Circular dichroic investigations of secondary structure in synthetic peptide inhibitors for cAMP-dependent protein kinase: a model for inhibitory potential

Abstract

The structure of the inhibitory domain of the inhibitor protein of the cAMP-dependent protein kinase has been assessed by circular dichroism studies of synthetic inhibitory peptides. Using the inhibitory peptide PKI(5-22)amide (Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile22) [Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992] and shorter peptides of this sequence, it has been estimated that this parent peptide is composed of approximately 30% alpha-helix with the remainder being random coil with one beta-turn. The pseudosubstrate arginine cluster (Arg15-Arg19) is within the suggested region of random coil and beta-turn, representing one critical region of binding recognition by the protein kinase. The alpha-helix region proposed between Thr6 and Ile11 likewise contributes to the full biological potency and specificity of the inhibitor peptide and inhibitor protein. The removal of the two N-terminal threonines, for example, causes both a marked conformational change in the peptide and a diminishment by an order of magnitude of inhibitory activity. It is proposed that this alpha-helix region could serve one of several possibilities, including that it may provide a suitable constraint on the Tyr7 such that the hydroxyl is oriented in a position proximal to the pseudosubstrate domain, and/or may allow the optimal location of other protein kinase recognition signals. These data provide an initial description of some of the structural features of the inhibitor protein that could contribute to its high biological potency.

Conformationally constrained analogs of protein kinase inhibitor(6–22)amide: Effect of turn structures in the center of the peptide on inhibition of cAMP‐dependent protein kinase

“…As indicated in Figure 6, the CD spectrum of PKI(6-22)amide at 222 nm confirms the presence of some cr-helix as originally reported by Reed et al (1987) for PKI(5-22)amide. The spectra of these two peptides were similar, indicating that the shortening of PKI(5-22)amide by one residue to yield the 17-residue peptide used in this study did not greatly alter the conformations that it populates in solution.…”

“…(Le~~~,Ile~~)PK1(5-22)amide had a K, value of 130 nM (246 nM when repeated in this study) as compared to the potent PKI(5-22)amide with a K, of 3.1 nM (Glass et al, 1989a). These data showing appreciable loss of inhibitory activity, along with the results of physical studies of PKI peptides (Reed et al, 1987(Reed et al, , 1989, promoted us to investigate further the possibility of turn structures in the central part of the molecule. Peptides were designed to alter and constrain po- Corrected for 5-10% destruction during acid hydrolysis.…”

NMR study of a 34‐residue N‐terminal fragment of the parathyroid‐hormone‐related protein secreted during humoral hypercalcemia of malignancy

“…An extended coil such as the peptide Kemptide, the model substrate of the CAMP-dependent protein kinase, displays a negative ellipticity which peaks at 198 nm. At 190 nm, the ellipticity remains negative and in general it is positive around 215 -220 nm [16]. The results in Fig.…”

“…The sequence 4 -9 (Glu-His-Gln-Leu-Leu-His) has a very high probability of forming an a-helix [27] with a < P a > = 1.31. The CD spectra were not used to analyze other secondary structures such as p-turns since their identification is too heavily reliant on curve fitting [16,28,291 and inferior to information gained from the NMR studies.…”

“…Table 2 shows that the temperature coefficient of the Arg-19 NH is extremely low (-3.09 ppb/K), indicating that it is well shielded from the solvent. This result remains unaffected by the removal of residues 1 -14 (leaving 15 -34) or 21 -34 (leaving residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], The shielding therefore is not a consequence of the Arg-19 NH being buried within another part of the structure. The vicinal coupling constants for Asp-17 and Leu-18 in Table 2 are 4.8 and 7.2 Hz respectively.…”