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/

UnaG, a new green fluorescent protein from japanese eel

Unagi is a often seen dish in japanese cuisine, yet it never occurred to me that the eel can be fluorescent. Well, indeed it is. As Atsushi Miyawaki discovered this bilirubin binding fluorescent protein in eel muscle, for the very first time, a fluorescent protein in vertebrate is identified.

The green fluorescence of UnaG is from binding of a endogenous chromophore ligand called bilirubin(BR). This is very different from Jellyfish Green Fluorescent Protein(GFP), which is self-sufficient to form a chromophore by a series of autocatalytic post-translational modification. Thus GFP and its homologues are also referred as auto fluorescent proteins (AFP).

Apart from AFPs, yet exists another type of fluorescent proteins I would like to call chromophore-binding fluorescent protein, such as IFP and iRFP. They are both near-infared fluorescent proteins engineered from biliverdin (BV) binding bacterial phytochrome. Much like UnaG, these proteins show no fluorescence at apo state, upon proper ligand binding, intensive fluorescence will be induced.

Compare to GFP, which is oxygen-dependent in chromophore formation, the advantage of UnaG is that it can be used under both aerobic and anaerobic conditions. And the brightness of UnaG in ligand bound state is also better than EGFP on numbers. These make UnaG a tempting alternative of GFP in some research areas. Due to the high-affinity and high specificity bilirubin binding property of UnaG, it was developed as a bilirubin sensor in the study.

Looking into the future, it is almost certain that a lot more fluorescent proteins like UnaG will be identified in vertebrates. As for UnaG itself, one can imaging it will be served as a prototype for engineering bight, stable fluorescent proteins, even with alternative colours.

Reference:

1.Kumagai, A. et al. A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 1–10 (2013). doi:10.1016/j.cell.2013.05.038