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Title: Structural changes of photoactive yellow protein
Author: Ramachandran, Pradeep L.
ISNI:       0000 0004 2720 3166
Awarding Body: Oxford University
Current Institution: University of Oxford
Date of Award: 2010
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Photoactive Yellow Protein (PYP) belongs to a class of sensory proteins, within the Xanthopsin family. Like other sensory proteins which provide the initial protective response to environmental factors such as osmotic pressure, chemotaxis, and circadian events, PYP, is thought to be responsible for the negative phototactic response of Halorhodospira halophila to near UV and blue light. Understanding the mechanism by which light energy elicits a physiological response through photo active proteins is crucial in understanding their role in biology. The signalling state of PYP that is formed microseconds after light activation and has a life time of milliseconds to seconds is of particular interest, since formation of this state initiates downstream signalling events which cause the negative phototactic response of the organism. In this study, I present evidence for structural changes of PYP which occur upon illumination. By combining long distance restraints, derived from an electron paramagnetic resonance (EPR) method called double electron-electron resonance (DEER) spectroscopy, with short distance restraints derived from nuclear magnetic resonance (NMR) spectroscopy, a low-resolution structure for the signalling state of PYP is obtained. A large number of simulated annealing calculations were conducted to assess the quality of long range EPR distances used. Spin labels were modelled on to structures and calculations were repeated to determine statistical distribution of labels and compared to the experimental distributions from DEER. The structure of this state has not been resolved until now. The calculated structural ensemble of the signalling state includes an open conformation for the chromophore binding site which is contacted by the N-terminal domain. Transient absorption spectroscopy was used to observe solvatochromic effects of fluorescein conjugated to single cysteine mutants of PYP. The fluorescein absorption band is red-shifted when converted to the signalling state of PYP. I present this new observation as a new method in which fluorescein may be used to probe for local changes in structure. The EPR and NMR studies show that large structural changes are present during the P to h' transition. The large structural changes occur on the N- terminus, and the chromophore binding site of PYP. These changes include a relocation of the N-terminus to a position that is close to and possibly in contact with an exposed chromophore binding site.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available