Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

Allele Biotech has just made a news announcement indicating that researchers from Dr. Campbell’s lab at the University of Alberta, Canada, and scientists at Allele Biotech Drs. Nathan Shaner and Jiwu Wang published a paper in the Journal of Molecular Biology on July 5th introducing a new photoconvertible fluorescent protein mClavGR.

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and overcome the limitations imposed by the oligomeric structure of the natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 (ICAM-1) both when the membrane is in contact with a T lymphocyte expressing leukocyte function-associated antigen 1 (LFA-1) and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein sub-populations and subsequent tracking of dynamic changes in localization in living cells.

Compared with previously available photoconvertible FPs, mClavGR2 has much improved photostability of the red state under confocal illumination conditions, 3644 over mEOS2’s 2700 and Dendra2’s 2420. Most notable among other advantages of mClavGR2 is its monomeric structure, its highly optimized and relatively rapid folding efficiency, and its high photoconversion effi ciency due to the high pKa of the green state. Its brightness in both the green and the red states is similar to the popular mCherry.

In regard to monomeric state, the monomeric variant of EosFP, known as mEos, was created through the introduction of two point mutations that disrupted the protein-protein interfaces of the tetrameric species. Expression of mEos at temperatures of greater than 30 °C is problematic, but an effectively monomeric tandem dimer variant does express well at 37 °C. mEos2 has been reported to retain some propensity for dimer formation.

We anticipate that this new addition to the toolbox of engineered FPs will be of great utility in imaging of fast protein dynamics in live cells. Experiments to determine whether the advantages of mClavGR2 translate to improved performance in super-resolution imaging applications have been initiated.

Hiofan Hoi(a), Nathan C. Shaner(b), Michael W. Davidson(c), Christopher W. Cairo(a), d, Jiwu Wang(b) and Robert E. Campbell(a)
a University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada T6G 2G2
b Allele Biotechnology, 9924 Mesa Rim Road, San Diego, California 92121
c National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310
d Alberta Ingenuity Centre for Carbohydrate Science
Received 20 February 2010; revised 15 June 2010; accepted 25 June 2010. Available online 5 July 2010.

New Product of the Week 070510-071110: mClavGR greeen-to-red photoconvertible fluorescent protein, catalogue number to be created

Promotion of the Week 070510-071110: Purified lanYFP, bright even in SDS-PAGE gel WITHOUT dye or excitation, great for in gel marker.

Tags: Dendra2, dynamic imaging, FP, FPs, mCherry, mEOS, mKirGR, PALM, photoconvertible, photoswitchable, STORM