient intermediates.…”

Improved free-energy landscape reconstruction of bacteriorhodopsin highlights local variations in unfolding energy

“…S1 of the supplementary material). 28 Our work also documented that ∆G 0 per nm varied more than 15-fold, from 11 kcal/mol at the top of the ED helix pair to 0.6 kcal/mol at the bottom of the same helix pair. While it has long been known that hydrophobicity of the amino-acid sequence within the lipid bilayer and inter-and intra-helix interactions contribute to this variation, 29 recent work also shows variations in the strength of hydrogen bonds within TM helices.…”

Normal mode analysis and beyond

“…However, the origin of such patterns has not been fully understood yet. Recently, we have developed our original program, ppfc (PolyPeptide chain Force-distance Curve simulator), to study the forced unfolding mechanism of membrane proteins [33,34], and successfully reproduced experimentally observed FD curves. The program and user's manual are available on the web site [35].…”