Menu

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
 

 

September 2018

The Muscle Issue

The dynamic tissue reveals its secrets

Marketplace

Sponsored Product Updates

StemExpress LeukopakâNow Available in Frozen Format

StemExpress LeukopakâNow Available in Frozen Format

StemExpress, a Folsom, California based leading supplier of human biospecimens, announces the release of frozen Peripheral Blood Leukopaks. Leukopaks provide an enriched source of peripheral blood mononuclear cells (PBMCs) with low granulocyte and red blood cells that can be used in a variety of downstream cell-based applications.

New Antifade Mounting Media from Vector Laboratories Enhances Immunofluorescence Applications

New Antifade Mounting Media from Vector Laboratories Enhances Immunofluorescence Applications

Vector Laboratories, a leader in the development and manufacture of labeling and detection reagents for biomedical research, introduces VECTASHIELD® Vibrance™ – antifade mounting media that delivers significant improvements to the immunofluorescence workflow.

Best Practices for Sample Preparation and Lipid Extraction from Various Samples

Best Practices for Sample Preparation and Lipid Extraction from Various Samples

Download this white paper from Bertin Technologies to learn how to extract and analyze lipid samples from various models!

Bio-Rad Launches CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin for Process Protein Purification

Bio-Rad Launches CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin for Process Protein Purification

Bio-Rad Laboratories, Inc. (NYSE: BIO and BIOb), a global leader of life science research and clinical diagnostic products, today announced the launch of two new chromatography media for process protein purification: CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin.