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

Abstract

<jats:p>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 <jats:sup>1</jats:sup>H,<jats:sup>15</jats:sup>N NMR spectroscopy. All the backbone <jats:sup>1</jats:sup>H and <jats:sup>15</jats:sup>N assignments and most of the <jats:sup>1</jats:sup>H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity‐enhanced <jats:sup>15</jats:sup>N‐NOESY‐HSQC and sensitivity‐enhanced <jats:sup>15</jats:sup>N‐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.</jats:p>