Solution structure and backbone dynamics of long-[Arg3]insulin-like growth factor-I

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of N-15-labeled Long-[Arg(3)]IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 Angstrom root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 Angstrom root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using N-15 nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE), There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average {H-1}N-15 NOE of 0.55 for the regions, The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus, Despite these data indicating conformational flexibity for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.