Tag Archives: GFPmut2

Folding and maturation, or how to evolve your own GFP (part 2)

In part 1 I discussed the directed evolution of fast-folding GFPs. These were developed for specific purposes of improving the solubility, stability and folding of the protein. Now, I will discuss the maturation step and how it was measured for a variety of GFPs.

As mentioned in the first part, maturation is the final step in the transformation of GFP from a chain of amino acids to a fluorescent protein – the creation of the chromofore. The chromofore has a very long name (p-hydroxybenzylideneimidazolidone) which in wild type  GFP is formed from amino acids S65, Y66 and G67. In this process, the amide nitrogen of G67 backbone performs a nucleophilic attack on the carbonyl carbon of S65. Oxidation with atmospheric O2 and dehydration reactions create the imidazolinone ring, which is conjugated to the side chain of Y66.

In most papers that measure GFP maturation, the authors do not distinguish folding from maturation. In many cases, the experiments involve complete denaturation of the protein in vitro (e.g. by urea or guanidinium chloride) and then measuring fluorescence following re-folding. In vivo, it would be difficult to assess folding kinetics (because you do not really have an exact time zero for when the protein is being translated).  [In our lab, we are trying to develop a system to visualize translation in vivo in real time by imaging techniques.  If it works, it would be useful for determining exact folding & maturation rates in vivo of a single fluorescent protein].

In any case, it is easier to measure the kinetics of the maturation step, both in vivo and in vitro, simply by removing and adding oxygen. I mentioned in part 1 that this is how the maturation rate of GFP-S65 was calculated in vivo.  A new paper from 2011 from the lab of Funatsu analyzed the maturation rates of several GFPs in vitro. Their idea was simple – using a commercial kit, the performed in vitro transcription-translation reaction of the FPs at anaerobic conditions (they added catalase and glucose oxidase to get 0.1mg/l of oxygen). They then stopped the reaction and added oxygen, then followed fluorescence.

The proteins they assayed were: wild-type GFP, GFP-S65T, GFP-S65T/S147P, EGFP, sfGFP, Emerald, GFPm, GFPmut2, GFPmut3, sgGFP, “cycle 3”(a.k.a GFPuv), frGFP (a.k.a GFPuv3), GFPuv4 and 5 and two yellow FPs: EYFP and Venus..

Their results are interesting. As expected, the S65T mutation improved the maturation rate by 3.2 fold. However, EGFP matured only 20% faster than the S65T alone. The S65T/S147P is a variant that is stable under a range of temperatures. This mutant matured faster than EGFP, at a rate 40% faster than S65T alone. GFPmut2 and GFPmut3 (S65G/S72A) both showed a 7-fold faster maturation than WT GFP (>2-fold the S65T alone). sgGFP (SuperGloGFP, F64L/S65C/I167T, from Qbiogene) showed improved maturation rate compared to S65T (70%  faster).  Emerald, which contains 4 mutations on top of the EGFP mutations showed only a slight increase in maturation rate (similar to S65T/S147P). “cycle 3” was only slightly better than WT GFP, frGFP (which is cycle 3 +EGFP mutations combined) showed a maturation rate which was 10% less than EGFP.  Addition of the I167T mutation to create GFPuv5 increased the maturation rate by ~70% (just like in sgGFP).

Most interesting is the super-folder GFP (sfGFP), which showed a maturation rate of only 2.3-fold over the WT (that is ~70% of that of S65T). Thus, though this protein may be more stable and may fold very fast compared to other variants, the important step of maturation is the slowest among all variants tested (except the WT). Since folding assays measure fluorescence as the output for a mature protein, it means that the folding step (prior to maturation) is much faster than previously appreciated.

GFPm is a weird case.  GFPm, developed by David Tirrell, is a variant with mutations from cycle 3 and GFPmut3. However, Tirrell tried something unique –  to replace the leucines in the protein with 5,5,5- tri-fluoroleucine (tfl). The fluoreinated form proved to be insoluble, and fluorescent of the cells (E. coli) was 500-fold less than the regular protein. They then went on with mutagenesis, developing new variants which were brighter (up to 650-fold over GFPm). Personally, I do not understand how this can be of much use, since using tfl will probably have major effects on every aspect of cell biology we are interested in. Anyway, the maturation rate of GFPm (not fluoreinated) is itself pretty high – almost 3-fold over S65T and is actually the fastest maturing GFP variant tested. Oddly, they do not discuss this result anywhere in the text. Perhaps there is a technical issue they wanted to avoid?

Last but not least, the two YFPs showed maturation rates which are similar to WT GFP (Venus) or ~2.3-fold better (EYFP).

So how does all this information help us?

First, if we know which mutations enhance maturation and which slow it down, we can design faster-maturing proteins.

Second, we can use this data to estimate translation or translocation rates in vivo. However, we should remember that the data obtained in vitro (at 37C) does not neccessarily agree with actual maturation rates in vivo in every cell type. for instance, yeast or fly grow in colder temperatures which may affect maturation. Oxygen levels in tissue culture dish are differnt than in entire animals, and also differnt in differnt organs.  Also, if the GFP is fused to another protein, it may also affect folding as we learned in part 1, as well as maturation. Finally, the in vitro environment in the tube lacks many biomolecules (proteins other than used for translation, small molecules, ions, oxidizing molecules, antioxidants etc) which can affect oxygen availability in the immediate environment of the newly translated GFP.

Third, we can use such data to design cool experiments. So… stay tuned for part 3, which I will dedicate to biological timers.

ResearchBlogging.orgIizuka R, Yamagishi-Shirasaki M, & Funatsu T (2011). Kinetic study of de novo chromophore maturation of fluorescent proteins. Analytical biochemistry, 414 (2), 173-8 PMID: 21459075
Yoo TH, Link AJ, & Tirrell DA (2007). Evolution of a fluorinated green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 104 (35), 13887-90 PMID: 17717085

Folding and maturation, or how to evolve your own GFP (part 1)

GFP is one of the most widely used proteins in research. Its usefulness has advanced our understanding of biology in huge leaps forward. One of the greatest advantages of GFP is that the chromophore is formed in an autocatalytyic manner, no need for an enzyme or cofactor assistance. All that is required is atmospheric oxygen.

GFP has sort of a barrel shape, with the chromophore inside. The chromophore is formed from residues serine 65 (S65), tyrosine 66 (Y66) and glycine 67 (G67). The process of creating the structure of the protein is called folding, whereas the process of creating the chromophore is termed maturation. Maturation occurs after the protein folds to near native conformation.

However, the natural, wild type, GFP is not the best tool. It is rather dim and not very stable at 37°C  (Since the jellyfish lives at low temperatures). Also, it matures rather slowly. Early on, a mutation was introduced, S65T, that increased the brightness of the GFP and also shifted its excitation peak (from a major peak at 395 and a minor at 475, to a single major peak at 488).  In vivo, it was shown that maturation of GFP-S65T takes 27 minutes (this was measured by expressing GFP in E. coli bacteria under anaerobic conditions, then supplementing the bacteria with O2. Therefore, they only measured the last few steps of the maturation process). EGFP, the most common variant used in research labs, contains a second mutation, F64L. This mutation stabilizes the protein at 37°C and was suggested to increase the maturation rate.

Over the years, GFP has been a subject for further “directed evolution” to achieve required traits like different colors, brightness, pH stability and photostability, oligomerization state, as well as folding and maturation.

Why do we care about folding and maturation?

Well, GFP is often fused to other proteins. In some cases, that protein is misfolding, or folding slowly. This can affect the folding of GFP. This is particularly true when expressing fused proteins in E. coli, where in many cases the ectopically expressed protein is aggregating in inclusion bodies. Since GFP itself has a slight tendency to dimerize, any aggregation of the fusion protein may amplify the dimerization of the GFP. Thus, it would be advantageous to have a better folding GFP, with a lower tendency to dimerize.

As to maturation, having a fast-maturing GFP would be very beneficial for studies of translation, or translation-coupled localization.  Because, if the GFP takes 20-30 minutes to mature after it is being translated, we cannot really say what happened and where was the GFP or any fused protein, in that time.

Our story begins in 1996, when the lab of Willem Stemmer tried to improve GFP by a technique called DNA shuffling. Their goal was to improve whole cell fluorescent in E. coli cells expressing GFP. After three cycles of DNA shuffling, and selection based on fluorescence intensity with UV excitation, they isolated a clone (named simply ‘GFP cycle 3’) which showed 45-fold increased fluorescence compared to the then commercial Clontech pGFP. However, the spectral characteristics were unchanged, and the maturation rate (T1/2 = 95min at 37°C in whole cells) also seemed unchanged).  Mutations found in GFP cycle 3 compared to their starting GFP showed that three hydrophobic amino acids were replaced by hydrophilic amino acids. Thus, the improved fluorescence is probably due to reduction in protein aggregation by the hydrophobic surfaces, and increased solubility of the GFP.

CHO cells expressing wt GFP (A) or GFP cycle 3 (B). Source: Crameri et al. (1996) Nat. Biotech. 14:315.

Ten years later, the superfolder GFP (sfGFP) was engineered by the lab of Geoffrey Waldo for the particular purpose of creating a protein with less tendency to misfold when fused to poorly folded proteins in E. coli.

How was this protein engineered?

They started with a folding reporter GFP  (frGFP) which contained the cycle 3 & EGFP mutations.

They fused frGFP to a poorly folding protein, H subunit ferritin, which is insoluble when expressed in E. coli. After several rounds of mutagenesis and screening for fluorescence, they obtained the highly fluorescent sfGFP which bears six new mutations.  Ferritin-sfGFP was found to be 50-fold more fluorescent than the starting fusion protein. When expressed alone, cells expressing sfGFP were twice as bright as with frGFP. Importantly, the excitation & emission spectra , QY and EC were similar in both proteins.

Measuring the folding rate was done by first denaturing the proteins with urea. Then washing the urea and measuring the time it takes the GFP variant to regain fluorescence.  This method actually measures folding and maturation and this should be taken into account. In any case, though both proteins showed 95% yield within 4 minutes, sfGFP showed a 3.5-fold faster initial rate. sfGFP also tolerated higher urea levels, and showed better fluorescence with a bunch of different fused proteins with different characteristics (expression level, solubility etc…).

fusion of frGFP or sfGFP to 18 differnt proteins shows sfGFP much brighter in all cases. 0.125s, 1s: exposure time. below the images of the bacteria are Western blot images of the fusion proteins. Source: Pedelacq et al. (2006). Nat. Biotech. 24(1):79

They found that the most contributing mutation to the folding  kinetics and stability is the S30R mutation. The change from an oxygen group (that forms a hydrogen bond with E17) to a positively charged side chain mediates an electrostatically charged network of the β-barrel which seems to create a global stability to the structure. A second mutation, Y39N, slightly changes the angle of the backbone, and allows a hydrogen bond with D36, further stabilizing the structure. The other four mutations slightly contribute to the stability.

Although sfGFP seems to fold fast (though no comparison to EGFP was made in this paper), the assays here did not really differentiate folding from maturation.

A year later, the group of David Liu used sfGFP  in an effort to create a new GFP protein that is highly soluble. In order to do that, they replaced multiple neutral amino acids on the outer side of the protein with either positively or negatively charged amino acids to produce super-charged GFP variants GFP(+36) and GFP(-30) [compared to the net charge of sfGFP which is (-7) or GFP which is (-8)]. This scGFP was highly soluble, and also highly resistant to denaturing, even by boiling to protein at 100°C. The spectral characteristics of the scGFPs remained similar to those of GFP and sfGFP indicating that the structure of the outside “barrel” does not affect the chromophore features inside the barrel.

In 2008, Fisher & DeLisa produced yet another variant of GFP called superfast GFP. By exploiting the cell’s protein secretion quality control mechanisms, they screened for new super folding variants. The secretion mechanism exports unfolded proteins to the periplasm of E. coli. In the periplasm, GFP is apparently non-fluorescent. If the protein folds fast enough, the quality control mechanism prevents its export and the protein remains in the cytoplasm.  Their starting GFP was GFPmut2, a variant that was previously optimized for FACS analysis, harboring the mutations S65A/V68L/S72A. Following several round of selection, they isolated a clone which they named superfast GFP. Based on their analysis (using guanidinium chloride (GdnCl) to unfold the proteins and then diluting the GdnCl to refold the proteins, the T1/2 for superfast GFP refolding (resumed fluorescence) was 11 minutes, compared to 33 min (GFPmut2), 20min (sfGFP) and 73min (frGFP).

Graph showing folding of GFP proteins over time. black: square – frGFP; circle – sfGFP; triangle – GFPmut2. open circle – superfast GFP; triangle, square – two other mutants. Source: Fisher AC & DeLisa MP. (2008). PLoS One. 3(6):e2351.

So, far we discussed fast folding GFP proteins. However, the methods to measure folding of these proteins do not discriminate folding from maturation.  Furthermore, the in vitro results do not necessarily represent the in vivo folding rates (i.e. 11 min in vitro does not mean 11 min in vivo).

In the next post I will discuss the issue of maturation.

ResearchBlogging.orgCrameri A, Whitehorn EA, Tate E, & Stemmer WP (1996). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature biotechnology, 14 (3), 315-9 PMID: 9630892
Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, & Waldo GS (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology, 24 (1), 79-88 PMID: 16369541
Lawrence MS, Phillips KJ, & Liu DR (2007). Supercharging proteins can impart unusual resilience. Journal of the American Chemical Society, 129 (33), 10110-2 PMID: 17665911
Fisher AC, & DeLisa MP (2008). Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control. PloS one, 3 (6) PMID: 18545653