New technology illuminates protein interactions in living cells

Nov 09, 2007
New technology illuminates protein interactions in living cells
Schematic of fluorescent detector: When a target protein is folded correctly, "tags" come together so that the dye binds with high affinity and fluoresces brightly; misfolded proteins have low affinity for the dye. Credit: Schepartz/Nature Chemical Biology

While fluorescence has long been used to tag biological molecules, a new technology developed at Yale allows researchers to use tiny fluorescent probes to rapidly detect and identify protein interactions within living cells while avoiding the biological disruption of existing methods, according to a report in Nature Chemical Biology.

Proteins are commonly tagged using variants of the “green fluorescent protein” (GFP), but these proteins are very large and are often toxic to live cells. They also tend to aggregate, making them difficult to work with and monitor. This new methodology uses the fluorescence emitted by a small molecule, rather than a large protein. It gives researchers a less disruptive way to capture images of the intricate contacts between folded regions of an individual protein or the partnerships between proteins in a live cell.

“Our approach bypasses many of the problems associated with fluorescent proteins, so that we can image protein interactions in living cells,” said senior author Alanna Schepartz, the Milton Harris Professor of Chemistry, and Howard Hughes Medical Institute Professor at Yale. “Using these molecules we can differentiate alternative or misfolded proteins from those that are folded correctly and also detect protein partnerships in live cells.”

Each protein is a three-dimensional structure created by “folding” its linear chain of amino acids. Usually only one shape “works” for each protein. The particular shape a protein takes depends on its amino acids and on other processes within the cell.

Schepartz and her team devised their new tagging system using small molecules, called “profluorescent” biarsenal dyes. These molecules easily enter cells and become fluorescent when they bind to a specific amino acid tag sequence within a protein. While these compounds have been used for about a decade to bind single proteins, this is the first time they have been used to identify interactions between proteins.

The researchers’ strategy was to split the amino acid tag for the dye into two pieces, locating each piece of the tag far apart in the chain of a protein they genetically engineered and expressed in the cells. Then they monitored cells exposed to the dye. Where the protein folded correctly, the two parts of the tag came together and the fluorescent compound bound and lit up. There was no signal unless the protein folded normally.

“This method of detection can provide important insights into how proteins choose their partners within the cell — choices that may be very different from those made in a test tube,” said Schepartz. She emphasizes that this technology does not monitor the process of protein folding — but, rather “sees” the protein conformations that exist at a given time.

“In theory, our technique could be used to target and selectively inactivate specific protein complexes in the cell, as therapy, or to visualize conformations at very high resolution for diagnostic purposes,” said Schepartz. She speculates that the technology could be applied to detection strategies that identify protein misfolding in neurodegenerative diseases like Alzheimer’s or Parkinson’s.

Source: Yale University

Explore further: Leave that iguana in the jungle, expert tells Costa Rica

add to favorites email to friend print save as pdf

Related Stories

Light of life

3 hours ago

A fluorescent microscopic view of cells from a type of bone cancer, being studied for a future trip to deep space – aiming to sharpen our understanding of the hazardous radiation prevailing out there.

How plant cell compartments change with cell growth

Aug 22, 2014

A research team led by Kiminori Toyooka from the RIKEN Center for Sustainable Resource Science has developed a sophisticated microscopy technique that for the first time captures the detailed movement of ...

Сalculations with nanoscale smart particles

Aug 19, 2014

Researchers from the Institute of General Physics of the Russian Academy of Sciences, the Institute of Bioorganic Chemistry of the Russian Academy of Sciences and MIPT have made an important step towards ...

Elusive viral 'machine' architecture finally rendered

Aug 11, 2014

For half a century biologists have studied the way that the lambda virus parks DNA in the chromosome of a host E. coli bacterium and later extracts it as a model reaction of genetic recombination. But fo ...

Recommended for you

Team defines new biodiversity metric

Aug 29, 2014

To understand how the repeated climatic shifts over the last 120,000 years may have influenced today's patterns of genetic diversity, a team of researchers led by City College of New York biologist Dr. Ana ...

Danish museum discovers unique gift from Charles Darwin

Aug 29, 2014

The Natural History Museum of Denmark recently discovered a unique gift from one of the greatest-ever scientists. In 1854, Charles Darwin – father of the theory of evolution – sent a gift to his Danish ...

User comments : 0