Design and characterization of a synthetic electron-transfer protein

被引:45
作者
Kornilova, AY
Wishart, JF
Xiao, WZ
Lasey, RC
Fedorova, A
Shin, YK
Ogawa, MY [1 ]
机构
[1] Bowling Green State Univ, Dept Chem, Bowling Green, OH 43403 USA
[2] Bowling Green State Univ, Ctr Photochem Sci, Bowling Green, OH 43403 USA
[3] Brookhaven Natl Lab, Dept Chem, Upton, NY 11973 USA
[4] Univ Calif Berkeley, Lawrence Berkeley Lab, Dept Chem, Berkeley, CA 94720 USA
[5] Univ Calif Berkeley, Lawrence Berkeley Lab, Div Struct Biol, Berkeley, CA 94720 USA
关键词
D O I
10.1021/ja0006954
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
A 30-residue polypeptide [H21(30-mer)] with the sequence Ac-K(IEALEGK)(2)(IEALEHK)-(IEALEGK)G-NH2 was synthesized. The circular dichroism (CD) spectrum of the peptide shows minima at 208 and 222 nm and theta(222)/theta(208) = 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 (K-d = 1.5 +/- 0.4 mu M and theta(max) = -23 800 +/- 130 deg cm(2) 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-Delta(3)-pyrroline-3-methyl-methanethiosulfonate attached to C21 of the 30-mer. Fourier deconvolution analysis of the dipolar line broadening of the electron paramagnetic resonance (EPR) spectrum yields a measure of the interchain C alpha-C alpha distance of 13.5 +/- 0.9 Angstrom at position 21 of the coiled-coil, which is nearly identical to those distances observed for the isostructural family of bZip proteins. Two metallohomodimers, [Ru(trpy)(bpy)-H21 (30-mer)](2) and [Ru(NH3)(5)-H21(30-mer)](2), in which the ruthenium complexes were coordinated with the H21 site of the 30-mer, were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), chemical cross-linking studies, and analytical ultracentrifugation show that the peptides exist as a dimeric coiled-coil with a molecular weight of similar to 7.5 kDa. The electron transfer (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 similar to 24 Angstrom across the noncovalent peptide interface. Pulse radiolysis was used to measure an ET rate constant of k(et) = 380 +/- 80 s(-1) for the intracomplex electron transfer (Delta G degrees = -1.11 eV) from the Ru-II(NH3)(5)-H21 donor to the Ru-III(trpy)(bpy)-H21 acceptor. The value for k(et) falls within the range reported for modified proteins over comparable distances and supersedes the one reported in an earlier communication.
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页码:7999 / 8006
页数:8
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