mNeonGreen, the brightest monomeric green fluorescent protein

Somewhat like smartphones, the spec competitions of fluorescent proteins are also quite intensive these days. From time to time, the crown of brightest fluorescent protein falls onto another newly engineered fluorescent protein. Today let’s take a look at the currently brightest GFP: mNeonGreen¹, meanwhile also briefly review the former kings of GFPs.

mNeonGreen is an engineered monomeric green-yellow fluorescent protein(Ex 506nm/Em 517nm) from LanYFP, which is bright tetrameric yellow fluorescent protein (Ex 513nm/Em 524nm) originated from lancelet, rather than the conventional origins like jellyfish or coral.

When evaluating a FP, brightness, photostability and maturation time are  usually the top three specs to consider. The product of extinction coefficient and fluorescence quantum yield determines brightness. Photostability is the time to photobleach 50% fluorescence intensity under widefield arc lamp. Maturation time means the time for fluorescence to reach its half-maximal value after exposure to oxygen at 37 °C. In the case for mNeonGreen, it excels in all these three aspects.  Comparing to EGFP, mNeonGreen is 3X brighter, 2X faster for maturation with comparable photostability, yet EGFP is the golden standard of photostability for All FPs. By the numbers, mNeonGreen indeed sets a new bar for FP specs.

From the table above, it is clear that mNeonGreen is the new owner of GFP throne. But it doesn’t necessarily mean that the former Kings are useless now, in fact, all these FPs will probably still play important roles as they used to be.

        o   EGFP: the all time first choice of GFP.  It has being used and tested for over a decade without any problem,  EGFP is still the standard today.

o   mEmerald²: the predecessor of EGFP. It folds more efficiently than EGFP but with a fast photobleaching component, mEmerald is also wide used today.

o   mWasabi³: derived from mTFP1 (by our group!). With 2X brightness compared with EGFP, mWasabi was the brightest GFP for quite a few years till Clover came out.

o   Clover⁴: a recent bright variant of EGFP. Clover is paired with mRuby to form the new standard FP FRET pair for the replacement of CFP/YFP.

o   sfGFP(superfolder)⁵: as the name implies, sfGFP folds extremely fast, and is also extremely stable. It is mostly used to construct split GFP.

Maybe mNeonGreen is capable of replacing and surpassing the GFPs above in various situations, but until it is widely spread out and used, nothing is sure. We will have to wait and see. 

Nathan Shaner, who is also the main contributor of the widely used mFruits red fluorescent proteins^6,7, does the engineering work of mNeonGreen. This work of monomerization and improvement is also a textbook-like example for fluorescent protein engineering.

Interestingly, if you navigate through Allele biotech (distributor of mNeonGreen, mTFP1 and mWasabi) website, you will notice there is a red fluorescent protein from Lancelet named as LanRFP⁸(Ex 520nm/Em 600nm).  My guess is that LanRFP is also a tetramer and Nathan is trying to monomerize it. After it is optimized, one can imaging that it to be paired with mNeonGreen to be a new FRET pair.

Well, which fluorescent protein is next to be crowned? 

Reference:

1.     Lambert, G. G. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 1–8 (2013). doi:10.1038/nmeth.2413

2.     Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nat Methods 2, 905–909 (2005).

3.     Ai, H.-W., Olenych, S. G., Wong, P., Davidson, M. W. & Campbell, R. E. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging. BMC Biol 6, 13 (2008).

4.     Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).

5.     Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24, 79–88 (2005).

6.     Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572 (2004).

7.     Shaner, N. C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5, 545–551 (2008).

8.      http://www.allelebiotech.com/lanRFP/