THE 2.8 Å CRYSTAL STRUCTURE OF A CONSTITUTIVELY ACTIVE ALPHA TRANSDUCIN SUBUNIT
| dc.contributor.author | Hillpot, Eric C | |
| dc.contributor.chair | Zax, David B. | |
| dc.contributor.committeeMember | Davis, Harry Floyd | |
| dc.date.accessioned | 2019-10-15T16:51:14Z | |
| dc.date.available | 2019-10-15T16:51:14Z | |
| dc.date.issued | 2019-08-30 | |
| dc.description.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. | |
| dc.identifier.doi | https://doi.org/10.7298/hd0h-r243 | |
| dc.identifier.other | Hillpot_cornell_0058O_10702 | |
| dc.identifier.other | http://dissertations.umi.com/cornell:10702 | |
| dc.identifier.other | bibid: 11050736 | |
| dc.identifier.uri | https://hdl.handle.net/1813/67750 | |
| dc.language.iso | en_US | |
| dc.subject | Transducin | |
| dc.subject | Cellular biology | |
| dc.subject | G protein coupled receptor | |
| dc.subject | G proteins | |
| dc.subject | Signaling | |
| dc.subject | vision | |
| dc.subject | x-ray crystallography | |
| dc.subject | Biology | |
| dc.subject | Biochemistry | |
| dc.title | THE 2.8 Å CRYSTAL STRUCTURE OF A CONSTITUTIVELY ACTIVE ALPHA TRANSDUCIN SUBUNIT | |
| dc.type | dissertation or thesis | |
| dcterms.license | https://hdl.handle.net/1813/59810 | |
| thesis.degree.discipline | Chemistry and Chemical Biology | |
| thesis.degree.grantor | Cornell University | |
| thesis.degree.level | Master of Science | |
| thesis.degree.name | M.S., Chemistry and Chemical Biology |
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