Sequential Unfolding of Individual Helices of Bacterioopsin Observed in Molecular Dynamics Simulations of Extraction from the Purple Membrane
Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its ?-helices unfold individually. In the first metastable state observed in the simulations, helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt-bridge network consisting of Asp-212 and Lys-216 on helix G and Arg-82 and Asp-85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. Inversely, helix F has to flip by 180° to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro-186. Mutation of Pro-186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way to helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un)folding of bacterioopsin proceeds through metastable bundles of fewer than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.
INTRODUCTION
Integral membrane proteins are involved in a wide variety of functions like photosynthesis, transport of ions and small molecules, and signal transduction. They either consist of a varying number of ?-helices (e.g., G-protein coupled receptors (1), aquaporin (2), and the ammonia channel (3)) or they adopt a ?-barrel fold containing between 8 and 22 ?-strands (4). The former are much more common than the latter, which are exclusively found in the outer membrane of Gram-negative bacteria. However, despite the relative abundance of membrane proteins among all proteins and despite the fact that they represent the majority of the targets for existing drugs (5,6), only a few structures have been solved so far. Moreover, the mechanism of folding and assembly within the membrane is not clear (7).
Bacteriorhodopsin (BR) is one of the most extensively studied integral membrane proteins (8-10). BR is a light-driven proton pump and its photoactive retinal, which is bound covalently through the Schiff base to Lys-216, is embedded in seven closely packed transmembrane ?-helices (termed A-G) arranged in an up-and-down topology (Fig. 1, (top). In the purple membrane BR adopts a trimeric state stabilized by the presence of lipids in the central compartment, which has a nearly cylindrical shape (11). High-resolution atomic force microscopy (AFM) topography of the cytoplasmic surface of a wild-type purple membrane shows that trimeric BR molecules arrange in a hexagonal lattice (12).
The forced unfolding and extraction from the purple membrane of BR and of its retinal-free form, bacterioopsin (BO), have been investigated in depth by combining AFM imaging with single-molecule force spectroseopy (12-15). AFM is a powerful method to shed light on mechanical protein unfolding or unbinding of a protein-ligand complex at the single molecule level, removing the averaging over large ensembles of molecules implied in other biophysical/ biochemical approaches. Two different AFM techniques are available to probe the mechanical resistance of biomolecules. In the force-ramp method, a time-dependent force is applied (16), while in the so-called force-clamp method, the force is held constant (17). Based on the force-ramp method, dynamic force spectroseopy (18) has provided a deep insight into the unbinding mechanism of a variety of biological complexes, such as the (strept)avidin-biotin complex (19) and the complex between L-selectin and various binding partners (20).
However, it is desirable to relate the information on unfolding or unbinding provided by the AFM techniques to the changes in tertiary and secondary structure. For this purpose, AFM observations can be complemented with molecular dynamics (MD) simulations, which describe the behavior of individual molecules at an atomic level of detail. Constant-velocity MD (termed also steered-MD and abbreviated as SMD) and constant force MD (CFMD) simulations mimic the force-ramp and the force-clamp method of AFM, respectively, and have been widely used to study protein-ligand unbinding (21-25) and protein unfolding (26-29). Very different timescales are involved in AFM experiments and SMD/CFMD simulations because force spectroscopy experiments are typically carried out on the millisecond timescale or slower while simulations are currently limited to nanoseconds. Nevertheless, simulations have helped to interpret consistently experimental observations and have been even used to formulate predictions subsequently verified by in vitro experiments (18,27,30-36).