Proteomics, Protein Dynamics, Supramolecular Complexes

bbothnerchemistry.montana.edu

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. Post Doctoral: Dr. Walid Maaty. Graduate student: Pavel Tarlykov. Undergraduate: Joshua Heinemann.

Selected Publications

Maaty WS, Wiedenheft B, Tarlykov P, Schaff N, Heinemann J, Robison-Cox J,Valenzuela J, Dougherty A, Blum P, Lawrence CM, Douglas T, Young MJ, Bothner B.:
Something old, something new, something borrowed; how the thermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress.
PubMed PMID: 19759909; PubMed Central PMCID: PMC2739297. Sep16;4(9):e6964 (2009)

Keywords:
Analytical, Biochemistry, Chemical Biology, Proteomics

Viruses are obligate cellular pathogens and therefore many cellular proteins are critical for viral infection, replication, and release from a host cell. Using cutting edge proteomics approaches, we are seeking to identify cellular pathways that are involved with the infection process. The significance of this work is two-fold: the basic biology of viruses can be elucidated and novel targets for antiviral agents can be identified.
Noroviruses are a serious health and economic concern world-wide and are responsible for > 23 million infections per year in the USA alone. Despite the obvious importance of this group of viruses, much remains to be learned about their biology. This is largely due to a lack of animal and tissue culture model systems. The recent development of a murine norovirus (MNV) that can be cultured was a major breakthrough. We have adopted this system and are using a two-level proteomics approach to elucidate pathways and proteins involved with MNV infection. At the systems level, 2D differential gel electrophoresis (2D-DIGE) is being applied to generate an overview of the global changes in the host proteome during infection. This is being complemented by activity-based screening of enzyme classes. Activity-based protein profiling (ABPP) is currently the only technique that can directly measure specific protein activity across a biological system. Current research is directed toward understanding the role of apoptosis in MNV infected cells at the level of the system and individual proteins that become activated during programmed cell death. Graduate student: Linnzi Furman. Undergraduate: Lena Peterson.

Selected Publications

Ortmann, AC, Brumfield SK, Walther J, McInnerney K, Brouns SJ, van de Werken HJ, Bothner B, Douglas T, van de Oost J, Young MJ:
Transcriptome analysis of infection of the archaeon Sulfolobus solfataricus with STIV
J Virol. 82(10):4874-4883, 2008.

Maaty, W.S.A, Ortmann, A.C., DlakiÄ, M., Schulstad K., Hilmer, J.K., Liepold, L., Weidenheft, B., Douglas,T., Young, M., and Bothner, B.:
Characterization of the Archaeal thermophile Sulfolobus Turreted Icosahedral Virus validates an evolutionary link among dsDNA viruses from all domains of life
J. Virology 80(15):7625-7635. (2006)

Furman LM, Maaty WS, Petersen LK, Ettayebi K, Hardy ME, Bothner B.:
Cysteine protease activation and apoptosis in Murine norovirus infection
Virol J. 2009 Sep 10;6:139

Keywords:
Biochemistry, Analytical, Chemical Biology, Proteomics

The solution-phase protein motion that is part of a multi-component complex 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 the 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 additional methods applied to the quantitative analysis of virus particle stability and dynamics. Graduate students: Jonathan Hilmer and Vamseedhar Rayaprolu. Undergraduates: Lena, Petersen and Tim Potter. Technical assistance: Geoff Blatter

Selected Publications

Bothner B, Taylor DJ, Jun B, Lee KK, Siuzdak G, Schultz CP, Johnson JE :
Maturation of a tetravirus capsid alters the dynamic properties and creates a metastable complex
Virology 334(1):17-27 (2005)

Lee KK, Tang J, Taylor D, Bothner B, Johnson JE :
Small Compounds Targeted to Subunit Interfaces Arrest Maturation in a Nonenveloped, Icosahedral Animal Virus
J. Virology 78(13): 7208-7216 (2004)

Taylor DJ, Wang Q, Bothner B, Natarajan P, Finn MG, Johnson JE:
Correlation of chemical reactivity of Nudaurelia capensis omega virus with a pH-induced conformational change
Chem Commun (Camb). 22: 2770-2771 (2003)

Bothner B, Schneemann A, Marshall D, Reddy V, Johnson JE, Siuzdak G:
Crystallographically identical virus capsids display different properties in solution
Nature Struct. Biol. 2:114-116 (1999)

Lewis JK, Bothner B, Smith TJ, Siuzdak G:
Antiviral agent blocks breathing of the common cold virus
Proc Natl Acad Sci U S A 95(12):6774-6778 (1998)

Bothner B, Dong XF, Bibbs L, Johnson JE, Siuzdak G:
Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry
J Biol Chem 273(2):673-676 (1998)

Speir JA, Bothner B, Qu C, Willits DA, Young MJ, Johnson JE:
Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid that Maintains Infectivity and Capsid Dynamics
J. Virology 80(7):3582-3591. (2006)

Hilmer JK, Zlotnick A, Bothner B.:
Conformational equilibria and rates of localized motion within hepatitis B virus capsids
J Mol Biol. 2008 Jan11;375(2):581-94. Epub 2007 Oct 22. PubMed PMID: 18022640; PubMed Central PMCID: PMC2238684. (2008)

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.
Post Doctoral: Dr. Walid Maaty. Graduate students: Jonathan Hilmer and Vamseedhar Rayaprolu. Undergraduates: Joshua Heinemann and Tim Potter.

Keywords:
Analytical, Biochemistry, Biophysical, Protein Chemistry, Structure

Research in the Bothner lab is directed toward understanding the assembly, cell entry, and infection process of icosahedral viruses. This research takes us from the atomic scale provided by high resolution structural models to the complex interaction networks of nucleic acids, metabolites, and proteins that make up a living system. A diverse set of analytical, biophysical, biochemical, and cell biology techniques are used in the discovery process. The Bothner lab is part of the Center for Bio-Inspired Nanomaterials and the Thermal Biology Institute

Keywords:
Biochemistry, Biophysical, Chemical Biology, Proteomics

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