title: Estimation of helix-helix association free energy from partial unfolding of bacterioopsin.
abstract: To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).
authors.affiliation: Department of Biology, University of Texas at San Antonio, San Antonio, Texas 78249, USA.
authors.forename: Suraj John
authors.initials: SJ
authors.lastname: Nannepaga
authors.forename: Ravikumar
authors.initials: R
authors.lastname: Gawalapu
authors.forename: Daniel
authors.initials: D
authors.lastname: Velasquez
authors.forename: Robert
authors.initials: R
authors.lastname: Renthal
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005960:Glucosides / Q000737:chemistry; D019617:Halobacterium salinarum; D007700:Kinetics; D008027:Light; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008823:Micelles; D008956:Models, Chemical; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D012997:Solvents; D014364:Tryptophan / Q000737:chemistry
chemical_list: D003619:Dansyl Compounds; D005960:Glucosides; D008823:Micelles; D012997:Solvents; C036851:dansyllysine; C018619:octyl-beta-D-glucoside; D000431:Ethanol; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D014364:Tryptophan; D008239:Lysine