title: Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Jansson
authors.forename: M
authors.initials: M
authors.lastname: Uhlen
authors.forename: B
authors.initials: B
authors.lastname: Nilsson
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002942:Circular Dichroism; D017931:DNA Primers; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D017931:DNA Primers; D018970:Insulin-Like Growth Factor Binding Protein 1; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D010434:Pepsin A