title: Membrane insertion pathway of annexin B12: thermodynamic and kinetic characterization by fluorescence correlation spectroscopy and fluorescence quenching.
abstract: Experimental determination of the free energy stabilizing the structure of membrane proteins in their native lipid environment is undermined by the lack of appropriate methods and suitable model systems. Annexin B12 (ANX) is a soluble protein which reversibly inserts into lipid membranes under mildly acidic conditions, which makes it a good experimental model for thermodynamic studies of folding and stability of membrane proteins. Here we apply fluorescence correlation spectroscopy for quantitative analysis of ANX partitioning into large unilamellar vesicles containing either 25% or 75% anionic lipids. Membrane binding of ANX results in changes of autocorrelation time and amplitude, both of which are used in quantitative analysis. The thermodynamic scheme describing acid-induced membrane interactions of ANX considers two independent processes: pH-dependent formation of a membrane-competent form near the membrane interface and its insertion into the lipid bilayer. Our novel fluorescence lifetime topology method demonstrates that the insertion proceeds via an interfacial refolded intermediate state, which can be stabilized by anionic lipids. Lipid titration measurements are used to determine the free energy of both transmembrane insertion and interfacial penetration, which are found to be similar, approximately -10-12 kcal/mol. The formation of the membrane-competent form, examined in a lipid saturation experiment, was found to depend on the local proton concentration near the membrane interface, occurring with pK = 4.3 and involving the protonation of two residues. Our results demonstrate that fluorescence correlation spectroscopy is a convenient tool for the quantitative characterization of the energetics of transmembrane insertion and that pH-triggered ANX insertion is a useful model for studying the thermodynamic stability of membrane proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160-7421, USA.
authors.forename: Yevgen O
authors.initials: YO
authors.lastname: Posokhov
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.forename: Lucy
authors.initials: L
authors.lastname: Lu
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry; D017302:Annexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D000077732:Fidaxomicin; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D000617:Aminoglycosides; D017302:Annexins; D000077732:Fidaxomicin