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Design and Characterization of A Synthetic Electron-Transfer Protein
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Section:Abstract
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 θ222/θ208 = 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 μM and θmax = −23 800 ± 130 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-Δ3-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 Cα
Cα distance of 13.5 ± 0.9 Å 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 
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 24 Å across the noncovalent peptide interface. Pulse radiolysis was used to measure an ET rate constant of ket = 380 ± 80 s-1 for the intracomplex electron transfer (ΔG° = −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 one reported in an earlier communication.
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History
- Published In Issue August 23, 2000
- Received February 18, 2000
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