Ongoing Projects

Mechanism of iron and sulfur assimilation by microorganisms

Given the substantial scientific efforts that have been directed toward enzymes containing metal sulfide clusters, it is surprising that large gaps of knowledge exist in how cells accumulate the components iron (Fe) and sulfur (S) for metal clusters and how the elements are assembled into protein reactive centers. We have selected two model organisms, the cysteine auxotroph Methanococcus maripaludis and the methanogen Methanosarcina acetivorans to address the most significant of the outstanding questions. Our work is be guided by the central hypothesis that soluble FeSaq molecular clusters released by enzymatic FeS2 reduction are assimilated to meet Fe and S demands for metallocluster biosynthesis in methanogens. This work has applications in biomining of rare metals and development of antimicrobial agents. Collaborators at MSU include Eric Boyd, Joan Broderick, Will Broderick, Jen DuBois, Eric Sheppard, and David Mogk.

Health impacts of dietary change

This area of research is at the intersection of metabolism and health and involves examining dynamic responses to metabolic challenges. We use inflammation assessments, metabolomics, and the analysis of gut microbiota to elucidate the influence of diet and physical activity on health. The overarching goal is to develop effective strategies to lower risk of diseases. Inflammation plays an active part in the development of cardiovascular disease, type 2 diabetes mellitus, cancer, and neurodegenerative diseases such as Alzheimer’s disease. While delaying the onset of chronic diseases relating to inflammation is an important means of enhancing well-being, the onset of these diseases occurs and younger and younger ages as obesity, physical inactivity, stresses, and other factors relating to lifestyle become more prevalent. Current projects include assessing metabolic and inflammatory responses to the controlled consumption of Aronia berries, lentls, cottonseed oil, and grass-feed beef.

Metabolomics and Proteomics

Metabolomics refers to the parallel analysis of all the small molecules of biological origin (the metabolome) in a sample. Proteomics is the global study of the protein content in an organ, cell, or cellular compartment. A wide range of samples can be analyzed using mass spectrometry-based omics approaches including cells, tissues, hot springs, and bodily fluids. Comparative analyses of proteomic data provides information on cellular processes and signaling pathways that integral to a specific experimental question. Quantitative analysis of the metabolome provides a systems view of the biochemical status of a cell or organism. Metabolomics is particularly attractive for gaining insight into the complex biological processes that together define a system at a given time point because, it provides a direct read-out of cellular physiology and biochemical activity. Our combined proteomics and metabolomics approach is motivated by the hypothesis that multi-level omics data provides the most complete and sensitive measure of physiology and will enhance the development of network models of cellular function. We are currently using LCMS, GCMS, NMR, and chemical tagging of biomolcules to study cellular response to stress in archaea, bacteria, and eukaryotic model systems. Specific projects include arsenic uptake and conversion in cells, role of the microbiome in arsenic induced cancer, and antibiotic resistance of bacteria in biofilms.

The Interplay of stability and Dynamics on Protein Function

Solution-phase protein motions that are critical  to functionality of multi-component complexes can not always be inferred from the three-dimensional structure. For example, in contrast to the still-life representation of viral capsids in models based on cryo-electron microscopy and X-ray crystallography, these supramolecular protein complexes are highly dynamic in solution. The range and frequency of capsid protein dynamics are poorly understood, despite evidence that assembly and infectivity of animal viruses requires conformational freedom. Protein function is intimately connected to dynamics and therefore knowledge of the frequency, range, and coordination of motion by supramolecular complexes is critical to understanding how they function. Our lab uses viruses as a paradigm for studying protein dynamics in supramolecular complexes. With the use of kinetic hydrolysis and quantitative mass spectrometry, we are determining the free energy and rates of large scale protein motion within viral particles. These are the first quantitative measurements for protein dynamics in a megadalton complex. Hydrogen-deuterium exchange, chemical labeling, and quartz crystal microbalance measurements are a few of the methods applied to the quantitative analysis of virus particle stability and dynamics.

Previous Projects 

BETCy

The mission of the Biological Electron Transfer and Catalysis (BETCy) Energy Frontiers Research Center is to define the molecular mechanisms controlling electron flow in coupling electrochemical potential energy to chemical bond formation. The BETCy EFRC examines the mechanisms of Electron Bifurcation (Combining exergonic and endergonic electron transfer reactions for the efficient coupling of electrochemical potential to chemical bond formation), Nucleotide Driven Electron Transfer (Combining energy stored in chemical bonds with electrochemical potential in electron transfer reactions to efficiently drive difficult chemical bond forming reactions) and Catalytic Bias (Mechanisms for controlling directional catalytic rates in proton coupled electron transfer.  Research groups from seven universities are part of the center which is lead by Montana State University.  The Bothner lab is specifically involved in characterizing the active form of multi-component bifurcating enzyme complexes that contain numerous metal centers and cofactors.  High resolution native mass spectrometry, chemical cross-linking, proteolysis, and hydrogen deuterium exchange experiments are now underway.

Biological to digital conversion

The world’s population is rapidly growing and aging. It is projected that the number of elderly will increase from 600 million to 1.5 billion by the year 2050.  The associated increase in demand for health care and the current trend of increasing healthcare costs poses serious challenges to sustainability.  Therefore, to maintain and hopefully improve the current status quo of health, more efficient strategies for personalized healthcare are needed. Long-term monitoring of physiology is an important step in this direction and is already being used to preempt disease and stress2.  Personalized medicine has the potential to reduce healthcare costs by predictively identifying health problems before symptoms arise, allowing preemptive intervention and avoiding costly procedures.   An automated biological to digital converter (BDC) has been developed at MSU which utilizes a robotic microfluidics device to facilitate in-depth real-time metabolic profiling.  A working prototype of this device is in use, but further development is needed. The technology has the potential to directly connect humans, animals, and cells in culture to powerful analytical instrumentation such as a mass spectrometer for monitoring biomarkers and metabolic changes in real-time.  In doing so, this technology allows us to analyze physiological changes with a time resolution that is orders of magnitude higher than current technology.  If developed further, this technology could be used to automate metabolic biomarker analysis in people’s everyday life, increasing quality of life and reducing healthcare costs.  This is an interdisciplinary project with the Montana Microfabrication Facility, Computer Science, and Mathematics Departments at MSU.

Stress Response in an Extremophile

The idea that life is a delicate balance of chemical processes that can occur only within a narrow range of conditions is changing as scientists continue to discover life in extreme environments. The thermal features of Yellowstone National Park are one example. Pools of nearly boiling acid, once thought to be void of life, are now known to contain thriving populations of unicellular organisms and their viruses. Of the three domains of life (Eukarya, Bacteria, and Archaea), the Archaea are the least understood. Many of the organisms that are classified as extremophiles are members the archaeal domain of life. Currently these organisms are the focus of intense research because of our lack of understanding their ability to thrive in conditions once thought uninhabitable and the possibility of isolating enzymes that can with stand harsh industrial conditions. The specific objectives of this project are two-fold: 1) learn about viruses from extreme environments. 2) understand the Sulfolbus solfataricus response to stress. Cutting edge proteomics and activity-based protein profiling (ABPP) are being applied to these studies. Among the many exciting findings from this work is the extensive use of protein post-translational modification in Archaea. The relatively small genome size of Sulfolobus makes this an ideal organism for systems biology studies. This is being pursued in conjunction with other MSU research groups within the Thermal Biology Institute.

Protein Cages as Nanomaterials

Nature has evolved active bio-architectures that are both dynamic and responsive individually as well as collectively when assembled into hierarchical structures. In fact, dynamic protein regions are responsible for biological mineral nucleation, surface recognition, chemical reactivity, and targeting. The concerted protein motion that is part of a multi-component biomolecular complex is rarely obvious from the high resolution three-dimensional structure. Protein function is intimately connected to dynamics and therefore knowledge of the frequency, range, and coordination of motion by supramolecular complexes is critical to understanding function and the development of bio-inspired nanomaterials. The extremely large size and icosahedral architecture of virus capsids limit the use of many standard techniques for studying protein motion such as NMR and FRET. To overcome these problems, we employ an array of biophysical techniques to study the solution phase behavior of viruses. Kinetic hydrolysis, an approach being developed in our lab, is a straight-forward and powerful technique for identifying the dynamic regions within a single protein or in the context of a multi-component complex. Protein dynamics is being investigated at three levels: the dynamics of the subunit, the assembled cage architecture, and the dynamics associated with higher order particle/particle and surface/particle interactions. The long-term goal of this effort is to understand dynamics of the nanoparticle/cage system at each distinct level of complexity so that the underlying mechanism of nucleation, recognition, and functionality can be elucidated and exploited. This work is being conducted in collaboration with other research groups in the Center for Bio-Inspired Nanomaterials.