B51D-01 08:00h
Biogeoscience from a Metallomic and Proteomic Perspective
In the wake of the genomics revolution, life scientists are expanding their focus from the genome to the "proteome" - the assemblage of all proteins in a cell - and the "metallome" - the distribution of inorganic species in a cell. The proteome and metallome are tightly connected because proteins and protein products are intimately involved in the transport and homeostasis of inorganic elements, and because many enzymes depend on inorganic elements for catalytic activity. Together, they are at the heart of metabolic function. Unlike the relatively static genome, the proteome and metallome are extremely dynamic, changing rapidly in response to environmental cues. They are substantially more complex than the genome; for example, in humans, some 30,000 genes code for approximately 500,000 proteins. Metaphorically, the proteome and metallome constitute the complex, dynamic "language" by which the genome and the environment communicate. Therefore biogeochemists, like life scientists, are moving beyond a strictly genomic perspective. Research guided by proteomic and metallomic perspectives and methodologies should provide new insights into the connections between life and the inorganic Earth in modern environments, and the evolution of these connections through time. For example, biogeochemical research in modern environments, such as Yellowstone hot springs, is hindered by the gap between genomic determinations of metabolic potential in ecosystems and geochemical characterizations of the energetic boundary conditions faced by these ecosystems; genomics tells us "who is there" and geochemistry tells us "what they might be doing", but neither genomics nor geochemistry easily provide quantitative information about which metabolisms are actually active or a framework for understanding why ecosystems do not fully exploit the energy available in their surroundings. Such questions are fundamentally kinetic rather than thermodynamic and therefore demand that we characterize and understand the proteins and inorganic elements used by organisms to catalyze reactions and capture energy from their surroundings. Similar challenges are faced when attempting to map the evolutionary relationships inferred from phylogenetic analyses of genomes to ecological histories determined by geochemists and paleobiologists - for example, ongoing efforts to understand the evolutionary history of eukaryotes and metazoa - because the driving forces for the evolution and ecological radiation of organisms lie at the intersection of metabolism and environment, and hence in the gap between genomes and geochemistry. Future progress in understanding the biogeochemistry of modern and ancient environments will be spurred by integrating proteomic and metallomic methods and perspectives.
B51D-02 INVITED 08:15h
Microbial Metallomics: A Bioinorganic Perspective
The term "metallome", as described by R.J.P. Williams is synonymous with the instantaneously determined concentration of all inorganic species within a cellular system [1]. Here, we refine this definition to specify that metallomics should reflect not only the overall content of inorganic species in cells, but the chemical identity of such species (i.e., in both ligated and unligated states) a well as their location. Of particular interest to the metallobiochemist are the roles inorganic species play in the up- and down-regulation of protein expression in cells. Thus, we consider that a fully realized "metallome" will reflect the biochemical pathways associated with metal ion speciation and localization within cells. Here we will describe currently proposed strategy and methodology that is used by the Elliott Lab of Boston University to capture snapshots of the proteomic and metallomic landscape of a variety of microbial systems. Two such systems will be described in some detail. The first is Methylococcus capsulatus (Bath), a methanotrophic organism that is known to undergo dramatic morphological changes upon the introduction of varying concentrations of Fe and Cu in the growth medium. In particular, the introduction of increasing concentrations of copper induces the expression of extensive intracytosolic membranes [2]. The Fe:Cu ratio also controls the expression of gene transcripts for soluble and particulate methane monooxygenase enzymes. This provides a unique opportunity to explore metal sensing, uptake, speciation and localization in methanotrophs. A developing model of protein expression and metal input will be discussed. Specific metallomic experiments regarding Shewanella oneidensis will also be discussed, in the context of an ongoing effort to understand how this organism can utilize a dazzling array of electron acceptors, including Fe(III), Mn(IV), nitrite, DMSO, TMAO, and fumarate. [1] R.J.P. Williams, Coord. Chem. Rev., 216:583-595 (2001). [2] C.A. Brantner, et al. Arch. Microbiol. 178: 59-64 (2002).
B51D-03 08:30h
Chemical potential constraints on the composition and subcellular localization of proteins
The distribution and speciation of the metallome in organisms is amenable to study using thermodynamic calculations that take into account the chemical potentials obtaining in living cells. In particular, subcellular spatial gradients of the negative logarithms of the activities of the electron and proton (pe and pH, respectively) strongly influence the speciation of aqueous metals and other inorganic species, as well as aqueous organic and biomacromolecular species. Although pe-pH diagrams are commonly used to describe speciation in inorganic aqueous systems, they have not been applied to assess and quantify the relative stabilities of biomacromolecules in living organisms. Nevertheless, there is much to be gained by doing so. The purpose of the present communication is to demonstrate this by generating pe-pH and other equilibrium activity diagrams for proteins in the system C-H-N-O-S. The relative abundances of amino acid residues in the proteins considered are representative of proteins found in different subcellular locations. For example, the boundaries of the stability fields for extracellular, cytoplasmic, and nuclear proteins can be assessed and portrayed on pe-pH diagrams. By overlaying pe-pH diagrams for proteins with those for metals, one can predict the oxidation states of metals compatible with the proteins found in the different subcellular locations. The standard molal thermodynamic properties of these proteins can be estimated from group additivity algorithms that include provision for protein ionization as a function of solution pH. The temperature and pressure dependence of these properties can be computed with the aid of the revised HKF equations of state. Because quantifying the relative stabilities of proteins is a multidimensional problem, a Gibbs free energy minimization software package was used to carry out a plethora of computer experiments for specified temperatures, pressures, and bulk compositions. Plotting the results of the Gibbs free energy minimization computer experiments on pe-pH diagram results in relative stability fields for extracellular, cytoplasmic, and nuclear proteins at ambient as well as hydrothermal temperatures and pressures. Unlike the lines of equal activity for inorganic species with constant charge, the boundaries of the metastability fields of proteins are curved because the proteins ionization states vary with pH. Reconnaisance calculations indicate that the calculated ranges of pe and pH for these locations compare favorably with reported physiological values at 25 $^\circ$C and 1 bar. The results of this research represent a first step toward quantifying the relative metastabilities of proteins and their interaction with each other in living cells.
B51D-04 08:45h
The interplay of pH and CO$_{2}$ on intracellular metal content in {\it Thalassiosira weissflogii}
How does the metal composition of phytoplankton relate to the metal composition of their ambient environment? Are there other aspects of water chemistry or growth conditions that substantially influence the overall metal composition of living cells? How does the magnitude of the resulting variability compare to differences in the metal contents among species and major phytoplankton taxonomic groups? To address these questions, we will present a study of how pH and CO$_{2}$ (aq) interplay on the uptake of bioactive trace metals Fe, Mn, Cu, Zn, Co, Ni, Zn, Cd and Mo, adding to the more well documented influences of absolute and relative metal availability. First we compare intracellular metal content in {\it Thalassiosira weissflogii} (Tw) grown in modified Aquil media with high and low Zn and Cd and bubbled with different CO$_{2}$-air mixtures. Then we examine the temporal variations in intracellular metal content of Tw during metal buffered batch culture without pH and pCO$_{2}$ control. In total, we argue that the metal composition of a diatom is dependent on multiple interacting aspects of the growth conditions. The links and disconnects between Fe, Mn and Cu content and photosynthetic efficiency, and between Zn, Cd and Co content and carbon acquisition enzymes will be discussed.
B51D-05 INVITED 09:00h
New Tools & Techniques for the Metallomics Revolution
The metallome has been defined as the complete complement of metals and metal moieties in a biological cell, tissue, or system. This definition is akin to that of the genome (genes), proteome (proteins), and metabolome (metabolites). Metallomics accordingly is the study of metals and metal species, and their interactions, transformations, and functions in biological systems. While traditional bioinorganic chemistry has focused on the role and interactions of a single (or few) metals in a protein or enzyme system, metallomics purports to study global, multi-element interactions and relationships. The metallomics challenges for analytical chemistry and biochemical characterization are significant. This paper will discuss these challenges and the emergent techniques and tools that are being developed to address them. Mass spectrometry will play an important and pivotal role. Two approaches are currently being developed in the authors' laboratories. At Pacific Northwest National Laboratory, an extremely high-resolution approach using Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICRMS) is under development. At Indiana University, a rapid, dual-reflectron Time-of-Flight mass spectrometry (TOFMS) technique is being developed. Both approaches rely on dual inductively coupled plasma (ICP) and electrospray ionization (ESI) sources for elemental and biomolecular ion generation. The initial development of these techniques, and their potential application to systems biology and environmental characterization, will be discussed.
B51D-06 09:15h
Simultaneous Fe, S and P Speciation Using HPLC=HR-ICPMS
Iron, sulphur and phosphorus-containing biomolecules play important roles in biochemistry and proteomics and are part of the metallome of an organism. Analysis of HPLC peaks by ICPMS for these metals is a difficult analytical challenge because of polyatomic interferences: carbon, oxygen and nitrogen based interferences (NO+, NOH+, COH+, H3CO+, O2+) are the principal limitation in P and S detection and ArO+ is the principal interference on Fe. Polyatomic interferences formed by the elements in the different mobile phases can be separated from P and S in the molecules of interest, allowing the use of gradient elution. With high resolution ICPMS (HR-ICPMS), P and S can be completely resolved from these interferences even in 100 % acetonitrile. Protein phosphorylation can be determined accurately by HR-ICPMS, either on HPLC peaks or by laser ablation of gel electrophoresis spots. The high power magnet field regulator of the Finnigan ELEMENT2 enables rapid mass scanning, e.g. a duty cycle of 99% is obtained for the simultaneous detection of P and S. In order to show the potential of HR-ICPMS for metallomics, mixtures of deoxyribonucleotides (dAMP, dTMP, dGMP, dCMP) and peptides have been separated by HPLC with simultaneous on-line quantitation of P and S, and hemoglobin and myoglobin were analyzed for Fe/S ratios and quantitative determination of Fe.
B51D-07 INVITED 09:30h
Metals as Selective Agents in the Evolution of Metabolic Pathways
The evolution of coupled redox reactions which form the basic metabolic pathways on Earth was driven by the availability but not the catalytic efficiency of transition metals. Regardless whether the metals are contained within secondary prosthetic group (e.g., porphyrins), their catalytic selectivity is determined by the protein moieties. In complex metabolic pathways, such as photosynthesis, nitrogen fixation, and aerobic respiration, the protein moieties cooperatively interact and the protein-protein interactions constrain the rate of evolution of the ensemble. The protein-protein interactions were, in turn, constrained by the metal or prosthetic group binding and chemistry. Hence, metal cofactors became a major agent that set the tempo of evolution in many metabolic pathways - the result being conservation of pathways, often at the cost of efficiency.