Abstract:
Membrane proteins play a range of important roles in biological systems, yet they
are underrepresented in the data base of high-resolution structures of all proteins. There is
intense interest in developing new methodologies for studying membrane proteins. An
essential step to membrane protein method development is devising reliable membrane
mimics in which to embed membrane proteins.
The goal of this thesis was to develop and apply nanodisc membrane mimics to
the study of an influenza A membrane protein called M2. Nanodiscs provide a lipid
bilayer environment with access to both sides of the bilayer and are smaller than
commonly used liposome model membranes whose size provides challenges for some
biophysical methods. This thesis shows how the sample composition of M2 containing
nanodiscs was optimized. Dynamic light scattering and size exclusion chromatography
was used to characterize M2-nanodiscs.
Electrophysiological and budding assays showed that M2 in liposomes were in a
functionally relevant conformation. Extensive previous work has been done on studying
M2 protein in spherical liposome using site-directed spin label electron paramagnetic
resonance (SDSL-EPR). We carried out SDSL-EPR studies of M2-nanodiscs and
compared them to published work on M2 in liposomes. Our EPR data is consistent with
M2 protein in nanodiscs having a similar conformation, mobility and membrane topology
as that seen in previously published M2-liposome work. Furthermore, we probed the
ability of nanodiscs to allow for conformational exchange by comparing the impact of
drug binding on M2-nanodiscs with M2-liposomes.
