Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Assignment of a human serum glycoprotein SP-40,40 gene (CLI) to chromosome 8.

SP-40,40 is a serum glycoprotein consisting of two different subunits (alpha and beta) assembled into a dimer by disulfide bonds. Northern blot hybridization, using total RNA from several cell lines, showed that SP-40,40 is expressed in glioblastoma and testicular tumor cells, as well as hepatoma cells. Spot blot hybridization of flow-sorted human chromosomes, using a SP-40,40 cDNA fragment as a probe, localized the gene for SP-40,40 to human chromosome 8. This gene has been given the designation CLI, for complement lysis inhibitor, by the Human Gene Nomenclature Committee.     

NMR study of isoleucine transfer RNA from Thermus thermophilus

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.