Heinrich Michael C, Capraro Benjamin R, Tian Aiwei, Isas Jose M, Langen Ralf, Baumgart Tobias
University of Pennsylvania, Department of Chemistry.
J Phys Chem Lett. 2010 Nov 16;1(23):3401-3406. doi: 10.1021/jz101403q.
Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.
生物膜功能与膜曲率相关联，其调节通常涉及膜相关蛋白。发动蛋白的膜结合含N端两亲性螺旋的BIN/发动蛋白/Rvs（N-BAR）结构域与曲率的产生和维持有关。要深入了解N-BAR结构域对膜曲率的调节机制，需要进行定量实验表征。我们测量了与从微量移液器吸出的巨型囊泡中拉出的管状膜结合的果蝇发动蛋白（DA-N-BAR）的N-BAR结构域对管拉力的调节作用。我们观察到，与相连的低曲率囊泡膜相比，荧光标记的DA-N-BAR在小管上显示出显著更高的蛋白质密度。此外，我们发现平衡管拉力系统地依赖于DA-N-BAR的水溶液浓度，从而首次对自发曲率的产生进行了定量评估。在足够高的蛋白质浓度下，拉出的管不需要外力来维持机械平衡，这与先前报道的发动蛋白的定性自发成管现象一致。
