Effect Of Surface Charges On The Rates Of Intermolecular Electron-transfer Between De Novo Designed Metalloproteins

A de novo designed coiled-coil metalloprotein was prepared that uses electrostatic interactions to control both its conformational and bimolecular electron-transfer properties. The title protein exists as a coiled-coil heterodimer of the [Ru(trpy)(bpy)-KK(37-mer)] and [Ru(NH3)(5)-EE(37-mer)] polypeptides which is formed by interhelix electrostatic attractions. Circular dichroism studies show that the electrostatic heterodimer has Kd 0.19 +/- 0.03 muM and is 96% helical at high concentrations. Intercomplex electron-transfer reactions were studied that involve the [Ru(NH3)(5)-H21](2+) electron-donor and the [Ru(trpy)(bpy)H21](3+) electron-acceptor belonging to different electrostatic dimers. An important feature of the designed metalloprotein is its two cationic redox centers embedded within protein surfaces having opposite charge. Thus, the Ru-II(NH3)5-H21 site was placed on the surface of one chain of the coiled-coil which was made to be positively charged, and the Ru-III(trpy)(bpy)-H21 site was placed on the surface of the other chain which was negatively charged. The rates of intermolecular electron-transfer increased from (1.9 +/- 0.4) x 10(7) M-1 s(-1) to (3.7 +/- 0.5) x 107 M-1 s(-1) as the ionic strength was increased from 0.01 to 0.20 M. This indicates that the electrostatic repulsion between the ruthenium centers dominates the kinetics of these reactions. However, the presence of the oppositely charged protein surfaces in the coiled-coils creates an electrostatic recognition domain that substantially ameliorates the effects of this repulsion

High-Field Magic Angle Spinning Dynamic Nuclear Polarization Using Radicals Created by γ-Irradiation

High-field magic angle spinning dynamic nuclear polarization (MAS DNP) is often used to enhance the sensitivity of solid-state nuclear magnetic resonance experiments by transferring spin polarization from electron spins to nuclear spins. Here, we demonstrate that γ-irradiation induces the formation of stable radicals in inorganic solids, such as fused quartz and borosilicate glasses, as well as organic solids, such as glucose, cellulose, and a urea/polyethylene polymer. The radicals were then used to polarize 29Si or 1H spins in the core of some of these materials. Significant MAS DNP enhancements (ε) of more than 400 and 30 were obtained for fused quartz and glucose, respectively. For other samples, negligible values of ε were obtained, likely due to low concentrations of radicals or the presence of abundant quadrupolar spins. These results demonstrate that ionizing radiation is a promising alternative method for generating stable radicals that are suitable for high-field MAS DNP experiments

Design And Characterization Of A Synthetic Electron-transfer Protein

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

1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 with Ca2+ bound at EF1, EF3 and EF4

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP3Rs) and a variety of voltage-gated Ca2+ channels in the brain. We report complete NMR chemical shift assignments of the Ca2+-saturated form of CaBP1 with Ca2+ bound at EF1, EF3 and EF4 (residues 1–167, BMRB no. 16862)

Photo-detrapping of solvated electrons in an ionic liquid

金沢大学理工研究域自然システム学系We studied the dynamics of photo-detrapped solvated electrons in the ionic liquid trimethyl-N-propylammonium bis(trifluoromethanesulfonyl)imide (TMPA-TFSI) using laser flash photolysis. The solvated electrons were produced by the electron photodetachment from iodide via a 248 nm KrF excimer laser. The solvated electron decayed by first-order kinetics with a lifetime of about 240 ns. The spectrum of the solvated electron in the ionic liquid TMPA-TFSI is very broad with a peak around 1100 nm. After the 248 nm pulse, a 532 nm pulse was used to subsequently detrap the solvated electrons. After the detrapping pulse, quasi-permanent bleaching was observed. The relative magnitude of the bleaching in the solvated electron absorbance was measured from 500 to 1000 nm. The amount of bleaching depends on the probe wavelength. The fraction of bleached absorbance was larger at 500 nm than that at 1000 nm, suggesting that there are at least two species that absorb 532 nm light. We discuss the present results from viewpoint of the heterogeneity of ionic liquids. © 2009 Elsevier Ltd

Large Deviations of the Maximum Eigenvalue in Wishart Random Matrices

We compute analytically the probability of large fluctuations to the left of the mean of the largest eigenvalue in the Wishart (Laguerre) ensemble of positive definite random matrices. We show that the probability that all the eigenvalues of a (N x N) Wishart matrix W=X^T X (where X is a rectangular M x N matrix with independent Gaussian entries) are smaller than the mean value =N/c decreases for large N as $\sim \exp[-\frac{\beta}{2}N^2 \Phi_{-}(\frac{2}{\sqrt{c}}+1;c)]$, where \beta=1,2 correspond respectively to real and complex Wishart matrices, c=N/M < 1 and \Phi_{-}(x;c) is a large deviation function that we compute explicitly. The result for the Anti-Wishart case (M < N) simply follows by exchanging M and N. We also analytically determine the average spectral density of an ensemble of constrained Wishart matrices whose eigenvalues are forced to be smaller than a fixed barrier. The numerical simulations are in excellent agreement with the analytical predictions.Comment: Published version. References and appendix adde

Exact Minimum Eigenvalue Distribution of an Entangled Random Pure State

A recent conjecture regarding the average of the minimum eigenvalue of the reduced density matrix of a random complex state is proved. In fact, the full distribution of the minimum eigenvalue is derived exactly for both the cases of a random real and a random complex state. Our results are relevant to the entanglement properties of eigenvectors of the orthogonal and unitary ensembles of random matrix theory and quantum chaotic systems. They also provide a rare exactly solvable case for the distribution of the minimum of a set of N {\em strongly correlated} random variables for all values of N (and not just for large N).Comment: 13 pages, 2 figures included; typos corrected; to appear in J. Stat. Phy