Folding Research Of Bacteriorhodopsin

Membrane proteins, integrating into lipid bilayer, play essential roles in almost allintracellular activities. This kind of protein is very important to related disease treatment andrepresents the majority of cellular drug targets. Due to its excellent stability and reversibleunfolding, bactriorhodopsin (bR), which is the only protein in purple membrane ofHalobacterium, has served as a popular model for the folding of α-helical membrane proteins.In this study, several chemical and physical methods have been applied into bR foldingresearch and detailed folding thermodynamic and kinetic information in liposome has beenrevealed, which has laid important foundations for following single molecule fluorescencestudy and other membrane protein research.Applying single molecule fluorescent technologies into membrane protein foldingresearch is a great innovation of this paper.9site-directed mutants have been desighed andprepared for this purpose. Native bR has been obtained by construction of the pBA2/MPK409expression system. Obvious fluorescence resonance energy transfer (FRET) has beenoberseved in the labelled mutant0C-249C between the fluorphore pair of AF488and AF647.DDM, as a mild surfactant, has proved to have no negative influence on separation of labeledbR and free dyes, which is very useful for purification after labeling.Several kinds of liposome, such as POPC, have been prepared to simulate nativeenvironment of membrame proteins. bR has been efficiently transferred into lipid vesicles.Thermodynamic study of bR in lipid bilayer has been conducted, which has provided directexperimental evidence for relationship between liposome curvature stress and stability ofmembrane protein. Increase of curvature stress enhanced the stability of bR. What is more,detailed folding kinetic study of bR in liposome has also contributed a lot for building of bRfolding model. Interestingly, we found that bR stability increased with decrease of vesicle size.Associted with drop of liposome size, interactions and connections between lipid and proteinhave been strengthened. Unfolding rates and regeneration yield also decreased due to theenhanced interactions. This is the first report of relationship between bR stability and foldingkinetics and liposome size. In addition, Unfolding kinetics of bR under different temperaturesindicated there was no other intermediates observed in the given temperature range. At lest4reactions and3intermediates have been detected in bR folding.In this paper, detailed thermodynamic and kinetc information laid essential foundationsfor following single molecule fluorescence research and provided useful references to othermembrane protein folding study.