DYNAMIC MOTIONS OF THE ALPHA SUBUNIT OF HETEROTRIMERIC G PROTEINS IN THE NUCLEOTIDE- AND RIC-8A-BOUND STATES
Year of Award
Doctor of Philosophy (PhD)
Other Degree Name/Area of Focus
Department or School/College
Department of Chemistry and Biochemistry
J. B. Alexander Ross, Stephen R. Sprang
Edward Rosenberg, Christopher Palmer, Bruce Bowler
dynamics, G protein, Ric-8A
The University of Montana
The conformational changes and segmental dynamics involved in the G alpha-subunit of heterotrimeric G proteins have been investigated for 3 separate binding states by using binning-time-dependent single-molecule F÷rster resonance energy transfer (smFRET) of freely diffusing proteins and time-resolved fluorescence anisotropy. The bound states include Ric-8A (Resistance to Inhibitors of Cholinesterase-8A (Miller, Emerson et al. 2000; Miller and Rand 2000; Tall, Krumins et al. 2003)), a guanine nucleotide exchange factor (GEF), and nucleotides GDP and GTP. To analyze the smFRET data, energy transfer efficiency histograms were constructed at binning times varied from 1000 to 2,500 ╡s. Then the conformational equilibria and rates of conformational change between end states (GTP-bound, GDP-bound, and Ric-8A-bound) were extracted using the 3-Gaussian model, developed by Gopich and Szabo (Gopich and Szabo 2007; Gopich and Szabo 2010). Using this model, we determined that intra- and inter-domain dynamics occur on the ms time scale. The Helical-Helical conformational changes are relatively small (< 5 ┼), without observable influence from the binding partner (nucleotide or GEF). The intra Ras-like domain conformational changes are somewhat larger (>5 ┼), and have distinct, multiple states regardless of binding partner (GTP, GDP and Ric-8A). The inter-domain conformational changes are much larger (>40 ┼), and likewise exhibit distinct, multiple states that are binding-partner dependent.
Black, Labe Adam, "DYNAMIC MOTIONS OF THE ALPHA SUBUNIT OF HETEROTRIMERIC G PROTEINS IN THE NUCLEOTIDE- AND RIC-8A-BOUND STATES" (2015). Graduate Student Theses, Dissertations, & Professional Papers. 4610.
© Copyright 2015 Labe Adam Black