Solution structure and backbone dynamics of long-[Arg(3)]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 (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A 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 (15)N 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 ((1)H)(15)N 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 flexibility 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.

Secondary structure determination of 15N-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy.

Insulin-like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long-[Arg-3]-IGF-I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213-223), a potent analogue of IGF-I, which has a Glu-3 to Arg-3 substitution and a hydrophobic, thirteen amino acid N-terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity-enhanced 15N-NOESY-HSQC and sensitivity-enhanced 15N-HSQC-NOESY-HSQC spectra. The IGF-I domain of Long-[Arg-3]-IGF-I was found to have an almost identical structure to IGF-I. The N-terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn-like structure that is spatially close to the beginning of helix 1 in the IGF-I domain. Hydrogen-deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF-I domain are either in the helical or the extended structural elements. Many of the amide protons in the N-terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long-[Arg-3]-IGF-I and the decreased binding to insulin-like growth factor binding proteins.