Reconstructing The Free Energy Landscape Of Bacteriorhodopsin Folding By Single-molecule Force Spectroscopy

Membrane proteins are important functional structures in cells.Because native membrane protein resides in a free energy minimum dictated by their sequences and lipid environment,fundamental to the central goals of understanding membrane protein folding and conformational change is the reconstruction of its folding free energy landscape.The conventional Bulk methods measuring membrane protein folding free energy involves the manipulations of solution environment that drive membrane proteins unfold(refold)while monitoring the content of its structure,which uncover the knowledge of free energy landscape.However,these methods are confronted with difficulties in:(1)measuring the free energy landscape of single membrane protein in the native lipid environment,(2)finding a common reference unfolding state for free energy comparing and(3)suitable solution environment to achieve reversible equilibrium conditions.The membrane protein free energy landscape reconstruction methods,based on the single molecular force spectroscopy(AFM)technology,overcome the difficulties of bulk methods.However,the free energy landscape reconstruction practices lack of comparative analysis between different means thus may neglect the limitations of single method and bring controversy to the free energy landscape results.Meanwhile,the previous works are limited to the free energy measurement of the whole or main secondary structure unfolding which were performed far from equilibrium of membrane protein,making it difficult to recover equilibrium thermodynamic quantities.Finally,these earlier free energy results did not account for the work of stretching the unfolded polypeptides.In this thesis,we conducted free energy landscape reconstruction with methods including force-dependent transition kinetics(Bell model and Zhang-Dudko model),Inverse Boltzmann and Inverse Weierstrass Transform from the single molecule force spectroscopy(SMFS)data of bacteriorhodopsin initial 8 amino acids(LLILGFGV)unfolding(refolding)from its G helix.And the derived(35)G₀ results met a general agreement(17±4 kcal/mol),bringing validation to these means.And we drew a conclusion about the advantages,insufficiencies and scope of the above four methods.In the meantime,we went through details in our data analyze model and algorithm of AFM SMFS-based free energy landscape reconstruction,and completed the data analyze procedure.Finally,this thesis provided a validation for membrane protein free energy reconstruction methods,and laid a foundation for future studies of point-mutant free-energy changes,of reconstituted membrane protein in controlled lipid samples and of qualifying membrane-protein–ligand interactions.