Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.
1997 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 14, 4108-4117 p.Article in journal (Refereed) Published
Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.
Place, publisher, year, edition, pages
1997. Vol. 36, no 14, 4108-4117 p.
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-13474DOI: 10.1021/bi961553iPubMedID: 9100004OAI: oai:DiVA.org:kth-13474DiVA: diva2:325490
QC 201006182010-06-182010-06-182011-11-08Bibliographically approved