Summary: | Thesis (Ph. D.)--Boston University === The major aim of this thesis is to investigate the molecular basis for the function
of several types of rhodopsins with special emphasis on their application to the new
field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7
a-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a
light driven proton pump similar to the extensively studied bacteriorhodopsin (BR);
channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII),
a light-sensing protein that modulates phototaxis used in archaebacteria; and squid
rhodopsins (sRho), the major photopigment in squid vision and a model for human
melanopsin, which controls circadian rythms.
The primary techniques used in these studies were FTIR difference spectroscopy
and resonance Raman spectroscopy. These techniques, in combination with site directed
mutagenesis and other biochemical methodologies produced new knowledge
regarding the structural changes of the retinal chromophore, the location and function
of internal water molecules as well as specific amino acids and peptide backbone.
Specialized techniques were developed that allowed rhodopsins to be studied in intact
membrane environments and in some cases in vivo measurements were made on
rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting
proteins and membrane potential to be investigated.
Evidence was found that the local environment of one or more internal water
molecules in SRII is altered by interaction with its cognate transducer, Htrii, and is
also affected by the local lipid environment. In the case of AR3, many of the broad
IR continuum absorption changes below 3000 cm-1, assigned to networks of water
molecules involved in proton transport through cytoplasmic and extracellular portions
in BR, were found to be very similar to BR. Bands assigned to water molecules
near the Schiff base postulated to be involved in proton transport were, however,
shifted or absent. Structural changes of internal water molecules and possible bands
associated with the interaction with ,8-arrestins were also detected in photoactivated
squid rhodopsin when transformed to the acid Meta intermediate. Near-IR confocal
resonance Raman measurements were performed both on AR3 reconstituted into E.
coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of
a negative transmembrane potential. On the basis of these measurements, a model
is proposed which provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.
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