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EXPANDING THE ROLE OF GAS-PHASE METHODS IN STRUCTURAL BIOLOGY: CHARACTERIZATION OF PROTEIN QUATERNARY STRUCTURE AND DYNAMICS BY TANDEM MASS SPECTROMETRY AND ION MOBILITY

Blackwell, AE;

This dissertation presents efforts to expand the role of mass spectrometry (MS) in
structural biology. Determination of quaternary structure of a protein complex has been
hindered by limited fragmentation from collision-induced dissociation (CID). As an
alternative, surface-induced dissociation (SID) was implemented for a quadrupole –
time of-flight instrument in the Wysocki laboratory. This research tested the hypothesis that
SID should produce fragmentation reflective of subunit organization. Furthermore, ion
mobility (IM) was used to prove the direct relationship between precursor conformation
and observed dissociation patterns, and the relationship between activation and product
ion conformation.
The structure and dynamics of a dimeric small heat shock protein (sHSP) with no
solved structure was investigated. The importance of N- and C-terminal domains for
dimerization was determined, and the dimers were shown to exchange subunits. From
exchange kinetics it is proposed that subunit exchange is unrelated to heat shock activity.
SID was used to elucidate the subunit architecture of heterogeneous protein
assemblies, including one previously solved protein structure and two formerly
uncharacterized proteins. The heterohexamer toyocamycin nitrile hydratase dissociated
into trimers, revealing the hexamer to be a dimer of trimers. The bacterial ribonuclease
toxin:antitoxin tetramer was shown to have an antitoxin dimer at its core, with flanking
individual toxin subunits. The examples presented here are the first clear proof that SID
results can clearly indicate the substructure of a protein assembly.
PREVIEW
22
IM was used to study the conformation of precursor and product ions. A greater
understanding of the relationship between precursor conformation and observed
dissociation patterns was developed. Different charge states of a dodecameric sHSP were
found to have significantly different conformations, which were directly reflected in SID
spectra. IM comparison of CID and SID product ions showed that the same charge state
of a product ion from either method has the same CCS. This suggests the product ion
conformation is dependent upon ion charge state, and independent of activation method
and collision energy. The cause and effect relationship between precursor conformation
and MS/MS patterns, and activation and product ion conformation were clearly
illustrated.
Together, this body of research expands the role of MS for structural biology.