Fourier transform infrared difference spectroscopy of bacteriorhodopsin

We have used the technique of Fourier transform infrared difference spectroscopy to study the photocycle and proton pump of bacteriorhodopsin (bR). By applying this method to the study of native bR and bR with modified chromophores, we have confirmed and extended the earlier resonance Raman results concerning the configuration of the chromophore of bR and its intermediates. These studies also demonstrated that the all-trans to 13-cis photoisomerization of the chromophore is essential to the functioning of the protein. This tecnnique has also allowed us to explore the role of the opsin in the protein's proton pump. Large scale motions of the opsin during the photocycle were detected and a simple model to interpret these motions was formulated. The model involves the rotation of the protein's a helices towards the membrane normal during the formation of the M intermediate. The role of three amino acids, tyrosine, glutamate and aspartate, in the proton pump was investigated by comparing the difference spectra of native bR with those of bR grown on isotopically labelled analogues of these compounds. This comparison revealed that the sidechains of these amino acids undergo changes in protonation and/or environment during the photocycle, functioning, as had been thought, as proton donors and acceptors. However, we were not able to construct a synoptic model of the role of these groups in the proton pump. Finally, we have built and tested a device to be used to take kinetic infrared data on bR. The device is based on the optical spinning sample technique and is placed inside a conventional Fourier transform infrared spectrometer. It can be used to acquire data in the entire midinfrared region and has a maximum time resolution of 40 ps. This time resolution has not yet been achieved. Preliminary data taken with this device are presented.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Year dc:date

2011

Author dc:creator

• Dollinger, Gavin David

Contributors dc:contributor

• Frauenfelder, Hans

• Fourier transform infrared difference spectroscopy
• photocycle
• proton pump
• bacteriorhodopsin
• chromophores
• photoisomerization

Statement dc:rights

• 1985 Gavin David Dollinger

Language dc:language

en

Identifier

864921

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/25284