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Phosphorylation at the Flick of a Switch

Incorporating light-controlled dimerization domains into kinases provides tight regulation of these enzymes.

May 1, 2017
Ruth Williams

FLIPPING OUT: Researchers have designed kinases that can be inactivated and activated by light. In violet light, engineered green fluorescent domains (called pdDronpa) dimerize, glow, and block the enzyme’s active site (top). In blue light, the domains break into monomers, lose their fluorescence, and uncage the kinase’s active site (bottom).© GEORGE RETSECK

Controlling a protein’s activity with light enables spatial and temporal regulation that would be practically impossible otherwise. Such fine control is desirable for teasing out the molecular details of cellular processes and for initiating the actions of therapeutic proteins in precise locations in the body.

Molecular biologists, including Michael Lin of Stanford University, are hard at work developing and improving such protein technology. And Lin’s latest approach is “particularly remarkable,” says Harald Janovjak of the Institute of Science and Technology in Austria.

The principal component of Lin’s system is an engineered protein dimer (a green fluorescence protein) that, upon exposure to blue light (500 nm), converts to two monomers. Upon violet light (400 nm) exposure, the monomers revert to the dimeric form. Without violet light, the monomers will slowly dimerize in approximately 15 minutes, says Lin.

By encoding these monomers as domains on either side of the active-site sequences of kinases, Lin’s team has created enzymes that are inactive when the domains are dimerized and active when the domains separate into monomers. Janovjak likens these engineered kinases to people folding and unfolding their arms. “If I were to cross my arms in front of me, that makes interactions with other people more difficult,” he says.

What’s more, the fluorescent protein itself changes brightness from high to low as it switches from dimer to monomer, providing a visual indication of kinase activation, Lin explains.

Using this dimerization technique, Lin’s team has made four photo-switchable kinases, which the researchers have shown work as well as the endogenous enzymes in both cells and animals. But, the concept could be readily applied to other types of proteins, says Lin. (Science, 355:836-42, 2017) 

KINASE PHOTO-ACTIVATION TECHNIQUE
 
HOW IT WORKS ENGINEERING REQUIRED REVERSIBLE ACTIVATION SUITABLE TARGETS
Light-induced kinase
activator localization
 
The system uses the light-induced binding of a phytochrome (Phy) to phytochrome interaction factors (PIF), all found naturally in photosynthetic organisms. A kinase activator is engineered to contain a PIF domain, and a Phy domain is tethered to a location of interest—say, the plasma membrane. Light prompts recruitment of the activator to the desired site (by Phy-PIF interaction) and consequent kinase activation.
 
Two recombinant proteins Yes Kinases regulated by localization activators
 
Single-chain photoswitchable kinases
 
A kinase is engineered to contain two monomer domains that interact to form a dimer. This dimerization folds and inactivates the kinase. Blue light separates the monomers, thus unfolding and activating the enzyme.
 
One recombinant protein Yes In theory, all kinases
 

 

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