Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

AbstractA novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction

Marked changes in electron transport through the blue copper protein azurin in the solid state upon deuteration

Measuring electron transport (ETp) across proteins in the solid-state offers a way to study electron transfer (ET) mechanism(s) that minimizes solvation effects on the process. Solid state ETp is sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurement technique extends the use of ETp meas-urements down to low temperatures and the concomitant lower current densities, because the larger area still yields measurable currents. Thus, we reported previously a surprising lack of temperature-dependence for ETp via the blue copper protein azurin (Az), from 80K till denaturation, while ETp via apo-(Cu-free) Az was found to be temperature de-pendent \geq 200K. H/D substitution (deuteration) can provide a potentially powerful means to unravel factors that affect the ETp mechanism at a molecular level. Therefore, we measured and report here the kinetic deuterium isotope effect (KIE) on ETp through holo-Az as a function of temperature (30-340K). We find that deuteration has a striking effect in that it changes ETp from temperature independent to temperature dependent above 180K. This change is expressed in KIE values between 1.8 at 340K and 9.1 at \leq 180K. These values are particularly remarkable in light of the previously reported inverse KIE on the ET in Az in solution. The high values that we obtain for the KIE on the ETp process across the protein monolayer are consistent with a transport mechanism that involves through-(H-containing)-bonds of the {\beta}-sheet structure of Az, likely those of am-ide groups.Comment: 15 pages, 3 figures, 2 Supplementary figure

Coherent Electron Transport across a 3 nm Bioelectronic Junction Made of Multi-Heme Proteins

Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+Σ current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices

Negative Regulation of FcεRI-mediated Degranulation by CD81

Signaling through the high affinity receptor for immunoglobulin E (FcεRI) results in the coordinate activation of tyrosine kinases before calcium mobilization. Receptors capable of interfering with the signaling of antigen receptors, such as FcεRI, recruit tyrosine and inositol phosphatases that results in diminished calcium mobilization. Here, we show that antibodies recognizing CD81 inhibit FcεRI-mediated mast cell degranulation but, surprisingly, without affecting aggregation-dependent tyrosine phosphorylation, calcium mobilization, or leukotriene synthesis. Furthermore, CD81 antibodies also inhibit mast cell degranulation in vivo as measured by reduced passive cutaneous anaphylaxis responses. These results reveal an unsuspected calcium-independent pathway of antigen receptor regulation, which is accessible to engagement by membrane proteins and on which novel therapeutic approaches to allergic diseases could be based

Temperature and force dependence of nanoscale electron transport via the Cu protein Azurin

The mechanisms of solid-state electron transport (ETp) via a monolayer of immobilized Azurin (Az) was examined by conducting probe atomic force microscopy (CP-AFM), both as function of temperature (248 - 373K) and of applied tip force (6-12 nN). By varying both temperature and force in CP-AFM, we find that the ETp mechanism can alter with a change in the force applied via the tip to the proteins. As the applied force increases, ETp via Az changes from temperature-independent to thermally activated at high temperatures. This is in contrast to the Cu-depleted form of Az (apo-Az), where increasing the applied force causes only small quantitative effects, that fit with a decrease in electrode spacing. At low force ETp via holo-Az is temperature-independent and thermally activated via apo-Az. This observation agrees with macroscopic-scale measurements, thus confirming that the difference in ETp dependence on temperature between holo- and apo-Az is an inherent one that may reflect a difference in rigidity between the two forms. An important implication of these results, which depend on CP-AFM measurements over a significant temperature range, is that for ETp measurements on floppy systems, such as proteins, the stress applied to the sample should be kept constant or, at least controlled during measurement.Comment: 24 pages, 6 figures, plus Supporting Information with 4 pages and 2 figure

Strength of Hydrogen Bond Network Takes Crucial Roles in the Dissociation Process of Inhibitors from the HIV-1 Protease Binding Pocket

To understand the underlying mechanisms of significant differences in dissociation rate constant among different inhibitors for HIV-1 protease, we performed steered molecular dynamics (SMD) simulations to analyze the entire dissociation processes of inhibitors from the binding pocket of protease at atomistic details. We found that the strength of hydrogen bond network between inhibitor and the protease takes crucial roles in the dissociation process. We showed that the hydrogen bond network in the cyclic urea inhibitors AHA001/XK263 is less stable than that of the approved inhibitor ABT538 because of their large differences in the structures of the networks. In the cyclic urea inhibitor bound complex, the hydrogen bonds often distribute at the flap tips and the active site. In contrast, there are additional accessorial hydrogen bonds formed at the lateral sides of the flaps and the active site in the ABT538 bound complex, which take crucial roles in stabilizing the hydrogen bond network. In addition, the water molecule W301 also plays important roles in stabilizing the hydrogen bond network through its flexible movement by acting as a collision buffer and helping the rebinding of hydrogen bonds at the flap tips. Because of its high stability, the hydrogen bond network of ABT538 complex can work together with the hydrophobic clusters to resist the dissociation, resulting in much lower dissociation rate constant than those of cyclic urea inhibitor complexes. This study may provide useful guidelines for design of novel potent inhibitors with optimized interactions