Mini-proinsulin and Mini-IGF-I: Homologous Protein Sequences Encoding Non-homologous Structures Qing-Xin Hua 1 , Shi-Quan Hu 2 , Wenhua Jia 1 , Ying-Chi Chu 2 G. Thompson Burke 2 {, Shu-Hua Wang 2 , Run-Ying Wang 2 Panayotis G. Katsoyannis 2 * and Michael A. Weiss 1,3 * 1 Center for Molecular Oncology and Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 USA 2 Department of Biochemistry Mount Sinai School of Medicine, New York NY 10029, USA 3 Department of Chemistry The University of Chicago Chicago, IL 60637, USA Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T 3 R 3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corre- sponding two-chain analogue. The two proteins each contain substi- tutions in the B-chain (His B10 ! Asp and Pro B28 ! Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between Lys B29 and Gly A1 . The structures are essentially identical, resembling in each case the T- state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 a-helix (variable among crystal struc- tures) is less well-ordered in mini-proinsulin than in the two-chain var- iant. The single-chain analogue is not completely inactive: its af®nity for the insulin receptor is 1500-fold lower than that of the two-chain ana- logue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29±A1 peptide bond, optimizes af®nity but is not inte- gral to the mechanism of transmembrane signaling. Surprisingly, the ter- tiary structure of mini-proinsulin differs from that of mini-IGF-I (main- chain rms deviation 4.5 A Ê ) despite strict conservation of non-polar resi- dues in their respective hydrophobic cores (side-chain rms deviation 4.9 A Ê ). Three-dimensional pro®le scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures. # 1998 Academic Press Limited Keywords: protein folding; insulin; insulin receptor; transmembrane signaling; NMR *Corresponding authors {Deceased. Abbreviations used: DG, distance geometry; DQF-COSY, double-quantum ®ltered correlated spectroscopy; IGF-I, insulin-like growth factor I; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; rms, root-mean-square; rmsd, rms difference; rp-HPLC, reverse-phase high-performance liquid chromatography; SA, simulated annealing; TOCSY, total correlated spectroscopy; 2D-NMR, two-dimensional NMR. Amino acids are designated by standard three-letter code in the text and by one-letter code in the Figures. ``Native'' elements of structure designate features of crystal structures and may or may not correspond to the functional conformation in a receptor complex. J. Mol. Biol. (1998) 277, 103±118 0022±2836/98/110103±16 $25.00/0/mb971574 # 1998 Academic Press Limited