Solid-state nuclear magnetic resonance of rhodopsin and its photointermediates

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads<br/>to a highly energetic species called bathorhodopsin, which is stable at temperatures<br/>below 125 K. Bathorhodopsin stores about 2/3 of the absorbed photon energy but<br/>the mechanisms with which this energy is stored is not completely understood. A<br/>new insight into these mechanisms by means of low-temperature solid-state NMR<br/>is both subject and aim of this Ph.D. thesis. The issue of the energy storage has<br/>been investigated by a solid state magic angle spinning technique which combines<br/>modern symmetry-based recoupling techniques with in situ cooling of the sample.<br/>Production of bathorhodopsin is also done in situ in a customized NMR probe.<br/>Three kind of experiments are discussed: chemical shift, distance and torsional<br/>angle measurements. The first kind of experiments led to carbon chemical shifts<br/>values for almost all the carbons along the retinylidene chain of the retinal chromophore<br/>of bathorhodopsin. Our measurements show a significant perturbations of<br/>the 13C chemical shifts in bathorhodopsin which is interpreted in terms of charge<br/>delocalization along the chain and therefore indicates a participation of an electrostatic<br/>mechanism to the energy storage. This is at variance with an earlier solid<br/>state NMR study where only minor perturbations of the electronic structure in the<br/>isomerized retinylidene chain were observed. We believe that these data incorrectly<br/>refer to bathorhodopsin because of the incorrect conditions of temperature and illumination<br/>applied. To sample for other local mechanisms that may contribute to the<br/>energy storage, the C-C distance of the last two carbons of the retinylidene chain,<br/>at the link with the protein opsin, was also measured but no significant differences<br/>with rhodopsin have been found. Finally, the H-C=C-H torsional angle at the double<br/>bound where the isomerization takes place was measured in a double-quantum<br/>heteronuclear local field spectroscopy (2Q-HLF) experiment. Results indicate a<br/>deviation from planarity of at least 40? about this double bond in bathorhodopsin<br/>suggesting an unquantified amount of torsional strain acting as a further energy<br/>storage mechanism. In addition to these very interesting results, this thesis reports<br/>methods, equipment and procedures ready to be used for the study of other similar<br/>light-triggered processes.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2010

Author dc:creator

• Concistre, Maria

Advisor dc:contributor.advisor

• Levitt, Malcolm H.