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THE 2.8 Å CRYSTAL STRUCTURE OF A CONSTITUTIVELY ACTIVE ALPHA TRANSDUCIN SUBUNIT

Author
Hillpot, Eric C
Abstract
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.
Date Issued
2019-08-30Subject
Transducin; Cellular biology; G protein coupled receptor; G proteins; Signaling; vision; x-ray crystallography; Biology; Biochemistry
Committee Chair
Zax, David B.
Committee Member
Davis, Harry Floyd
Degree Discipline
Chemistry and Chemical Biology
Degree Name
M.S., Chemistry and Chemical Biology
Degree Level
Master of Science
Type
dissertation or thesis