THE 2.8 Å CRYSTAL STRUCTURE OF A CONSTITUTIVELY ACTIVE ALPHA TRANSDUCIN SUBUNIT
Hillpot, Eric C
G protein coupled receptors play important roles in cellular signaling and are stimulated by a multitude of extracellular signals. These proteins initiate signal transduction cascades that elicit a variety of cellular responses. Once activated, G protein coupled receptors activate G proteins which further stimulate target proteins to elicit cellular responses such as enzyme activity, translation and ion channels60. In this study, we set out to solve the crystal structure of a constitutively active form of the alpha subunit of the retinal G protein, transducin. This activated G protein, designated as αT* SFD QLRC, elicits the ability to stimulate its effector molecule, PDE, to levels comparable to activated native retinal alpha. Crystals of αT* SFD QLRC were grown and x-ray diffraction data sets were observed at low resolution. Following insights from a recent paper by Hu et al, 2018, a C210S mutant was created to help improve diffraction. Data sets of αT* SFD QLRC C210S were collected to 2.8 Å and compared to previously solved GDP-bound (1TAG) and GTPγS-bound (1TND) native αT subunits. The crystal structure of αT* SFD QLRC C210S reveals that despite being fully active in vitro, GDP, and not GTP, is bound in the structure. As a result, Switch II and Switch III appear to be in a transition state between the inactive GDP-bound state and the active GTP-bound state and are un-modelled due to low electron density. Future work involves improving crystallization of αT* SFD QLRC C210S to obtain a GTP-bound structure so that the structure can be used to elucidate the mechanism by which the transducin α subunit, especially the phenylalanine residue of the SFD mutation, confers maximum PDE activity.
Transducin; Cellular biology; G protein coupled receptor; G proteins; Signaling; vision; x-ray crystallography; Biology; Biochemistry
Zax, David B.
Davis, Harry Floyd
Chemistry and Chemical Biology
M.S., Chemistry and Chemical Biology
Master of Science
dissertation or thesis