Verlag des Forschungszentrums Jülich
JUEL-4208
Moukhametzianov, Rouslan
X-ray Crystallographic Study on the Mechanisms of Bacteriorhodopsin and the Sensory Rhodopsin / Transducer Complex
129 S., 2006
Microbial rhodopsins belong to a family of seven-helical transmembrane retinal proteins which
are found in Bacteria, Archaea and Eukaryota. They are considered to be the archetypes for ion
transport and signal transduction, which use for these distinct functions a common structural
design. The ion pumps Bacteriorhodopsin (BR) and Halorhodopsin (HR) operate as energy
converters, whereas the photoreceptors Sensory rhodopsin I (SRI) and II (SRII) operate as light
sensors providing the initial signal which via associated receptor-speci½c transducers (HtrI and
HtrII) activate a two-component signalling cascade that moves the cell in response to light.
The archaeal rhodopsins are the best understood proteins among seven-helical receptors with
respect to structural information from X-ray crystallography. High resolution structures of Sen-
sory rhodopsin II from Natronobacterium pharaonis and Bacteriorhodopsin from Halobacterium
salinarum as well as Halorhodopsin have already been obtained. The X-ray structure of the
complex between N.Pharaonis SRII (pSRII) and HtrII at 1.94 Å resolution was reported.
In this thesis the structure of the ground state, the early K state and the signalling late
M state of Sensory rhodopsin II and its cognate transducer as well as the structure of the
ground state and the late M state of the Bacteriorhodopsin were investigated by means of X-
ray crystallography. Crystals were grown in the lipidic cubic phase which provided data to a
resolution of 1.9 Å for the ground state, 2.0 Å for K state and 2.2 Å for the late M state of
the Sensory rhodopsin II/transducer complex and 1.35 Å for the ground state, 1.5 Å for the
late M state of Bacteriorhodopsin. The occupancies of the intermediate states trapped in the
crystals at cryo temperatures were estimated from the crystallographic analysis. The structure
solution based on molecular replacement yielded atomic pictures of ground, K and M state of
the pSRII/transducer complex and ground and M state of bacteriorhodopsin. The refinement
scheme using simulated annealing for models consisting of two conformations (one accounting for
the ground state and the other for the intermediate state) with corresponding occupancies were
assessed and used for structure solution. Additionally it was investigated if experimental phases
obtained by isomorphous replacement and anomalous scattering would help to determine the
transducer structure including linker domain. Different models of the crystal were investigated
to extract structural data for the complete fold of the transducer. Results provide insights in
signal transfer from pSRII to the transducer in the membrane part of the protein complex and
reveal the details of the evolvement of the proton translocation channel in BR.
The observed structural changes allow to propose a mechanism for the light induced activation
of the complex: Upon light excitation retinal isomerization leads in K state to a rearrangement
of a water cluster that partially disconnects two helices of pSRII. In the transition to late M
the changes in the hydrogen bond network proceed further. The signalling state is established
by tertiary structural changes induced by the new hydrogen bond pattern and the changed
charge distribution. The two partially decoupled subdomains of the receptor show a relative
displacement that is most significant between helices F and G which form the interface to TM2
of the transducer. The transducer responses to the receptor activation by a clockwise rotation
of about 15 ° of helix TM2 and a displacement of this helix by 0.9 Å at the cytoplasmic surface.
The late M state structure of the wild type bacteriorhodopsin extends the knowledge of this
important intermediate of the photocycle reported previously. The achieved resolution of 1.5 Å
and the low twinning of the crystal enable a better definition of the model. Though this structure
is very similar to the reported one it still provides new information particularly concerning the
water molecule chain in the cytoplasmic part of the channel between Asp96 and Schiff base.
The revealed structures allow drawing the parallels between ion transport and sensory signalling by this family of proteins. Important structural diÿerences and similarities related to the
function of these proteins are underlined.
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