title: Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin.
abstract: Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20-28% of the initial fluorescence with (KSV + V) less than or equal to 0.55 M-1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka = 5 M-1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weber et al., 1989).
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: G P
authors.initials: GP
authors.lastname: Kurzban
authors.forename: G
authors.initials: G
authors.lastname: Gitlin
authors.forename: E A
authors.initials: EA
authors.lastname: Bayer
authors.forename: M
authors.initials: M
authors.lastname: Wilchek
authors.forename: P M
authors.initials: PM
authors.lastname: Horowitz
mesh_terms: D000178:Acrylamides; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D005453:Fluorescence; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019809:Streptavidin; D013306:Streptomycetaceae; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000178:Acrylamides; D001426:Bacterial Proteins; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin