Optical Control of Protein Activity by Fluorescent Protein Domains

Abstract

Fluorescent proteins (FPs) are widely used as optical sensors while other light-absorbing domains have been used for optical control of protein localization or activity. Here we describe a previously unknown capability of a mutant of the photochromic FP Dronpa – light-dependent dissociation and association – and use it to control protein activities with light. Fusion of mutant Dronpa domains to both termini of an enzyme domain creates a fluorescent light-inducible protein that is inactive in the dark but can be activated by light. Unlike other methods for optical control of single proteins, Dronpa-based fluorescent light-inducible proteins are simple to generate and have self-reporting abilities. Our findings extend the useful applications for FPs in biological experiments and engineered cellular systems from exclusively sensing functions to also encompass optogenetic control.

Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

“…56,57 In order to create a comprehensive data set with varying degrees of photobleaching or photoswitching to an off state, as a test whether macromolecular sedimentation properties could be modeled in all cases, the laser power of the 488 nm excitation beam was varied in consecutive experiments, using settings of 50.2 mW, 8.4 mW, and 2.1 mW. Lower laser power was partially compensated for by using higher photomultiplier voltages and gain settings to keep the signal/noise ratio high.…”

Self-organization of protrusions and polarity during eukaryotic chemotaxis

“…Tools such as atomic force microscopy have been employed to measure changes in membrane tension [77,78] and could potentially be used to interactively manipulate tension as well. Optogenetic [79–85] and chemical [86,87] tools may be used to rapidly modulate protein location/activity in living cells. Such approaches should provide further insight into the complex dance between signaling, forces, and the cytoskeleton that underlie the self-organization of protrusions and polarity.…”

Emerging fluorescent protein technologies

“…In 2012, Lin and co-workers [62 ] reported that the green fluorescent Dronpa FP variant underwent an illumination-dependent change in oligomerization state. Upon illumination with cyan light (500 nm), the tetrameric Dronpa 145N variant dissociates to form a non-fluorescent monomeric protein.…”