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Breakthrough for optogenetics

An international team has visualised the photoreceptor AR3 for the first time and at unprecedented resolution.

The light-sensitive AR3 protein is produced by Halorubrum sodomense, an organism that grows in the Dead Sea. It is best known for its applications in optogenetics experiments, in which it is used to silence individual neurons and to detect changes in cell membrane voltage. Work by researchers at NPL, Oxford University and Diamond Light Source now provides protein engineers with the ‘blueprints’ to this important photoreceptor, pinpointing the positions of individual atoms to ultra-high precision. These advances open the door to the rapid development of new tools and methodologies in neuroscience, cell biology and beyond.

Corresponding author Prof. Anthony Watts from Oxford University said: “The superb resolution that we have achieved for these AR3 structures, 1.07 Å for the ground state, is among the highest for a wild-type membrane protein deposited to date in the Protein Data Bank. This quality allows us to visualise directly the complex distribution of water molecules inside the receptor and to describe the functional significance of the intricate networks of hydrogen bonds that they form, something that is of fundamental importance in many biomolecules – not just photoreceptors.”

The team’s paper, published in Nature Communications, details the first ever structure of the ground state of AR3. In this state, the protein is configured to transport one H+ ion across the cell membrane for each photon absorbed. The scientists were also able to crystallise the photoreceptor in a second conformation, a desensitized state that is adopted by AR3 in the prolonged absence of light.

Dr Isabel Moraes, Principal Research Scientist, NPL said: “The very high-resolution diffraction data has enabled us to observe in great detail the freedom of motion of several key amino acids and individual water molecules inside AR3. Solving the structures was extremely challenging, because of the complexity of the features that we can distinguish at atomic resolution. Many amino acid side chains inside the photoreceptor were resolved in more than one position or orientation, indicating their importance for protein function. Knowledge of the dynamics of these groups was key to understanding how the processes of sensitisation and desensitisation take place.”

Obtaining such high-resolution crystal structures would not have been possible without the state-of-the-art microfocus beamline at the Diamond Light Source synchrotron, near Didcot in Oxfordshire.

Senior Beamline Scientist Dr Danny Axford said: “The microcrystals grown by the Oxford and NPL teams were perfectly suited to the capabilities of the I24 beamline. The combination of precise microfocus X-ray delivery and advanced data analysis enabled us to push the resolution of the resulting structures to the limits.”

Also contributing to the study was Prof. Carol Robinson, whose group, based in the Chemistry Department at Oxford University, works on native mass spectrometry of integral membranes proteins. Commenting on the publication, Professor Robinson said:

“Using nano electrospray ionisation methods, we released AR3 from detergent micelles and were able to confirm the post-translational modifications visible in the crystal structures, as well as lipid binding. We were particularly interested in the truncation of the first six residues of the protein, which occurs as a result of the removal of the N-terminal signal peptide and which is followed by the cyclisation of the exposed Gln7 to form a pyroglutamate residue. It was particularly exciting to see this group resolved so clearly in the electron density maps.”

27 Jan 2021