The photosynthetic light-harvesting-reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, PQ) of LH1-RC (from Rhodopseudomonas palustris) and enhances its activity in photocurrent generation. Steady-state fluorometric analysis demonstrated that PG enhances the formation of the PQ state at lower irradiances. The photocurrent generation activity was analyzed via Michaelis-Menten kinetics, revealing that 38% of LH1-RCs reconstituted into the PG membrane generated photocurrent at a turnover frequency of 46 s. PG molecules, which interact with LH1-RC in vivo, play the role of an active effector component for LH1-RC to enhance its function in the biohybrid system.
Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.
The KcsA potassium channel is a prototypical channel of bacterial origin, and the mechanism underlying the pH-dependent gating has been studied extensively. With the high-resolution atomic force microscopy (AFM), we have resolved functional open and closed gates of the KcsA channel under the membrane-embedded condition. Here we surprisingly found that the pH-dependent gating of the KcsA channels was associated with clustering-dispersion dynamics. At neutral pH, the resting, closed channels were coalesced, forming nanoclusters. At acidic pH, the open-gated channels were dispersed as singly isolated channels. Time-lapse AFM revealed reversible clustering-dispersion transitions upon pH changes. At acidic equilibrium, a small fraction of the channels was nanoclustered, in which the gate was apparently closed. Thus, it is suggested that opening of the gate and the dispersion are tightly linked. The interplay between the intramolecular conformational change and the supramolecular clustering-dispersion dynamics provides insights into understanding of unprecedented functional cooperativity of channels.
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