A metal carbonyl-protein needle composite designed for intracellular CO delivery to modulate NF-κB activity.

Carbon monoxide (CO) has been recognized as a messenger for signal transduction in living cells and tissues. For intracellular CO delivery, several metal carbonyl complexes have been used as CO-releasing molecules (CO-RMs). To improve the properties of CO-RMs, such as the stability and the CO release rate, ligands and carriers of the metal complexes have been exploited. Here we report the development of an efficient intracellular CO delivery system using a protein scaffold. We used a protein needle reconstructed from gene product 5 of bacteriophage T4, which has high cellular permeability and stability. When ruthenium carbonyl complexes are conjugated to the needle using a His-tag triad at the C-terminus, the resulting composite has a significantly higher cellular uptake efficiency of Ru carbonyl and a 12-fold prolonged CO release rate relative to Ru(CO)3Cl(glycinate), a widely used CO-RM. We demonstrate that CO delivered by the composite activates the transcriptional factor nuclear factor-kappaB (NF-κB), which in turn leads to significant induction of expression of its target genes, HO1, NQO1, and IL6, through generation of reactive oxygen species (ROS). The signaling pathway is distinct from that of tumor necrosis factor (TNF)-α-induced activation of NF-κB. The protein needle-based CO-RM can be exploited to elucidate the biological functions of CO and used in the development of protein-based organometallic tools for modulation of cellular signaling.

Conductance switching and organization of two structurally related molecular wires on gold.

The self-assembly and electron transfer properties of adsorbed organic molecules are of interest for the construction of miniaturized molecular circuitries. We have investigated with scanning probe microscopy the self-organization of two structurally related molecular wires embedded within a supportive alkanethiol matrix. Our results evidence heterogeneous adsorption patterns of the molecular wires on gold with either incommensurate unit cells driven into assembly by lateral interactions or a dynamic, commensurate distribution on gold, along with formation of distinct 2D phases. We also observed diffusion-based conductance switching for one of the molecular wires, due to its propensity toward weaker lateral interactions and Au-S adatom formation. We have further demonstrated through the use of scanning tunneling spectroscopy differential current-voltage response for each molecular wire, despite their close structural similarity. Such molecular wires embedded in alkanethiol matrix and exhibiting conductance-switching phenomena have the potential to be used for the functionalization of electrodes in bioelectronic devices.

Voltage-controlled fluorescence switching of a single redox protein.

Heterogeneous electron transfer (ET) of the redox protein, wild-type azurin (wt-Az) from Pseudomonas aeruginosa, was monitored at the single-molecule (SM) level by fluorescence resonance energy transfer (FRET), one electron at a time. Azurin molecules were labeled with an organic fluorophore (Cy5), and the FRET-coupling between Cy5 and the redox center (copper) was used to study ET to a semi-transparent, 10nm thin gold electrode in an optical configuration. By using a confocal microscope and a bipotentiostat for control of the electrode potential, the oxidation and reduction processes of individual Az-Cy5 molecules were monitored. In the oxidized state of the redox center of the azurin molecule, the fluorescence emission of the covalently attached Cy5 was largely quenched by FRET ('off'-state), whereas the emission was recovered upon reduction ('on'-state). The work presented here, shows directly controlled single redox switching events of an individual redox protein and its thermodynamic dispersion. We show that the distribution of midpoint potentials (E0) of individual azurin molecules peaks at 45.7±0.5 mV with a full width at half maximum of 15 mV vs saturated calomel electrode (SCE).

Chemically-induced redox switching of a metalloprotein reveals thermodynamic and kinetic heterogeneity, one molecule at a time.

Oxidation (off state) and reduction (on state) of a single azurin molecule is monitored, one electron at a time, which depend on the chemical redox potential. By analysing the fluorescence time traces from individual azurin molecules, reaction kinetics and redox thermodynamics were determined.

Plasma membrane translocation of a protein needle based on a triple-stranded ß-helix motif.

Plasma membrane translocation is challenging due to the barrier of the cell membrane. Contrary to the synthetic cell-penetrating materials, tailed bacteriophages use cell-puncturing protein needles to puncture the cell membranes as an initial step of the DNA injection process. Cell-puncturing protein needles are thought to remain functional in the native phages. In this paper, we found that a bacteriophage T4 derived protein needle of 16 nm length spontaneously translocates through the living cell membrane. The ß-helical protein needle (ß-PN) internalizes into human red blood cells that lack endocytic machinery. By comparing the cellular uptake of ß-PNs with modified surface charge, it is shown that the uptake efficiency is maximum when it has a negative charge corresponding to a zeta potential value of -16 mV. In HeLa cells, uptake of ß-PN incorporates endocytosis independent mechanisms with partial macropinocytosis dependence. The endocytosis dependence of the uptake increases when the surface charges of ß-PNs are modified to positive or negative. Thus, these results suggest that natural DNA injecting machinery can serve as an inspiration to design new class of cell-penetrating materials with a tailored mechanism.

Expanding coordination chemistry from protein to protein assembly.

Bioinorganic chemistry is of growing importance in the fields of nanomaterial science and biotechnology. Coordination of metals by biological systems is a crucial step in intricate enzymatic reactions such as photosynthesis, nitrogen fixation and biomineralization. Although such systems employ protein assemblies as molecular scaffolds, the important roles of protein assemblies in coordination chemistry have not been systematically investigated and characterized. Many researchers are joining the field of bioinorganic chemistry to investigate the inorganic chemistry of protein assemblies. This area is emerging as an important next-generation research field in bioinorganic chemistry. This article reviews recent progress in rational design of protein assemblies in coordination chemistry for integration of catalytic reactions using metal complexes, preparation of mineral biomimetics, and mechanistic investigations of biomineralization processes with protein assemblies. The unique chemical properties of protein assemblies in the form of cages, tubes, and crystals are described in this review.

Effect of polar solvents on the optical properties of water-dispersible thiol-capped cobalt nanoparticles.

We were able to stabilize cobalt nanoparticles dispersible in water by optimizing the synthetic procedure using small polar thiol containing compounds as the capping agents. The nanoparticles were found to be spherical. The optical properties of the cobalt nanoparticles were investigated by monitoring the changes in the surface plasmon resonance (SPR) spectrum in various polar solvents. The extent of solvent dependence of the SPR spectrum was found to be dependent on the nature of the capping agent, the size of the cobalt nanoparticles, as well as the nature of the solvent. The Drude model was applicable for the particles capped with mercaptopropionic acid, while the effect of variations in the free electron density in the particles at different solvents became predominant in the nanoparticles capped with mercaptoethanol. The absorption spectra of the Co nanoparticles were simulated with the help of the classical Mie theory, and the results supported the effect of free electron density due to different capping agents on the spectra of the particles.

Conformational dynamics coupled to protonation equilibrium at the CuA site of Thermus thermophilus: insights into the origin of thermostability.

An extremely slow pH dependent conformational equilibrium between a valence-delocalized and a valence-trapped species of the dinuclear CuA domain of cytochrome c oxidase from Thermus thermophilus has been identified and characterized using UV-visible absorption, circular dichroism, time-resolved fluorescence and electron paramagnetic resonance spectroscopy as well as by stopped-flow kinetic techniques. The results indicated that the nature of this pH dependent conformation change in the CuA domain in the Thermus protein was distinctly different from that observed in the mesophilic analogue from Paracoccus denitrificans and in the engineered CuA domain in azurin. pH jump kinetic studies suggested existence of a fast deprotonation equilibrium followed by slow conformational change in the protein, which is contrary to that observed in the case of the analogous protein from P. denitrificans. Continuous-flow electrospray mass spectral studies on H/D exchange in the TtCuA showed that approximately 75% of the protons are exchanged within the dead-time of the experiment supporting fast proton transfer kinetics in the protein. Analysis of temperature dependence of the kinetics of the conformational transition showed that the rigidity of the protein structure decreases with increase in temperature. The results indicated that though the rate of proton transfer at individual sites in the protein could be very fast, the conformational change that requires simultaneous breaking of several interactions in a segment of the structure might be slow in the thermostable protein.

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus.

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.