Aequorea victoria’s secrets

Gerard G. Lambert, Hadrien Depernet, Guillaume Gotthard, Darrin T. Schultz, Isabelle Navizet, Talley Lambert, Daphne S. Bindels, Vincent Levesque, Jennifer N. Moffatt, Anya Salih, Antoine Royant, Nathan C. Shaner

Preprint posted on June 28, 2019

Article now published in PLOS Biology at

‘Far beyond your average(Av)GFP - Aequorea victoria’s secret fluorophores revealed’. The Shaner Lab uncovers several new fluorophores from A. victoria and A. australis - including the brightest GFP homolog yet!

Selected by Marc Somssich


There can be no doubt that the engineering of Aequorea victoria’s green fluorescent protein (AvGFP) as a protein tag has been an unprecedented success for life science research1. First described as a side note in a 1962 paper, the AvGFP gene was eventually cloned and heterologously-expressed in 19922,3. The late Roger Tsien then used the original AvGFP in 1994 to evolve the GFP protein tag which would revolutionize biological imaging4. Today, the majority of commonly used fluorescent proteins (FP) is still derived from this original AvGFP, as the protein lineage tree on beautifully illustrates (,5. Now, a quarter century after AvGFP was cloned, the lab of Nathan Shaner has revisited A. victoria and its close relative A. australis to search for other FPs in their transcriptomes6.

Key findings

Gerard Lambert and colleagues used mRNA-Seq to analyze the transcriptome of A. australis. Using this approach they found the A. australis homolog of the original AvGFP (AausGFP), but also several other, more divergent AvGFP homologs. This surprise find prompted them to also revisit A. victoria using their experimental approach. Indeed, they were also able to identify several AvGFP orthologs in A. victoria, which so far went unnoticed due to their low expression levels, relative to AvGFP. Among the FPs identified are: (i) AausFP1, the brightest FP measured on a per-molecule basis, (ii) AvicFP1, a superior AvGFP version, resembling the engineered EGFP, (iii) AausFP2 and 3, two chromoproteins (CPs) with an unusual chromophore exhibiting a broad excitation spectrum with no detectable emission, (iv) AvicFP2 and 3, two weakly green fluorescent FPs which can be converted to non-fluorescent CPs by blue light, and (v) AausFP4, a photoswitchable Dreiklang-type FP.

Highlights of this Preprint

Given the importance and impact that AvGFP had on the life sciences as a whole, it is quite surprising that its close ortho- and homologs have never been identified. Hence, Nathan Shaner and his colleagues have to be commended on their idea to revisit Aequorea victoria. Their results, reported in this preprint, clearly reward them for it.

The significance of the FPs described in this publication does not lie in the proteins themselves, however, but in their unique features that hold the potential for them to serve as templates, or scaffolds, for the engineering of completely new lines of fluorescent probes of all types.

AausFP1 exhibits very narrow excitation/emission peaks, an almost perfect quantum yield and a brightness almost five-fold higher than EGFP, and almost double that of the already formidable mNeonGreen. It therefore holds great potential as it is, and Lambert and colleagues already report its successful monomerization, though we have to wait for the next preprint from the lab to read about the details. Additionally, given that the closest homolog to AausFP1, AvicFP4, is the dimmest FP in A. victoria, a comparison of these two FPs poses an interesting subject to further understand the evolution and underlying molecular properties governing brightness of a FP.

AvicFP1 basically is an optimized AvGFP which naturally carries some of the critical mutations identified to create EGFP, and furthermore exhibits improved folding and maturation properties. Based on the measurements described here, AvicFP1 is already superior to EGFP with only two mutations inserted, and it therefore holds the potential to become an optimal fluorescent tag. Successful monomerization and employment of AvicFP1 as a LifeAct or H2B-tag are already reported in this preprint as well.

And while these two FPs are already impressive, even more potential may lie in the unusual CPs of AausFP2/3 and the photoconversion- and switching properties of AvicFP2/3 and AausFP4: The crystal structure of the AausFP2 revealed that a conserved cysteine (C62) is covalently linked to the methylene bridge of a slightly twisted GFP-like chromophore. This could account for its unique absorbance and lack of any detectable emission. Similarly, its homolog AvicFP2 behaves like a GFP in darkness, but blue light exposure results in an attachment of C62 to the chromophore and a shift towards a 588 nm absorbance with no emission, thereby making it similar to AausFP2. These proteins harbor the potential to serve as scaffolds for new photoconvertible FPs, far-red FPs or dark FRET-acceptors.

Finally, AausFP4 is similar to the artificial Dreiklang fluorophore, in which excitation, as well as on and off switching are all directed by light of different wavelengths7. Both fluorophores are switched on by UV light and excited by cyan light, but different to Dreiklang, which is switched off by near-UV light, AausFP4 is switched off by bright blue light. AausFP4 is therefore the first naturally occurring Dreiklang-type FP described and may help to further build on this mechanism.

Future directions

Some obvious questions at this point of course concern the further engineering of AausFP1:
– Does it retain all of its amazing properties, especially the brightness, once it is monomerized? Is it functional as a protein-tag?

– What might be the biggest differences between AausFP1 and AvicFP4, making these two very similar proteins the brightest and dimmest FP in Aequorea respectively?

– Are the FPs and CPs identified also coupled to aequorin? And does this coupling influence the properties reported here, especially for the photoconvertible/switchable proteins, which appear to not emit any light?

– And on a related note: Could any other cofactors play a role in this regard, that would influence these proteins to emit any light?

The new FPs presented in this preprint are meant to serve as the basis for novel engineered FPs. Accordingly, the logical next steps are the more detailed characterization of each of these proteins. While the very first steps have already been done for AausFP1 (manuscript in preparation) and AvicFP1 (reported here), a better description and understanding of the molecular details of AausFP2/3, AvicFP2/3 and AausFP4 could prove even more significant in the long run. Once we have a better understanding of their properties, it will become clearer which of the countless possibilities one can envision now can actually be realized.

And if we now consider that the results reported here are only from two of over 50 fluorescent species the Shaner lab has collected on their 2017 trip to Heron Island, one can only imagine what is to come from this lab in the coming years.

Finally a short note on the preprint itself: The manuscript is clearly written for a high-profile journal, and accordingly, the data is compressed heavily. It is therefore highly recommended to get the full (almost 10 Mb) supplementary pdf, which is full of additional details for each of the proteins.


  1. Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, et al. The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci. 2017;42: 111–129. Available at doi:10.1016/j.tibs.2016.09.010
  2. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992;111: 229–33. Available at doi:10.1016/0378-1119(92)90691-H
  3. Johnson FH, Shimomura O, Saiga Y, Gershman LC, Reynolds GT, Waters JR. Quantum efficiency of Cypridina luminescence, with a note on that of Aequorea. J Cell Comp Physiol. 1962;60: 85–103. Available at doi:10.1002/jcp.1030600111
  4. Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A. 1994;91: 12501–4. Available at doi:10.1073/pnas.91.26.12501
  5. Lambert TJ. FPbase: a community-editable fluorescent protein database. Nat Methods. 2019; Available at doi:10.1038/s41592-019-0352-8
  6. Lambert GG, Depernet H, Gotthard G, Schultz DT, Lambert T, Bindels DS, et al. Aequorea victoria’s secrets. bioRxiv. 2019;: 1–14.
  7. Brakemann T, Stiel AC, Weber G, Andresen M, Testa I, Grotjohann T, et al. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol. Nature Publishing Group; 2011;29: 942–947. Available at doi:10.1038/nbt.1952

Tags: fluorescence, fluorophores, gfp, microscopy

Posted on: 7th July 2019


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