Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies

Abstract

Fourier-transforminfraredstudies have been carried out to investigate the secondarystructure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondarystructure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipidassemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondarystructure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondarystructure. The complexes of avidin with both biotin and membrane-bound DMBPE lipidassemblies display a large increase in thermal stability compared with the native protein.