| Summary: | Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure
of transmembrane (TM) segment IX of the Na+/H+ exchanger isoform 1 (NHE1)
in dodecylphosphocholine micelles. Studying isolated TM segments in this fashion
constitutes a well-established "divide and conquer" approach to the study of membrane
proteins, which are often extremely difficult to produce, purify, and reconstitute
in full-length polytopic form. A similar approach was combined with NMR spin relaxation
experiments to determine the peptide backbone
flexibility of NHE1 TM VII.
The combined NMR structural and dynamics studies are consistent with an important
role for TM segment
flexibility in the function of NHE1, a protein involved in
apoptosis and myocardial disease. The study of the rhomboid protease system is also
described from two perspectives: 1) I attempted to produce several TM constructs
of the substrate spitz or a related construct and the production and purification are
described in detail; and 2) I present coarse-grained molecular dynamics simulation
results for the E. coli rhomboid ecGlpG and a spitz TM construct. Spitz appears to
preferentially associate with rhomboid near TMs 1 and 3 rather than the proposed
substrate gate at TM 5. The two proteins primarily interact at the termini of helices
rather than within the hydrocarbon core of the bilayer. Finally, I present a detailed
analysis of coarse-grained molecular dynamics simulations of the fibroblast growth
factor receptor 3 TM domain dimerization. Specifically, algorithms are described for
analyzing critical features of wild-type and G380R mutant constructs. The G380R
mutation is the cause of achondroplasia, the most common form of human dwarfism.
The results suggest that the proximity of a residue to the dimer interface may impact
the severity of the mutant phenotype. Strikingly, heterodimer and mutant homodimer
constructs exhibit a secondary dimer interface which may explain the increased
signaling activity previously reported for the G380R mutation--the helices may rotate
with the introduction of G380R. The unifying theme of this work is the 'study
of membrane proteins' using complementary techniques from structural biology and
computational biochemistry.
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