The long term goal of our investigations is the elucidation of the relationship between chemical structure, conformation, receptor binding and biological activity (metabolic and growth-promoting) of insulin. Our studies thus far provided data suggesting: a) that within the insulin molecule there is one region primarily associated with receptor binding (""""""""binding region"""""""") and another region associated with the expression of biological activity (""""""""message region""""""""); b) that the C-terminal region of the A chain may be involved in the expression of the biological activity of insulin; and c) that insulin analogues exhibiting a wide range of biological activities, by circular dichroism reveal little if any conformational difference from monomeric insulin. It is our objective to identify those structural features or amino acid residues involved in the functional regions of insulin and to delineate the conformational differences betwen insulin and analogue which would account for the differences in their biological behavior.
The specific aims i nclude: (1) The synthesis of insulin analogues bearing modifications that will affect directly or indirectly the C-terminal region of the A chain; (2) The snythesis of analogues bearing modification that will affect the stability of the B20-B23 Beta-turn; (3) The snythesis of analogues in which selected peptide bonds, predicted by computer graphics to be involved in receptor binding, will be modified; (4) The synthesis of analogues incorporating unique structural features found in the strongly growth-promoting insulin-like growth factors and hystricomorph insulins. These latter modifications may lead to """"""""new insulins"""""""" with enhancd mitogenic activities; (5) Comparison of the conformation of the monomeric form of insulin with that of insulin analogues, known to be monomeric, by nanosecond time-resolved fluorescence. We will assess to what extent the conformation of insulin in solution is comparable to the conformation of the analogues by measuring the fluorescene lifetimes and anisotropy decay of their tyrosine resides; (6) Measure the time-resolved fluorescence of insulin analogues in which individual tyrosine residues are substituted with residues having special photophysical properties.
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