Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.
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.
Huang
Z
Z
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290.
Pearce
K H
KH
Thompson
N L
NL
D015060:1,2-Dipalmitoylphosphatidylcholine; D000818:Animals; D002417:Cattle; D004058:Diffusion; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D010446:Peptide Fragments / Q000737:chemistry*; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010777:Photochemistry; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry*; D011516:Prothrombin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013329:Structure-Activity Relationship
D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011498:Protein Precursors; D015060:1,2-Dipalmitoylphosphatidylcholine; C038694:dipalmitoylphosphatidylserine; C029733:prothrombin fragment 1; D011516:Prothrombin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine