pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

Green fluorescent protein based pH indicators for in vivo use: a review

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.     

A study of high transverse energy interactions of 150 and 300 GeV pions and protons on a hydrogen target using a large acceptance calorimeter

Interactions of 150 GeV pions and 300 GeV pions and protons on hydrogen which result in final states with large transverse energy in the central rapidity region ?0.88 < y < 0.67 were studied with a calorimeter trigger. The dependence of the cross section on the azimuthal acceptance of the trigger, on the beam energy and on the incident particle type and the jet properties of the events were investigated. No dominant two-jet structure was found. Nevertheless, the parton spectrum in the colliding hadrons appears to be relevant for large transverse energy reactions.     

Preparations, structures, and electrochemical studies of aryldiazene complexes of rhenium: syntheses of the first heterobinuclear and heterotrinuclear derivatives with bis(diazene) or bis(diazenido) bridging ligands

The mono- and binuclear aryldiazene complexes [Re(C6H5N=NH)(CO)5-nPn]BY4 (1-5) and [(Re(CO)5-nPn)2-(mu-HN=NAr-ArN=NH)](BY4)2 (6-12) [P = P(OEt)3, PPh(OEt)2, PPh2OEt; n = 1-4; Ar-Ar = 4,4'-C6H4-C6H4, 4,4'-(2-CH3)C6H3-C6H3(2-CH3), 4,4'-C6H4-CH2-C6H4; Y = F, Ph) were prepared by reacting the hydride species ReH(CO)5-nPn with the appropriate mono- and bis(aryldiazonium) cations. These compounds, as well as other prepared compounds, were characterized spectroscopically (IR; 1H, 31P, 13C, and 15N NMR data), and 1a was also characterized by an X-ray crystal structure determination. [Re(C6H5N=NH)(CO)(P(OEt)3)4]BPh4 (1a) crystallizes in space group P1 with a = 15.380(5) A, b = 13.037(5) A, c = 16.649(5) A, alpha = 90.33(5) degrees, beta = 91.2(1) degrees, gamma = 89.71(9) degrees, and Z = 2. The "diazene-diazonium" complexes [M(CO)3P2(HN=NAr-ArN identical to N)](BF4)2 (13-15, 17) [M = Re, Mn; P = PPh2OEt, PPh2OMe, PPh3; Ar-Ar = 4,4'-C6H4-C6H4, 4,4'-C6H4-CH2-C6H4] and [Re(CO)4(PPh2OEt)(4,4'-HN=NC6H4-C6H4N identical to N)](BF4)2 (16b) were synthesized by allowing the hydrides MH(CO)3P2 or ReH(CO)4P to react with equimolar amounts of bis(aryldiazonium) cations under appropriate conditions. Reactions of diazene-diazonium complexes 13-17 with the metal hydrides M2H2P'4 and M2'H(CO)5-nP"n afforded the heterobinuclear bis(aryldiazene) derivatives [M1(CO)3P2(mu-HN=NAr-ArN=NH)M2HP'4](BPh4)2 (ReFe, ReRu, ReOs, MnRu, MnOs) and [M1(CO)3P2(mu-HN=NAr-ArN=NH)M2'(CO)5-nP"n](BPh4)2 (ReMn, MnRe) [M1 = Re, Mn; M2 = Fe, Ru, Os; M2' = Mn, Re; P = PPh2OEt, PPh2OMe; P',P" = P(OEt)3, PPh(OEt)2; Ar-Ar = 4,4'-C6H4-C6H4, 4,4'-C6H4-CH2-C6H4; n = 1, 2]. The heterotrinuclear complexes [Re(CO)3(PPh2OEt)2(mu-4,4'-HN=NC6H4-C6H4N=NH)M(P(OEt)3)4(mu-4,4'-HN=NC6H4- C6H4N=NH)Mn(CO)3(PPh2OEt)2](BPh4)4 (M = Ru, Os) (ReRuMn, ReOsMn) were obtained by reacting the heterobinuclear complexes ReRu and ReOs with the appropriate diazene-diazonium cations. The heterobinuclear complex with a bis(aryldiazenido) bridging ligand [Mn(CO)2(PPh2OEt)2(mu-4,4'-N2C6H4-C6H4N2)Fe(P(OEt)3)4]BPh4 (MnFe) was prepared by deprotonating the bis(aryldiazene) compound [Mn(CO)3(PPh2OEt)2(mu-4,4'-HN=NC6H4-C6H4N=NH)Fe(4- CH3C6H4CN)(P(OEt)3)4](BPh4)3. Finally, the binuclear compound [Re(CO)3(PPh2OEt)2(mu-4,4'-HN=NC6H4-C6H4N2)Fe(CO)2(P(OPh)3)2](BPh4)2 (ReFe) containing a diazene-diazenido bridging ligand was prepared by reacting [Re(CO)3(PPh2OEt)2(4,4'-HN=NC6H4-C6H4N identical to N)]+ with the FeH2(CO)2(P(OPh)3)2 hydride derivative. The electrochemical reduction of mono- and binuclear aryldiazene complexes of both rhenium (1-12) and the manganese, as well as heterobinuclear ReRu and MnRu complexes, was studied by means of cyclic voltammetry and digital simulation techniques. The electrochemical oxidation of the mono- and binuclear aryldiazenido compounds Mn(C6H5N2)(CO)2P2 and (Mn(CO)2P2)2(mu-4,4'-N2C6H4-C6H4N2) (P = PPh2OEt) was also examined. Electrochemical data show that, for binuclear compounds, the diazene bridging unit allows delocalization of electrons between the two different redox centers of the same molecule, whereas the two metal centers behave independently in the presence of the diazenido bridging unit.     

Dynamic condensation of water at crack tips in fused silica glass

Water molecules play a fundamental role in the physics of slow crack propagation in glasses. It is commonly understood that, during stress-corrosion, water molecules that move in the crack cavity effectively reduce the bond strength at the strained crack tip and, thus, support crack propagation. Yet the details of the environmental condition at the crack tip in moist air are not well determined. In a previous work, we reported direct evidence of the presence of a 100 nm long liquid condensate at the crack tip in fused silica glass during very slow crack propagation (10^?9–10^?10 m/s). These observations are based on in situ AFM phase imaging techniques applied on DCDC glass specimens in controlled atmosphere. Here, we discuss the physical origin of the AFM phase contrast between the liquid condensate and the glass surface in relation to tip-sample adhesion induced by capillary bridges. We then report new experimental data on the water condensation length increase with relative humidity in the atmosphere. The measured condensation lengths were much larger than what predicted using the Kelvin equation and expected geometry of the crack tip.     

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.     

Protonation of a histidine copper ligand in fern plastocyanin

Plastocyanin is a small blue copper protein that shuttles electrons as part of the photosynthetic redox chain. Its redox behavior is changed at low pH as a result of protonation of the solvent-exposed copper-coordinating histidine. Protonation and subsequent redox inactivation could have a role in the down regulation of photosynthesis. As opposed to plastocyanin from other sources, in fern plastocyanin His90 protonation at low pH has been reported not to occur. Two possible reasons for that have been proposed: pi-pi stacking between Phe12 and His90 and lack of a hydrogen bond with the backbone oxygen of Gly36. We have produced this fern plastocyanin recombinantly and examined the properties of wild-type protein and mutants Phe12Leu, Gly36Pro, and the double mutant with NMR spectroscopy, X-ray crystallography, and cyclic voltammetry. The results demonstrate that, contrary to earlier reports, protonation of His90 in the wild-type protein does occur in solution with a pKa of 4.4 (+/-0.1). Neither the single mutants nor the double mutant exhibit a change in protonation behavior, indicating that the suggested interactions have no influence. The crystal structure at low pH of the Gly36Pro variant does not show His90 protonation, similar to what was found for the wild-type protein. The structure suggests that movement of the imidazole ring is hindered by crystal contacts. This study illustrates a significant difference between results obtained in solution by NMR and by crystallography.     

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.     

Diastereo- and enantioselective catalytic vinylogous Mukaiyama-Mannich reactions of pyrrole-based silyl dienolates with alkyl-substituted aldehydes

A reliable, catalytic asymmetric vinylogous Mukaiyama-Mannich reaction of pyrrole-based silyl dienolates is introduced that is particularly apt for alkyl- and α-alkoxyalkyl-substituted aldehydes. The reaction course is effectively orchestrated by the Hoveyda-Snapper amino acid-based chiral ligand/silver(I) catalyst combination to produce valuable vicinal diamino carbonyl compounds in high yields, with virtually complete γ-site- and anti-selectivity and significant catalyst-to-product chirality transfer. The utility of the Mannich products can be seen in the synthesis of an unprecedented perhydrofuro[3,2-b]pyrrolone product, an aza-analogue of naturally occurring (+)-goniofufurone.