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Miller, Susan M.

Publications and source records attributed to Miller, Susan M..

8 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Summers_FinalReport_ER64408

Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolic functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report

Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we have devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolic functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation). We have also discovered that acute Hg exposure severely disrupts thiol, iron and redox homeostases, and electrolyte balance (LaVoie, et al., 2015) Thus, we proposed to bring these techniques to bear on the central problem of identifying the cellular proteins involved in bacterial uptake and methylation of mercury and its release from the cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics (Final Report)

Hg is a wide-spread contaminant in the environment and is toxic in all of its various forms. Organomercurials (RHg + ) and mercuric ion (Hg 2+ ) directly inactivate proteins by binding to their cysteine or selenocysteine residues. In our previous collaborative project involving the Summers, Miller and Lipton laboratories, we developed a high-throughput, mass spectrometry-based global proteomics approach using the 7 stable isotopes of Hg to identify proteins from phenylmercury-exposed E. coli cells that form stable PhHg-adducts. The two overall objectives of this subsequent collaborative project were (1) to expand our novel organomercury-proteomics capability for use with inorganic ionic (HgX 2 ) exposure and (2) to use this new capability to identify Hg-protein adducts in methylating and non-methylating Desulfovibrio exposed to inorganic ionic HgX 2 and test whether those identified uniquely in methylating bacteria are involved in the methylation pathway. The specific objectives and accomplishments of the Miller lab in this project included: Development of algorithms for analysis of the Hg-proteomic mass spectrometry data to identify and quantify Hg-adducted peptides and other trends in the data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Defining the Molecular-Cellular-Field Continuum of Mercury Detoxification

Hg is of special interest to DOE due to past use at the Oak Ridge Reservation (ORR). Its facile redox [Hg 2+/0 ] chemistry, bonding to carbon [e.g. MeHg + ] and unique physical properties [e.g., Hg 0 volatility] underlie a complex global Hg cycle involving biotic and abiotic chemical and physical transport and transformations in soils, sediments, waterways and the atmosphere. Facultative and anaerobic bacteria make MeHg + , which is neurotoxic to wildlife and humans. Sustainable stewardship requires eliminating both MeHg + and even more toxic Hg 2+ , which is also the substrate for methylation. The proteins encoded by the mer locus in aerobic and facultative mercury resistant (HgR) bacteria convert soil or waterborne Hg 2+ or MeHg + to less toxic, gaseous Hg 0 . HgR microbes live in highly Hg-contaminated sites and depress MeHg + formation >500-fold in such zones. So, enhancing the capacity of natural HgR microbes to remove Hg 2+ /MeHg + from wetlands and waterways is a logical component of contaminated site stewardship. To apply enhancement in the field requires knowing how the HgR pathway works including the metabolic demands it makes on the cell, i.e., the entire cell is the relevant catalytic unit. HgR loci occur in metabolically diverse bacteria and unique mer-host co-evolution has been found. In this project we extended our previous studies of mer enzymes in γ-proteobacteria, which are abundant in high Hg areas of the ORR to include studies of mer enzymes from HgR α-proteobacteria and HgR actinobacteria, which also increase in the high Hg regions of the ORR. Specifically, we (1) examined interactions between structural components of MerA and MerB enzymes from γ-proteobacteria, (2) investigated effects of mutations on kinetic efficiency of Hg 2+ reduction by γ-proteobacterial MerA, (3) cloned and performed initial characterization of MerA and MerB enzymes from Streptomyces lividans, an actinobacterium, (4) cloned and performed initial characterization of a fused MerB-MerA protein from Ochrobactrum anthropi, an α-proteobacterium, (5) investigate the extent of Hg isotope fractionation that occurs with purified γ-proteobacterial MerA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Structure and dynamics of a compact state of a multidomain protein, the mercuric ion reductase

Here, the functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Molecular Mechanisms of Bacterial Mercury Transformation (Final Report)

The bacterial mercury resistance (mer) operon functions in Hg biogeochemistry and bioremediation by converting reactive inorganic Hg(II) and organic [RHg(II)] 1+ mercurials to relatively inert monoatomic mercury vapor, Hg(0). Its genes regulate operon expression (MerR, MerD, MerOP), import Hg(II) (MerT, MerP, and MerC), and demethylate (MerB) and reduce (MerA) mercurials. We focus on how these components interact with each other and with the host cell to allow cells to survive and detoxify Hg compounds. Understanding how this ubiquitous detoxification system fits into the biology and ecology of its bacterial host is essential to guide interventions that support and enhance Hg remediation. In the current overall project we focused on two aspects of this system: (1) investigations of the energetics of Hg(II)-ligand binding interactions, and (2) both experimental and computational approaches to investigating the molecular mechanisms of Hg(II) acquisition by MerA and intramolecular transfer of Hg(II) prior to reduction within the MerA enzyme active site. Computational work was led by Prof. Jeremy Smith and took place at the University of Tennessee, while experimental work on MerA was led by Prof. Susan Miller and took place at the University of California San Francisco.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2012

Identifying Biomarkers and Mechanisms of Toxic Metal Stress with Global Proteomics

Hg is a wide-spread contaminant in the environment and is toxic in all of its various forms. Data suggest that RHg + and Hg 2+ are toxic in two ways. At low levels, Hg species appear to disrupt membrane-bound respiration causing a burst of reactive oxygen species (ROS) that further damage the cell. At higher Hg concentrations, RHg + and Hg 2+ may form adducts with cysteine- and selenocysteine-containing proteins in all cellular compartments resulting in their inactivation. Although these mechanisms for toxicity are generally accepted, the most sensitive targets associated with these mechanisms are not well understood. In this collaborative project involving three laboratories at three institutions, the overall goal was to develop of a mass spectrometry-based global proteomics methodology that could be used to identify Hg-adducted (and ideally, ROS-damaged) proteins in order to address these types of questions. The two objectives of this overall collaborative project were (1) to identify, quantify, and compare ROS- and Hg-damaged proteins in cells treated with various Hg species and concentrations to test this model for two mechanisms of Hg toxicity, and (2) to define the cellular roles of the ubiquitous bacterial mercury resistance (mer) locus with regards to how the proteins of this pathway interact to protect other cell proteins from Hg damage. The specific objectives and accomplishments of the Miller lab in this project included: (1) Development of algorithms for analysis of the Hg-proteomic mass spectrometry data to identify mercury adducted peptides and other trends in the data. (2) Investigation of the role of mer operon proteins in scavenging Hg(II) from other mer pathway proteins as a means of protecting cellular proteins from damage.

59 BASIC BIOLOGICAL SCIENCES↗