Protein crystallography with a micrometre-sized synchrotron-radiation beam

For the first time, protein microcrystallography has been performed with a focused synchrotron-radiation beam of 1 µm using a goniometer with a sub-micrometre sphere of confusion. The crystal structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample

X-ray crystallographic study on the mechanisms of Bacteriorhodopsin and the Sensory Rhodopsin/Transducer Complex

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-specific 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 Sensory 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 $\mathring{A}$ 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 Xray crystallography. Crystals were grown in the lipidic cubic phase which provided data to a resolution of 1.9 $\mathring{A}$ for the ground state, 2.0 $\mathring{A}$ for K state and 2.2 $\mathring{A}$ for the late M state of the Sensory rhodopsin II/transducer complex and 1.35 $\mathring{A}$ for the ground state, 1.5 $\mathring{A}$ 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$\circ$ of helix TM2 and a displacement of this helix by 0.9 $\mathring{A}$ 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 $\mathring{A}$ 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 differences and similarities related to the function of these proteins are underlined

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota(1- 3). This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis(4). The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically 5 to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Angstrom resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface