The alpha-helix formed by the amino acid residues 9-19 of the B-chain of insulin is involved in the stabilization of its three-dimensional structure. We have shown that modification at positions B9, B10, B12, and B16 results in analogues possessing biological activities ranging from ca. 0.2% to ca. 500% relative to that of natural insulin. The lowest potency was displayed by [B12 Asn]insulin, in which the hydrophobic B12 Val residue was replaced by the hydrophilic Asn residue. We now report the synthesis of four insulin analogues in which hydrophobicity is retained, and only the spatial arrangement of atoms in the B12 region is altered. Substitution of B12 Val with alpha-aminoisobutyric acid (Aib), D-Ala, and Phe led to analogues possessing biological activities, in lipogenesis assays, of 8.5%, 2%, and 0.2%, respectively, relative to that of natural insulin. Inversion of the B11-B12 sequence, -Leu-Val-, led to an analogue displaying 3.3% activity. A synthetic B-chain in which the B11 Leu-B12 Val sequence was replaced by B11 Ala-B12 Ile was incapable of combining with the natural A-chain. We conclude that the Val residue in the B12 position in insulin fulfills special side-chain packing requirements involved in the stability of the structure of insulin. Even slight steric alteration at position B12 results in a distortion of the overall conformation of the B-chain which affects its ability to combine with the natural A-chain. This distortion is retained in the corresponding analogue, which is reflected in diminished biological potency.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.

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The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-lO of the B chain of insulin. We have synthesized a human insulin analogue, [AspBlqinsulin, corresponding to the mutant proinsulin and evaluated its biological activity. [AspBIl]Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 ± 146% relative to the patural hormone. In lipogenesis assays, the synthetic analogue exhibited a potency that was 435 + 144% relative to insulin, which is statistically not different from its binding affinity. Reversed-phase HPLC indicated that the synthetic analogue is more apolar than natural insulin. We suggest that the observed properties reflect changes in the conformation of the analogue relative to natural insulin, which result in a stronger interaction with the insulin receptor. Thus, a single substitution of an amino acid residue of human insulin has resulted in a superactive hormone.

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On p. 378. Fig. 6, the lower left axis shonld read Residue Rotation, [R'] ; the right axis On p. 394, procedure 6, line 6 should read m.p. (corr.) 194-195°C.; [a]'," = + 12.75' should read Molar Ellipticity, 0.(c = 1.17, glacial aretic acid). Some Optical Properties of Poly-1-benzyl-L-histidine snd Poly-L-histidineHiopolymers, 1, 277-294 (1sCi3)

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