Huang Z, Pearce K H, Thompson N L
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290.
Biophys J. 1994 Oct;67(4):1754-66. doi: 10.1016/S0006-3495(94)80650-2.
Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.
先前的研究表明，牛凝血酶原片段1以Ca(2+)特异性方式与由磷脂酰胆碱（PC）和磷脂酰丝氨酸（PS）组成的底物支持平面膜结合。表观平衡解离常数为1 - 15 microM，平均膜驻留时间约为0.25 s-1。在本研究中，采用瞬逝干涉图案荧光图案光漂白恢复技术（TIR-FPPR）来测量弱结合片段1的平移扩散系数。结果表明，在类流体PS/PC平面膜上的平移扩散系数约为10(-9) cm2/s，并且当片段1表面密度增加时会降低。对片段1在类固体PS/PC平面膜上进行了对照测量。解离动力学与在类流体膜上的相似，但未检测到蛋白质的平移流动性。TIR-FPPR还用于测量荧光脂质NBD-PC在类流体PS/PC平面膜中的扩散系数。在这些测量中，扩散系数约为10(-8) cm2/s，这与通过传统荧光图案光漂白恢复技术测量的结果一致。这项工作首次测量了弱结合于膜表面的蛋白质的平移扩散系数。
