1H-NMR Assignment and Secondary Structure of Human Insulin-Like Growth Factor-I(IGF-I) in Solution

Abstract

Human insulin-like growth factor-I (IGF-I) was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for all the backbone protons and almost all of the sidechain protons of the total 70 amino acid residues, using sequence-specific assignment procedures. The secondary structure elements of human IGF-I were identified by investigation of the sequential and medium range NOEs as a preliminary step in determining the three-dimensional structure of this protein by means of distance geometry calculations. The typical NOEs of d alpha beta(i,i + 3) and d alpha N(i,i + 3), as well as the successive strong NOEs of dNN connectivities and slowly exchanging amide protons confirmed the presence of three helical segments corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48, and Leu54-Cys61, and the existence of a beta-turn in the Gly19-Gly22 region. Our results definitely indicate that the secondary structure of human IGF-I in solution is consistent with that of insulin in the crystalline state.

The Insulin-like Growth Factor (IGF)Binding Protein 1 Binding Epitope on IGF-I Probed by Heteronuclear NMR Spectroscopy and Mutational Analysis

“…Others have reduced the IGF-I self-association at 2-10 mM protein concentration, and thereby improved the spectral quality, by the addition of 10% (8) or 20% (9) acetic acid. In this study, we have not used acetic acid, since it influences the IGF-I/IGFBP-1 binding affinity.…”

“…No assignments whatsoever could be made for residues 18, 34 -37, 50, 63, 68, and 69 in that study. For the studies performed with acetic acid present (6,8), virtually complete assignments were accomplished.…”

Solution Structure and Backbone Dynamics of Long-[Arg3]insulin-like Growth Factor-I

“…The number of restraints used in the refinement of the structure of Long-[Arg 3 ]IGF-I is significantly greater than used for the original IGF-I structures (9,10) and is comparable to the number used for subsequent work with IGF-II (13) and analogs of IGF-I (25,26). The solution structures of Long- [Arg 3 ]IGF-I display good convergence to a single fold in the hydrophobic core region containing the three ␣-helices (Fig.…”

“…Recent NMR studies have revealed that IGF-I has three ␣-helical regions surrounding a hydrophobic core (9,10). Residues 3-29 of IGF-I, which are homologous to the B-chain of insulin, contain helix-1 (Ala 8 to Cys 18 ).…”

“…Thus far, the only refined structures of proteins from the IGF family include two reports of the IGF-I structure, both obtained by homonuclear NMR techniques (9,10), two structures of IGF-II obtained by a combined homonuclear/heteronuclear NMR approach (13,14), and the structure of a mini-IGF-I, which has the C-domain deleted (15). Detailed analysis of the protein dynamics has not been reported for any of the IGF proteins.…”