Abstract
Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.
Highlights
Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied
The structural aspects of the bR reaction cycle have been studied by several techniques, some of them are time-resolved methods: X-ray crystallography[4,16,17,18], solid-state NMR19–21, cryo-EM crystallography[5,22,23,24,25], high speed atomic force microscopy (HS-AFM)[26,27,28,29,30,31,32] and, more recently, time-resolved serial femtosecond crystallography (TR-SFX) and X-ray free electron laser crystallography (XFELs)[3,7,33]
To measure single molecule kinetics of bR upon light activation, we integrated a green laser into our HS-AFM setup to provide 520-nm photons during defined duration and of defined intensity over ~15 μm diameter of the sample around the HS-AFM tip (Supplementary Fig. 1) while recording the protein dynamics (Fig. 1c)
Summary
Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. In these experiments a large ensemble of bR is exposed to stimulating light pulses and the spectroscopic properties of bR and/or pH-sensitive labels is measured According to these measurements, the K state and retinal isomerization occurs within picoseconds and the L state is reached after ~1.4 μs, a H+ is released on the extracellular side after ~630 μs, and the M state decays after ~4.7 ms. A closed state with 13-cis retinal accumulated in the 0–5 ms-structure, while an open state with large displacement of the E-F loop accumulated in the 10–15 ms-structure with a decay of 23.8 ms In these experiments the reverse conformational change to the O state could not be observed likely because the crystal contacts did not allow such rearrangements[7]. X-ray and cryo-EM crystallography and NMR based methods provide ensemble averaged data
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