Search T-Space Advanced Search
Home

Browse
Communities
& Collections

Issue Date
Author
Title
Subject

Sign on to:
Receive email
updates

My Account
authorized users

Edit Profile

Help
About T-Space
 Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/24835

 Title: Structure-based Design and Characterization of Genetically Encoded PhotoactivableE DNA-binding Proteins Based on S. cervisiae GCN4 and Hr. halophila PYP Authors: Morgan, Stacy-Anne Advisor: Woolley, Andrew Department: Chemistry Keywords: Photoactive Yellow ProteinPYPGCN4leucine zipperbZIPphoto-controlphoto-isomerizationphoto-activation Issue Date: 31-Aug-2010 Abstract: Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type. URI: http://hdl.handle.net/1807/24835 Appears in Collections: DoctoralDepartment of Chemistry - Doctoral theses

Files in This Item:

File Description SizeFormat
Morgan_Stacy-Anne_201006_PhD_thesis.pdf10.24 MBAdobe PDF
View/Open

Items in T-Space are protected by copyright, with all rights reserved, unless otherwise indicated.