A pair of pyrene groups as a conformational probe for designed two α-helix polypeptides

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Abstract
Peptides with two α-helix segments anchored on 2,2′-bipyridyl-4,4′-dicarboxylic acid and suberic acid have been designed and synthesized. L-1-Pyrenylalanine (Pya) was introduced near the centre of each α-helical segment as a CD and fluorescent probe to detect the arrangement of the two α-helix segments. The amphiphilic α-helical 14-peptide was designed with an amino acid sequence in which the hydrophobic amino acids, Leu and Pya, are deployed in the same manner as hydrophobic amino acids in coiled-coil proteins. The synthesis was carried out by a solid-phase synthesis on Kaiser's oxime resin and solution coupling with anchors. The probing behaviour of a pair of Pya residues was examined by CD and fluorescent measurements under various conditions, e.g. in aqueous and methanolic solutions, in the presence of guanidine hydrochloride (GuHCl) and under increasing temperature. The peptides showed a highly α-helical CD pattern in the far-UV region in aqueous solution. Furthermore, they showed strong split CD peaks with positive and negative maxima at longer and shorter wavelengths, respectively, at the pyrene absorption region (1La and 1Bb). These split CD signals decreased with the disruptions of the two α-helix structure by the addition of methanol, GuHCl and also with increasing temperature. On the other hand, the excimer emission of pyrene in fluorescence was very weak in aqueous solution, but it was transiently increased while the helix-helix interaction was relaxed to some extent with the addition of methanol. These facts indicate that the two pyrene groups are forced to be in close proximity and to be in a right-handed sense between the two α-helix structures in aqueous solution; whereas, the arrangement of the pyrene groups is loosened by the destruction of the 3D structure. Thus, the pyrene probe has been demonstrated to provide significant information about the arrangement of the two α-helix segments by the simple measurements of CD and fluorescence spectra.