Isolation of Acidic, Basic and Neutral Metalloproteins by QPNC-PAGE

A standard protocol for isolating metalloproteins in complex biological samples is presented using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE

Kinetics of the reduction of metalloproteins by chromous ion

The reduction of Cu(330) in Rhus vernicifera laccase by chromous ion is 30% faster than reduction of Cu(614) at room temperature [pH 4.8, µ = 0.1 (NaCl)], and two parallel first-order paths, attributed to heterogeneity of the protein, are observed at both wavelengths. The reactions of stellacyanin, spinach and French-bean plastocyanins, and cytochrome c with chromous ion under similar conditions are faster than that with laccase by factors of 102 to 104, and are first order in protein concentration. Comparison of rates and activation parameters for the reduction of "blue" copper in laccase, stellacyanin, and the two plastocyanins indicates that reduction of the Cu(614) site in laccase may occur by intramolecular electron transfer from one of the Cu(330) sites. Our value of ΔH (17.4 kcal/mol) for the chromous ion reduction of cytochrome c is consistent with a mechanism in which major conformational changes in the protein must accompany electron transfer

Quantitative Preparative Native Continuous Polyacrylamide Gel Electrophoresis (QPNC-PAGE)

QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point. This variant of gel electrophoresis is used by biologists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures

Great metalloclusters in enzymology

Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N2, CO, and H2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the copper-sulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined, and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis

The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia

Background: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb2+, Al+3, SeO2, Cu2+, AsO4, and Zn2+. With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. Results: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. Conclusions: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils

Ab initio analysis of the x-ray absorption spectrum of the myoglobin-carbon monoxide complex: Structure and vibrations

We present a comparison between Fe K-edge x-ray absorption spectra of carbonmonoxy-myoglobin and its simulation based on density-functional theory determination of the structure and vibrations and spectral simulation with multiple-scattering theory. An excellent comparison is obtained for the main part of the molecular structure without any structural fitting parameters. The geometry of the CO ligand is reliably determined using a synergic approach to data analysis. The methodology underlying this approach is expected to be especially useful in similar situations in which high-resolution data for structure and vibrations are available.Comment: 13 pages, 3 figure

NMR studies of metalloproteins

Metalloproteins represent a large share of the proteomes, with the intrinsic metal ions providing catalytic, regulatory, and structural roles critical to protein functions. Structural characterization of metalloproteins and identification of metal coordination features including numbers and types of ligands and metal-ligand geometry, and mapping the structural and dynamic changes upon metal binding are significant for understanding biological functions of metalloproteins. NMR spectroscopy has long been used as an invaluable tool for structure and dynamic studies of macromolecules. Here we focus on the application of NMR spectroscopy in characterization of metalloproteins, including structural studies and identification of metal coordination spheres by hetero-/homo-nuclear metal NMR spectroscopy. Paramagnetic NMR as well as (13)C directly detected protonless NMR spectroscopy will also be addressed for application to paramagnetic metalloproteins. Moreover, these techniques offer great potential for studies of other non-metal binding macromolecules.postprin

Accurate quantification of selenoproteins in human plasma/serum by isotope dilution ICP-MS : focus on selenoprotein P

Acknowledgements The research leading to these results was funded by the EMRP Joint Research Project “Metrology for metalloproteins” (HLT-05 2012). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.Peer reviewedPostprin

Conflicting interests in the pathogen-host tug of war : fungal micronutrient scavenging versus mammalian nutritional immunity

Funding: The authors are supported by the European Research Council (STRIFE project funded on grant number ERC-2009-AdG-249793, http://erc.europa.eu). AJPB is also supported by the Wellcome Trust (grant numbers 080088, 097377, www.wellcome.ac.uk) and the UK Biotechnology and Biological Sciences Research Council (grant number BB/F00513X/1, www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Toward quantum simulations of biological information flow

Recent advances in the spectroscopy of biomolecules have highlighted the possibility of quantum coherence playing an active role in biological energy transport. The revelation that quantum coherence can survive in the hot and wet environment of biology has generated a lively debate across both the physics and biology communities. In particular, it remains unclear to what extent non-trivial quantum effects are utilised in biology and what advantage, if any, they afford. We propose an analogue quantum simulator, based on currently available techniques in ultra-cold atom physics, to study a model of energy and electron transport based on the Holstein Hamiltonian By simulating the salient aspects of a biological system in a tunable laboratory setup, we hope to gain insight into the validity of several theoretical models of biological quantum transport in a variety of relevant parameter regimes.Comment: 8 Pages, 2 Figures, Non-technical contributing article for the Interface Focus Theme Issue `Computability and the Turning centenary'. Interface Focus http://rsfs.royalsocietypublishing.org/content/early/2012/03/22/rsfs.2011.0109.shor