Insulin-like growth factor-I (IGF-I): Solution properties and NMR chemical shift assignments near physiological pH Zhihe Kuang a,b , Shenggen Yao a , Kerrie A. McNeil b , Briony E. Forbes b , John C. Wallace b , Raymond S. Norton a, * a The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia b School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia 5005, Australia article info Article history: Received 8 August 2008 Revised 8 October 2008 Accepted 17 October 2008 Available online 3 December 2008 Keywords: Insulin-like growth factor Nuclear magnetic resonance Chemical shift Assignment Solution properties abstract Objective: Insulin-like growth factor-I (IGF-I) plays important roles in normal growth and development, as well as in disease states, and its structure and function have been studied extensively using nuclear magnetic resonance (NMR) spectroscopy. However, IGF-I typically gives poor quality NMR spectra con- taining many broad peaks, because of aggregation at the protein concentrations generally required for NMR experiments as well as the internal dynamics of the molecule. The present study was undertaken to determine a reliable set of assignments under more physiological conditions. Design: Several reports of chemical shift assignments have been published previously for IGF-I either bound to a ligand or at relatively low pH (3–4), but there are many contradictions among them, reflect- ing the poor behaviour of IGF-I. Low pH conditions are also suboptimal for the analysis of interactions between IGF-I and IGF binding proteins (IGFBP) or IGFBP fragments. Spectra were recorded at low con- centrations in order to identify conditions of temperature and pH where all peaks could be observed. Results: We show that good quality 2D 1 H– 15 N HSQC spectra of 15 N-labelled IGF-I can be obtained at pH 6 and 37 °C, much closer to physiological conditions, by using lower IGF-I concentrations (0.05 mM). Surprisingly, at this concentration and temperature, spectra were of better quality at pH 6 than at pH 4, in contrast to previous observations made at millimolar concentrations of IGF-I. We were then also able to assign the chemical shifts of IGF-I at pH 6 and 37 °C using 3D heteronuclear spectra recorded on a 0.7 mM 15 N/ 13 C-labelled IGF-I sample. Conclusion: These results provide a valuable resource for future studies of the structure, dynamics, folding, and binding interactions of IGF-I, as well as analogues thereof, by means of NMR spectroscopy. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Human insulin-like growth factors, IGF-I and IGF-II, are single- chain polypeptide hormones that contain 70 and 67 residues, respectively [1]. They exert mitogenic and metabolic effects by binding to cell surface IGF and insulin receptors and triggering intracellular signalling pathways [2]. The bioavailability and cellu- lar actions of IGFs are tightly regulated by a family of six high affin- ity IGF binding proteins (IGFBP-1 to IGFBP-6) [3,4]. Dysfunction of the IGF system is associated with many diseases such as growth disorders, diabetes, and cancer [5,6]. Given the important roles of IGFs in both normal growth and development, as well as in disease states, the structures and func- tions of these peptides have been studied extensively using a vari- ety of approaches, including nuclear magnetic resonance (NMR) spectroscopy [7–17]. Both IGF-I [7,8,11–15] and IGF-II [7,9,16,17] give poor NMR spectra, characterised by many broad peaks. This is believed to arise from a combination of aggregation at the pro- tein concentrations generally required for NMR experiments, to- gether with conformational flexibility of the molecules. In fact, chemical shift assignments of IGF-I have been obtained only at rel- atively low pH (3–4) [8,10–12,14] or when bound to a peptide derived from phage display [15]. It has also been noted that contra- dictory chemical shift assignments exist among those reported previously, and these did not seem to be due simply to pH or tem- perature difference, but rather to mis-assignments as a result of poor quality spectra and missing peaks [7]. We have been interested in analysing the binding of IGF-I to IGFBPs and their domain fragments by NMR using isotopically la- belled IGF-I. The low pH solution conditions (pH 3–4) used in pre- vious studies of IGF-I by NMR [8,10–12,14] were unsuitable for our binding studies because such low pH reduced the binding affinity. It has been shown that IGFBP-2 binding to IGF-I exhibited similar biosensor curves at pH 7.4, 6.4, and 5.4, but that dissociation was faster at pH 4.4 and significantly faster at pH 3.4 [7]. Moreover, 1096-6374/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2008.10.003 * Corresponding author. Tel.: +61 3 9345 2306; fax: +61 3 9345 2686. E-mail address: ray.norton@wehi.edu.au (R.S. Norton). Growth Hormone & IGF Research 19 (2009) 226–231 Contents lists available at ScienceDirect Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir