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Abstract
By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.
Highlights
MinalB-chaindomain of insulincontaining residuesB24, B25, and B26 packs against the equivalenrtegion of a second insulin molecule to form the insulin dimer in the asymmetric unit of the 2-Zn insulinhexamer [1, 2]
CD spectra for all samples were measured in 0.025 M Tris brought to pH 7.5 with HC1 at peptide concentrations of 17 p ~ Pe. ptide concentrations for all studies were determined by quantitative amino acid analysis and were verified by analytical reverse-phase HPLC
For receptor binding, with the D-configurationor the tyrosine hydroxyl group being either tolerated, not accepted, or even beneficial; [6] important structural differences exist between the Analyticel Radioiodination of Insulin and Insulin Analogs-Radiolabeling methods were based on previously published chloramine Tbased methods [20] but weremodified to restrict the extent of iodination to 0.01-0.03molof I/mol of peptide
Summary
At position B26, yielding insulinand [ ~ - T y r ~ ~ ~ ] insulin , a n 8 analog with 63% relative receptor binding potency 0.1% relative toinsulin(peptide 4, Fig. l b andTable I), replacement of PheBZ4by D-Tyr enhances binding potency t o 163% of that of insulin Terminal B-chain domain of insulin in the crystallographic structure of the 2-Zn insulin hexamer, it is notable that the introduction of D-Tyr (a residue which would be expected to perturb the aromatic packing arrangement ablusot to alter main chain conformatiosnignificantly, Ref. 23) at either of positions B26 or €324 results in no major loss in potency for receptor interaction. Since the truncated analog des-pentapeptide-(B26-B30)-[PheB'5-~-carboxamide]insulin(an analog in which theCOOH-terminalB-chain fromresidues
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