Conformational changes of beta-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles. A fluorescence and CD study.
The effect of beta-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (omega0). CD data underlined the distortion of the beta-sheet and a less constrained tertiary structure as the omega0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were tau1 = 1.28 ns and tau2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with omega0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in beta-lactoglobulin, induced by changes in omega0. The average bimolecular quenching constant <kq(CCl4)> was found not to depend on omega0, confirming the insolubility of this quencher in the aqueous interface, while <kq(arcylamide)> increases with omega0. The drastic decrease with omega0 of kq, associated with the longest lifetime kq2(CCl4), comparatively to the increase of kq2(acrylamide), emphasizes the location of beta-lactoglobulin in the aqueous interfacial region especially at omega0> or = 10. The fact that (omega0 = 30) >> kq2(acrylamide) (water) also confirms the important conformational changes of encapsulated beta-lactoglobulin.
Andrade
Suzana M
SM
Centro de Química Estrutural, Complexo 1, Instituto Superior Técnico, Lisboa, Portugal. sandrade@popsrv.ist.utl.pt
Carvalho
Teresa I
TI
Viseu
M Isabel
MI
Costa
Sílvia M B
SM
D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry*; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D008823:Micelles; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D014867:Water / Q000737:chemistry
D007782:Lactoglobulins; D008823:Micelles; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; D020106:Acrylamide; D014364:Tryptophan