Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.
Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.
Gitlin
G
G
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bayer
E A
EA
Wilchek
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D019809:Streptavidin; D014364:Tryptophan / Q000032:analysis
D000596:Amino Acids; D001426:Bacterial Proteins; D010446:Peptide Fragments; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin