This study shows that coupling to designed plasmonic nanoparticles can modulate the electrophysiological function of proteins in living mammalian cells. Nanostar-shaped particles, that are robust to biological noise, are designed to enable near-field-coupling to plasma membrane-localized mutated Archaerhodopsin proteins in live cells. The coupled rhodopsins exhibit enhanced fluorescence and an increased response speed to membrane voltage. Incorporating this plasmonic enhancement into a Markov chain photocycle model of the Archaerhodopsin mutant QuasAr6a, shows an increased fluorescence emission rate and manipulation of the protein dynamics through a combination of photocycle transition rate enhancements. The results show an improvement in fluorescence and voltage-response dynamics of the functional QuasAr6a Archaerhodopsin mutant, beyond what has been achievable through genetic engineering. This opens up possibilities for engineering the biological functionality of proteins through plasmonics: manipulating protein photocycles could improve light sensitivity, change optogenetic applications, and lead to fluorescent biosensors with enhanced dynamics.

Voltage imaging using genetically encoded voltage indicators (GEVIs) has taken the field of neuroscience by storm in the past decade. Its ability to create subcellular and network level readouts of electrical dynamics depends critically on the kinetics of the response to voltage of the indicator used. Engineered microbial rhodopsins form a GEVI subclass known for their high voltage sensitivity and fast response kinetics. Here we review the essential aspects of microbial rhodopsin photocycles that are critical to understanding the mechanisms of voltage sensitivity in these proteins and link them to insights from efforts to create faster, brighter and more sensitive microbial rhodopsin-based GEVIs.