A. Y. Kornilova, J. F. Wishart, W. Xiao, R. C. Lasey, A. Fedorova, Y.-K. Shin, and M. Y. Ogawa
J. Am. Chem. Soc. 122, 7999-8006 (2000) [Find paper at ACS Publications]
Abstract:
A 30-residue polypeptide, H21(30-mer), was synthesized having the sequence, Ac-K(IEALEGK)2(IEALEHK)(IEALEGK)G-NH2. The circular dichroism spectrum of the peptide shows minima at 208 and 222 nm, and q222/q208 = 1.06 which indicates the formation of a self-assembled coiled-coil when dissolved in aqueous solution. The concentration dependence of the CD data can be fit to an expression that describes a two-state monomer-dimer equilibrium for the apopeptide: Kd = 1.5 ± 0.4 mM and qmax = -23,804 ± 132 deg cm2 dmol-1, showing that it has a maximum helicity of 69%. A [MTSL-C21(30-mer)] dimer was also prepared in which MTSL is the thiol-specific nitroxide spin label 1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl-methanethiosulfonate attached to C21 of the 30-mer. Fourier deconvolution analysis of the dipolar line broadening of the EPR spectrum yields a measure of the inter-chain Ca-Ca distance of 13.5 ± 0.9 Å at position 21 of the coiled-coil which is nearly identical to those observed for the isostructural family of bZip proteins. The two metallohomodimers, [Ru(trpy)(bpy)-H21(30-mer)]2 and [Ru(NH3)5-H21(30-mer)]2 were prepared in which the ruthenium complexes were coordinated to the H21 site of the 30-mer. SDS-PAGE, chemical crosslinking studies, and analytical ultracentrifugation show that the peptides exist as a dimeric coiled-coil having a molecular weight of ca. 7.5 kDa. The ET heterodimer, [Ru(trpy)(bpy)-H21(30-mer)]/[Ru(NH3)5-H21(30-mer)] was prepared and molecular modeling shows that the two metal complexes are separated by a metal-to-metal distance of ca. 24 Å across the non-covalent, peptide interface. Pulse radiolysis was used to measure an ET rate constant of ket = 380 ± 80 s-1 for the intracomplex electron-transfer (DG0 = -1.11 eV) from the RuII(NH3)5-H21 donor to the RuIII(trpy)(bpy)-H21 acceptor. The value for ket falls within the range reported for modified proteins over comparable distances and supersedes the value reported in an earlier communication.