Kessler Max, Gottschalk Kay E, Janovjak Harald, Muller Daniel J, Gaub Hermann E
Chair of Applied Physics and Center for NanoScience, Ludwig-Maximilians Universität, Amalienstrasse 54, 80799 München, Germany.
J Mol Biol. 2006 Mar 24;357(2):644-54. doi: 10.1016/j.jmb.2005.12.065. Epub 2006 Jan 6.
Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of individual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First individual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.
尽管膜蛋白对细胞功能至关重要，但其折叠机制却鲜为人知。最近，基于原子力显微镜（AFM）的单分子力谱技术阐明了折叠能量景观的细节。在对单个膜蛋白进行展开和提取时，环和螺旋等结构元件中的能量屏障得以绘制并量化，精度可达几个氨基酸。在此，我们报告下一步合理的步骤：将单个蛋白质可控地重新折叠到膜中。首先，通过用AFM探针拉动细菌视紫红质单体的C末端，使其从紫膜中部分展开并提取出来。然后，通过逐渐降低探针，让蛋白质在记录折叠力的同时重新折叠到膜中。我们发现，在重新折叠过程中，某些螺旋会克服几十皮牛顿的相当大的外力被拉进膜中。根据螺旋在AFM悬臂上所做的机械功，我们得出了吉布斯自由折叠能的上限。随后的展开使我们能够分析展开屏障的模式，并证实蛋白质已重新折叠成天然状态。
