The biophysical properties, biological activity and function of macromolecular systems
are highly dependent on their structure. Structure-activity relationships of proteins and
their binding partners are critical for drug discovery, biochemical and medical research.
While the gas-phase environment might present as an unusual venue from which to
explore protein structure, for over the past two decades, nano-electrospray ionization
(nESI) coupled to mass spectrometry (MS) has been recognized as having great potential
for analysis of protein structure and protein non-covalent complexes. In conjunction
with related technique of ion mobility (IM), mass spectrometry (IM-MS) provides
insights into protein native-like conformations and any structural changes in may
undergo upon ligand binding or alternations induced via physical parameters such as
temperature, pressure or solution conditions. As most proteins tend to exist as multiple
domains; from the distribution of oligomeric states in the Protein Data Base (PDB) 86%
of proteins exist as oligomers; the work presented in this thesis focuses on application of
MS techniques to probe the tertiary and quaternary structure of various large and
multimeric protein complexes, their dynamics and/or conformational changes. Wherever
relevant, the gas-phase studies reported here are complemented by other techniques, such
as hydrogen deuterium exchange MS (HDX), molecular modelling (MD) and analytical
ultracentrifugation (AUC).
Firstly, the dynamics of intact monoclonal antibodies (mAbs) and their fragments are
explored with IM-MS. Variations observed in conformational landscapes occupied by
two mAb isotypes are rationalized by differences in disulfide linkages and non-covalent
interactions between the antibody peptide chains. Moreover, mAb intrinsic flexibility is
compared to other multimeric protein complexes in terms of collision cross section
distribution span. Secondly, variable temperature MS (VT-MS) and variable
temperature IM-MS (IM-MS) are used to probe unfolding and dissociation of four
standard multimeric protein complexes (TTR, avidin, conA and SAP) as a function of
the of analysis environment temperature. VT-MS is found to allow for decoupling of
their melting temperature (Tm) from the protein complex dissociation temperature (TGPD).
Whereas, VT-IM-MS is used to investigate structural changes of these protein
complexes at elevated temperatures and provide insights into the thermally induced
dissociation (TID) mechanism, as well as strength of the non-covalent interactions
between subunits. Thirdly, VT-(IM)-MS methodology is applied to study behaviour of
three mAbs: IgG1, IgG4 and an engineered IgG4 of increased thermal stability. Such
analysis shows to be promising for comparative thermal stability studies for proteins of
therapeutic interest. Lastly, the structure of ATP-phosphoribosyltransferase (MtATPPRT),
an enzyme catalysing the first step of the biosynthesis of L-histidine in
Mycobacterium tuberculosis, is explored. Conformational changes occurring upon
feedback allosteric inhibition by L-histidine are probed with MS, IM-MS, HDX-MS and
AUC. Reported results serve as the basis for IM-MS/HDX-MS based screening method
to be used for screening of a library of novel and promising anti-tuberculosis agents