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Bothner, Brian P.

Dr. Brian P. Bothner

Assistant Professor
Department of Chemistry and Biochemistry
Montana State University
Bozeman, MT
Protein Dynamics

The concerted protein motion that is part of a multi-component 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 how they function. My lab uses viruses as a paradigm for studying protein dynamics in supramolecular complexes. Viruses are also timely subjects for health, nanotechnology and bio-defense related research.

The life cycle of a typical virus traverses a range of environments. Assembly, maturation, cellular uptake, and genome delivery require numerous structural calisthenics. It is our goal to understand the role of protein dynamics in viral capsid protein function. 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. Limited proteolysis and peptide mass mapping is a straight-forward and powerful technique for determining the dynamic regions within a single protein or in the context of a multi-component complex. Site-directed chemical labeling using small molecule probes is a complementary method that allows the solvent accessible space of a protein or protein complex to be surveyed.

Viral Proteomics

Viruses are obligate cellular pathogens and therefore many cellular proteins are critical for viral infection, replication, and release from a host cell. Using state-of-the-art proteomics technology we are seeking to identify novel cellular proteins that are hijacked by viruses during the infection process. The significance of this work is two fold; the basic biology of viruses will be elucidated and new targets for antiviral agents will be identified. The Chemistry & Biochemistry Department at MSU has just added two new mass spectrometers, specifically for proteomics based experiments, to the existing facility expanding our capabilities of protein identifiction and post-translational modifications (MSU Mass Spectrometry Facility).

Protein identification, localization of protease cleavage sites and labeled amino acids relies on mass spectrometry measurements. Matrix assisted laser desorption (MALDI) with Time-of-Flight (TOF) mass analysis and Electrospray ionization (ESI) with ion-trap mass analysis are two methods that we regularly use (Intro to mass spectrometry). Identification of peptides based on their mass is known as peptide mass mapping or fingerprinting. This technique relies on knowledge of the amino acid sequence of the proteins being studied. Experimental mass spectrometry data is searched against a protein sequence data base that has been digested in silico and peptides are assigned to a protein. The high accuracy of mass analyzers and the ability to generate sequence information from peptides makes the identification of peptides straight forward.

The kinetics of protease digestion is highly dependent on the local protein backbone dynamics. In our experiments using proteolysis, a time course of the reaction allows us to identify the most dynamic protein regions based on the sites that are preferentially cleaved from the intact protein or complex. Identification of amino acids that are specifically labeled with small molecules is accomplished by completely digesting a labeled protein and searching for peptides that have a mass shift equal to the labeling reagent.


Our studies of small icosahedral RNA viruses have revealed that in solution, domains internal in the structural models can be exposed to the capsid surface. We were not the first group to propose such a radical idea. It was demonstrated that antibodies raised to poliovirus VP4 (which is internal next to the packaged nucleic acid) could reversibly neutralize infectivity (Q. Li et al. J. Virol.1994 68:3965). Our application of mass spectrometry based peptide mapping has dramatically improved the resolution and quantification of the protein dynamics.