Journal of Molecular Biology
CommunicationObserving Membrane Protein Diffusion at Subnanometer Resolution
Section snippets
Imaging loosely packed membrane proteins at subnanometer resolution
We have reconstituted ion driven rotors of llyobacter tartaricus ATP synthase into a lipid (palmitoyl-oleoyl phosphatidylcholine; POPC) bilayer16 and adsorbed the membranes onto freshly cleaved mica.17 After this, the sample was imaged at room temperature in buffer solution by contact mode AFM. To minimize the interaction between the AFM stylus and the sample, the force applied to the stylus was counterbalanced electrostatically to ≈25 pN.18 The AFM topograph revealed single rotors protruding
Observing the motion of single membrane proteins
Imaging the same membrane area after 90 seconds showed most rotors remained at their location (Figure 2(B)). A few rotors, however, changed their position. At the same time as some rotors disassembled, others assembled. Repeated imaging of the same area (Figure 2(C)) showed the continuous movement of individual proteins. Tracking individual rotors indicated that the molecules started and stopped their movement at any given time, independent of whether they had moved previously. The membrane
Analyzing the modes of motion
Individual rotors can undergo lateral and rotational movements, as apparent from shifts of their relative position (Figure 2). The uncertainty when determining the position of a single rotor was ≈1 nm, on average, which is about 30 times smaller than the accuracy of optical single molecule methods to detect molecular diffusion3., 4. and about 200–300 times smaller than the diffraction-limited width of an excellent optic. This positional accuracy combined with the high spatial resolution of the
Direct observation of membrane protein motion and membrane structure
Most importantly for molecular cell biologists, it has been demonstrated that individual biological molecules can be observed moving through a heterogeneous assembly of membrane proteins. As revealed from AFM topographs, the movement of single rotors was restricted by surrounding single motors and by rotors forming assemblies. Occasionally, the moving rotors reversibly joined either one of the obstacles. On the other hand, individual rotors moved freely through the lipid membrane without
Acknowledgements
We thank Kurt Anderson, Joe Howard, Kate Poole, Kai Simons, and Winchil Vaz for critical reading of the manuscript. This work was supported by the Maurice E. Müller and Swiss National Foundation (SNF), by the European Community (EFRE) and the Sächsische Ministerium für Wissenschaft und Kunst (SMWK), Germany.
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