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.
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
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.
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