Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120.
We report the functional characterization of site-directed biotin binding-site mutants of recombinant core streptavidin. The mutagenesis studies were aimed at characterizing the contributions of Trp residues known to contact biotin that have been postulated to control the exceptional binding affinity observed in this system. The functional properties of single site-directed mutants replacing Trp residues with Phe or Ala at positions 79, 108, and 120 were investigated by quantitating the EC50 binding parameters of these mutants to biotin and 2-iminobiotin in an ELISA format. The biotin EC50 for all mutants was the same as wild-type streptavidin, demonstrating that their delta Ka values relative to wild type were < 10(6). The conservative W79F and W108F mutants displayed only a 2- to 3-fold increase in EC50 for 2-iminobiotin, corresponding to an estimated delta Ka < 10, while the W120F mutant displayed a much greater alteration in 2-iminobiotin EC50, corresponding to an estimated delta Ka of 10(2). These delta Ka values are likely to reflect similar changes for biotin. The 2-iminobiotin EC50 values for the Ala mutants fell outside the accessible concentration range of the ELISA assay, demonstrating that these mutations lowered the Ka by a factor of 10(4) to 10(6). Direct estimation of biotin Ka values for W79A, W120A, and W120F in an ultrafiltration binding assay yielded Ka values of 4.3 x 10(7) M-1, 8.6 x 10(6) M-1, and > 5 x 10(9) M-1, respectively, in excellent agreement with the ELISA estimates of delta Ka with 2-iminobiotin as a reporter ligand. The results of these preliminary functional studies suggest that these aromatic side chains contribute significantly to the streptavidin-biotin binding free energy.
Chilkoti
A
A
Center for Bioengineering, University of Washington, Seattle 98195.
Tan
P H
PH
Stayton
P S
PS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D019809:Streptavidin; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin