Quantitative Proteomics Strategies To Explore The Saccharomyces cerevisiae Proteome

Quantitative proteomics aims at not just identifying, but accurately quantifying the cellular proteome, and while technological advances towards accurate and reliable quantification of proteins is advancing, this alone does not provide an accurate picture of a proteins role within a cell. There is a far greater level of functionality in the cellular environment than there are protein coding genes in the genome, owing partly to the organisation of individual proteins into larger assemblies. A single protein can form interactions with, potentially, a large number of other proteins, leading to a variety of different protein complexes, and subunits can move, break apart or combine depending on cellular conditions. This complex organisation is despite the normal proteomics strategies employing a destructive process, breaking protein structure down to the peptide level. Further difficulties in mapping the cellular proteome arise from the differential expression level of proteins, which in S.cerevisiae can span up to 5 orders of magnitude. This poses problems for the quantification of less abundant proteins in the cell, which can be masked by the more concentrated proteins. An attempt is made within this thesis to use quantitative proteomic techniques to build a picture of the S.cerevisiae cellular proteome. For the analysis of S.cerevisiae protein complexes ion exchange chromatography has been used to separate the cellular proteome into discrete fractions, each containing a different array of protein complexes. The aim here was to analyse the individual subunits of these complexes by LC-MS, with the use of label free quantification strategies. This enables the high throughput identification and quantification of 1800 proteins along with their potential interaction partners. However, for some of the complexes presented here the accuracy of the label free quantification is called into question, as complex subunits known to be equimolar are identified at different concentrations. In order to assess the accuracy of the label free data QconCATs were also designed to analyse the subunits of some complexes by label mediated quantification. In addition, an attempt is made to access proteins from the entire dynamic range of the cellular proteome using equaliser bead technology. This method uses a library of hexapeptide ligands bound to porous beads to bind, theoretically, every protein present in the sample to equal amounts. The beads are used here to bring up the less abundant proteins in the sample, while simultaneously reducing the amount of the abundant proteins. While this goal is achieved, it is also evident that certain proteins are able to bind the beads to a much larger extent than others, so rather than reducing the dynamic range of proteins identified, there is more of a shift in the dynamics, with previously mid-range proteins becoming highly abundant in the data presented here

Asymmetric Proteome Equalization of the Skeletal Muscle Proteome Using a Combinatorial Hexapeptide Library

Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method

Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens

© 2015 International Union of Biochemistry and Molecular Biology, Inc. This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron–sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645±16s−1. The optimal conditions for catalysis are in the pH range 6.5–8.0 and at the temperature of 50°C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox-CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox; upon reduction with H2, vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox-CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster

Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards

Medical Biochemistr

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/K009753/1, BB/K010220/1, BB/K009885/1, and BB/K00929X/1), the Engineering and Physical Sciences Research Council (EP/M001989/1, PhD studentship 1307196 to E.V.A.), a Royal Society Leverhulme Trust Senior Research Fellowship to J.N.B., the Christian Doppler Research Association, and OMV group

The bioinspired construction of an ordered carbon nitride array for photocatalytic mediated enzymatic reduction

A carbon nitride array (CNA) material has been constructed using a sacrificial diatom template. A regular carbon nitride nanorod array could be replicated from the periodic and regular nanochannel array of the template. The directional charge transport properties and high light harvesting capability of the CNA gives much better performance in splitting water to give hydrogen than its bulk counterpart. Furthermore, by combining with a rhodium complex as a mediator, the nicotinamide adenine dinucleotide (NADH) cofactor of many enzymes could be photocatalytically regenerated by the CNA. The rate of the in situ NADH regeneration is high enough to reverse the biological pathway of the three dehydrogenase enzymes, which then leads to the sustainable conversion of formaldehyde to methanol and also the reduction of carbon dioxide into methanol

Photosensitised Multiheme Cytochromes as Light‐Driven Molecular Wires and Resistors

Multiheme cytochromes possess closely packed redox‐active hemes arranged as chains spanning the tertiary structure. Here we describe five variants of a representative multiheme cytochrome engineered as biohybrid phototransducers for converting light into electricity. Each variant possesses a single Cys sulfhydryl group near a terminus of the heme chain, and this was efficiently labelled with a RuII(2,2′‐bipyridine)3 photosensitiser. When irradiated in the presence of a sacrificial electron donor (SED) the proteins exhibited different types of behaviour. Certain proteins were rapidly and fully reduced. Other proteins were rapidly semi‐reduced but resisted complete photoreduction. These findings reveal that photosensitised multiheme cytochromes can be engineered to act as resistors, with intrinsic regulation of light‐driven electron accumulation, and also as molecular wires with essentially unhindered photoreduction. It is proposed that the observed behaviour arises from interplay between the site of electron injection and the distribution of heme reduction potentials along the heme chain

Synthetic strategies to nanostructured photocatalysts for CO2 reduction to solar fuels and chemicals

Artificial photosynthesis represents one of the great scientific challenges of the 21st century, offering the possibility of clean energy through water photolysis and renewable chemicals through CO2 utilisation as a sustainable feedstock. Catalysis will undoubtedly play a key role in delivering technologies able to meet these goals, mediating solar energy via excited generate charge carriers to selectively activate molecular bonds under ambient conditions. This review describes recent synthetic approaches adopted to engineer nanostructured photocatalytic materials for efficient light harnessing, charge separation and the photoreduction of CO2 to higher hydrocarbons such as methane, methanol and even olefins

Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes

Photobiocatalysts are constituted by a semiconductor with or without a light harvester that activates an enzyme. A logical source of inspiration for the development of photobiocatalysts has been natural photosynthetic centers. In photobiocatalysis, the coupling of the semiconductor and the enzyme frequently requires a natural cofactor and a relay transferring charge carriers from the semiconductor. The most widely studied photobiocatalysts so far make use of conduction band electrons of excited semiconductors to promote enzymatic reductions mediated by NAD(+)/NADH and an electron relay. The present review presents the state of the art in the field and has been organized based on the semiconductor and the reaction type including oxidations, hydrogen generation, and CO2 reduction. The possibility of direct enzyme activation by the semiconductor and the influence of the nature of mediator are also discussed as well as the use of mimics of the enzyme active center in combination with the semiconductor. The final section summarizes the state of the art of photobiocatalysis and comments on our view on future developments of the field.Financial support by the Spanish Ministry of Economy and Competitiveness (Severo Ochoa and CTQ2012-32315) is gratefully acknowledged. J.A.M.-A. acknowledges the assistance of the CSIC for the award of a Postdoctoral JAE-Doc contract co-financed by the European Social Fund.Maciá Agulló, JA.; Corma Canós, A.; García Gómez, H. (2015). Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes. Chemistry - A European Journal. 21(31):10940-10959. https://doi.org/10.1002/chem.201406437S1094010959213

Methodologies for “Wiring” Redox Proteins/Enzymes to Electrode Surfaces

The immobilization of redox proteins or enzymes onto conductive surfaces has application in the analysis of biological processes, the fabrication of biosensors, and in the development of green technologies and biochemical synthetic approaches. This review evaluates the methods through which redox proteins can be attached to electrode surfaces in a “wired” configuration, that is, one that facilitates direct electron transfer. The feasibility of simple electroactive adsorption onto a range of electrode surfaces is illustrated, with a highlight on the recent advances that have been achieved in biotechnological device construction using carbon materials and metal oxides. The covalent crosslinking strategies commonly used for the modification and biofunctionalization of electrode surfaces are also evaluated. Recent innovations in harnessing chemical biology methods for electrically wiring redox biology to surfaces are emphasized