Biophysical studies on the structure and function of Archaerhodopsin-3

Light is indispensable for most life forms, and all organisms have evolved in order to harness or utilise its energy. Microbial rhodopsins are lightactivated proteins that generate an electrochemical gradient across the membrane for vital functions of organisms. Recently, the use of lightactivated proteins that generate an electrochemical gradient have gained attention in the field of optogenetics as tools to hyperpolarise or depolarise membranes, thus achieving neural silencing or as uorescent voltage indicators, as well as in bioelectronics to develop biosensors or photovoltaic cells. Here we explore the biophysical characteristics of a microbial rhodopsin, AR3, resolving its structure for the first time, to give new insights into activity, and permit development of a mutant library of AR3, identifying target residues to achieve spectral tuning. This biophysical characterisation allows the identification of key molecules and networks that enhance the photocurrent of AR3 transferrable to other homolog microbial rhodopsins, thus providing new insights into better protein design for optogenetics and bioelectronics. AR3 has been purified from its native organism (Halorubrum sodomense) and crystallised giving structures of the light-adapted (LA) state and the dark-adapted (DA) state with resolutions of 1.07 Å and 1.2 Å, respectively. The resolution of these structures has allowed the identification of complex hydrogen bonding networks and residues important in ion transport, as well as water molecules. The differences between the DA and LA states have been also shown, as well as identifying key differences between AR3 and bR internal rearrangements. Furthermore, we use high-speed AFM, serial crystallography and X-ray free electron lasers to identify conformational changes that take place in the protein during the photocycle in the early stages (using XFEL) and in the later stages (using HS-AFM). AR3 has recently gained importance in the world of microbial rhodopsins due to its high photocurrent change upon illumination enabling neural silencing in optogenetics applications. Here we study the photocurrent properties of AR3 using a novel droplet interface bilayer system. We study the activity of AR3 over long periods of illumination and address the role that chromophores bound to the protein, such as retinal or bacterioruberin, might have in the photocurrent and bleaching of the protein. Finally, we develop a mutant library of AR3 mutants with different spectral properties. We assess the proton pumping of the different mutants developed, as well as the photocurrent change and the action spectra, and study the effect in absorbance and photocurrent that different mutations have in key residues responsible for the proton pumping activity of the protein. Such mutants have potential applications for improved use in optogenetics, especially in red-shifted mutants.