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Previous studies on calcitonin gene related peptide (CGRP) have demonstrated that it has the characteristics of an amphiphilic peptide, and from an examination of the sequence, we have proposed that it contains an amphiphilic alpha-helix. We have synthesized two analogues of CGRP which have different lengths of idealized amphiphilic alpha-helical secondary structure. The first model, CGRM-1, has been substituted with residues generating an idealized amphiphilic alpha-helix in the region between residues 8 and 25, equivalent to approximately five turns of an alpha-helix. This peptide is not an agonist in any of our bioassays, but it does bind with low affinity to rCGRP receptors in crude liver membranes. Our second model, CGRM-2, has an idealized amphiphilic alpha-helix between residues 8 and 18, which is equivalent to approximately three turns of an alpha-helix. In an in vitro rat vas deferens assay, this peptide is an agonist with a potency one-fourth that of the native hormone. However, the potency of CGRM-2 in an adenylate cyclase assay is much lower, only 1/140th the potency of CGRP. Both model peptides display amphiphilic characteristics commensurate with their design. We conclude that there is an amphiphilic alpha-helix in rCGRP between residues 8 and 18 and that this helix terminates in the vicinity of residue 18.

Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the previous paper, N-methylated peptides were shown to be sensitive probes of substrate conformation within the adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) active site. While it has been shown that other protein kinases will catalyze the phosphorylation of the same peptide sequences as A-kinase, there is as yet little information as to whether the protein kinases differentiate between substrates on the basis of conformation. For this reason, the conformationally restricted N-methylated peptides were used to probe the active site of guanosine cyclic 3',5'-phosphate dependent protein kinase (G-kinase), which is homologous in sequence to [Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry 23, 4207-4218] and which has substrate specificities similar to [Lincoln, T. M., & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3239-3243] those of A-kinase. Although this enzyme appears to bind the peptides in a conformation resembling that of conformation A, it is more able to accommodate backbone methylation than is A-kinase. A peptide substrate at least 700-fold selective for G-kinase over A-kinase was found. Backbone methylation may, therefore, represent a way of making peptide substrates and inhibitors selective for a particular kinase.