What is the maturation time for fluorescent proteins?
Fluorescent proteins have become a dominant tool for the exploration of the dynamics and localization of the macromolecular contents of living cells. Given how pervasive the palette of different fluorescent proteins with their many colors (see Figure 1) and properties has become, it is incredible that we have really only seen a decade of concerted effort with these revolutionary tools. Indeed, it is difficult to imagine any part of biology that has not been touched in some way or another (and often deeply) by the use of fluorescent reporter proteins. However, as a tool for exploring the many facets of cellular dynamics, fluorescent proteins have both advantages and disadvantages. Once a fluorescent protein is expressed it has to go through several stages until it becomes functional as shown in Figure 2. These processes are together termed maturation. Until completion of the maturation process, the protein, even though already synthesized, is not fluorescent. To study dynamics, it is most useful if there is a separation of time scales between the reporter maturation process (which preferably should take place on “fast” time scales) and the dynamics of the process of real interest (that should be much slower than the maturation time). The first stage in the maturation process (not depicted) is the most intuitive and refers to the protein folding itself, which is relatively fast and should take less than a minute, assuming there is no aggregation. The next stage is a torsional rearrangement (Figure 2B, C) of what can be thought of as the active site of the fluorophore, the amino acids where the conjugated electrons that will fluoresce are located. The next step, known as cyclization (where a ring is formed between two amino acids, Figure 2 C, D), is longer but still fast in comparison to the final and rate-­‐
limiting step of oxidation. In this final oxidation step, molecular oxygen grabs electrons from the fluorophore, creating the final system of conjugated bonds. All these steps are a prerequisite to making the active site fluoresce. There are only a limited number of reliable measurements of the maturation time, and the values are still far from being completely agreed upon. One approach to measure fluorophore maturation is by moving from anaerobic growth where the fluorophore protein is expressed but cannot perform the slowest step of oxidation to aerobic conditions and watching the rate of fluorescent signal formation. More commonly, inducible promoters or cycloheximide induced translation arrest are used. Nagai et al. (BNID 103780) measure a time scale of less than 5 minutes for the maturation of YFP and 7 minutes for the corresponding maturation of GFP in E. coli. By way of contrast, Gordon et al. (BNID 102974) report a time scale of ≈40 minutes for the maturation of YFP and a very slow ≈50 minutes for the maturation of CFP though part of the difference can be explained by the fact that in this case the measurements were carried out in yeast at 25oC. The measurements were done by inducing expression and after 30 minutes inhibiting translation using cycloheximide. The dynamics of continued fluorophore accumulation even after no new proteins are synthesized was used to infer the maturation time scale. Note that for many of the processes that occur during a cell cycle such as expression of genes in response to environmental cues, the maturation time can be a substantial fraction of the time scale of the process of interest. If a marathon runner stops for a drink in the middle of a race, this will hardly affect the overall time of the racer’s performance. On the other hand, if the runner stops to have a massage, this will materially affect the time scale at which the racer completes the race. By analogy with the runner stopping at a restaurant, the maturation time can seriously plague our ability to accurately monitor the dynamics of a variety of cellular processes. Chromophore maturation effectively follows first-­‐order kinetics in most studies performed. As a result, this implies that we will find a small fraction of functional flurophores much earlier than the maturation time. Still, to have the majority of the population active, the characteristic time scale we need to wait is roughly the maturation time itself. This effect results in a built-­‐in delay in the reporting system and should be heeded when estimating response times based on fluorescent reporters. Similarly, if translation is being stopped (say by the use of a ribosome inhibitor such as cyclohexamide) one would still have a period of time where some proteins that were translated before the inhibition are coming “online” and add to the signal. This again should be taken into account when estimating degradation times. Another dynamical feature of these proteins that can make them tricky for precisely characterizing cellular dynamics is the existence of photobleaching. This process has a characteristic time scale of tens of seconds using standard levels of illumination and magnification. This value means that after a continuous exposure to illumination for several tens of seconds, the fluorescent intensity will have decayed to 1/e of its original value. Though sometimes a nuisance, recently this apparent disadvantage has been used as a trick both in the context of fluorescence recovery after photobleaching (FRAP) that allows inference about diffusion rates and in superresolution microscopy techniques where the bleaching of individual fluorophores makes it possible to localize these proteins with nanometer scale resolution. Differences in maturation times of different fluorophores were recently turned into a way to measure rates of degradation and translocation without the need for time course measurments (Khmelinskii et al., 2012). Fusing two fluorescent tags (fast maturing GFP, so called superfolder, and the slower maturing mCherry) to a protein of interest and measuring the ratio of intensities can serve as a built in timer. It helps show for example that daughter cells tend to get the old copies of some protein complexes such as spindle pole bodies and nuclear pore complexes while the mothers retain the newly formed copies. mCherry
mOrange
mBanana
mHoneydew
YFP (Citrine)
EGFP
ECFP
EBFP
Figure 1: Illustration of some of the palette of fluorescent proteins that
has revolutionized cell biology. (A) Fluorescent proteins spanning a
range of excitation and emission wavelengths. (B) Illustration of a petri
dish with bacteria harboring eight different colors of fluorescent protein
and used to “paint” an idyllic beach scene. (Adapted from: R. Y. Tsien,
Nobel lecture, Integr. Biol.,2, 77-93, (2010).)
Thr65
torsional
rearrangements
Gly67
Gly67
Tyr66
Tyr66
ring formation
and oxidation
Gly67
conjugated
fluorescent core
Gly67
Tyr66
Thr65
Tyr66
extended polypeptide
Thr65
Thr65
mature fluorophore
Figure 2: Schematic diagram of the chromophore formation in maturing enhanced green fluorescent protein
(EGFP). (A) The prematuration EGFP fluorophore tripeptide amino acid sequence (Thr65-Tyr66-Gly67)
stretched into a linear configuration. The first step in maturation is a series of torsional adjustments (B) and
(C). These torsional adjustments allow a nucleophilic attack that results in formation of a ring system (the
cyclization step). (D) Fluorescence occurs following oxidation of the tyrosine by molecular oxygen. The final
conjugated and fluorescent core atoms are shaded. (Adapted from: The Fluorescent Protein Color Palette,
Scott G. Olenych, Nathan S. Claxton, Gregory K. Ottenberg, Michael W. Davidson, 2007).
Table 1: Common fluorescent proteins maturation times. Because different approaches and conditions still
give quite different values one should be very careful in studies where the maturation time can affect the
conclusions. In mCherry there are indications of two time scales, the first leading to fluorescence at a different
wavelength regime (Khmelinskii et al., 2012). Values are rounded to one significant digit. Comprehensive table
can be found at Lizuka et al, 2011. For definitions of fluorophores via mutations relative to WT see Table S2 of
Shaner et al, 2005.
Fluorophore
Maturation
Cell type
Ref
BNID
S. cerevisiae
Gordon et al., 2007, 25 C
106883
time (min)
ECFP
50±10
o
GFP wildtype
50
In vitro
Lizuka et al., 2011
106892
sfGFP
6
E. coli
Khmelinskii et al., 2012
110546
7±0.6
E. coli
Megerle et al., 2008
102972
GFPmut3
7
In vitro
Lizuka et al., 2011
107004
EGFP
60
E. coli
Sniegowski et al., 2005
107001
EGFP
14
In vitro
Lizuka et al., 2011
107000
Emerald
12
In vitro
Lizuka et al., 2011
106893
5
In vitro
Katranidis et al., 2009
S. cerevisiae
Gordon et al., 2007, 25 C
102974
GFPmut3
GFPem
106887
o
EYFP
40±7
EYFP
20
In vitro
Lizuka et al., 2011
106891
Venus
40
In vitro
Lizuka et al., 2011
106890
mCherry
15
E. coli
Shaner et al., 2004
106877
mCherry
40
E. coli
Merzlyak et al, 2007
110551
mCherry
30
S. cerevisiae
Maeder et al., 2007
mCherry
17+30
S. cerevisiae
Khmelinskii et al., 2012
110552
mStrawberry
50
E. coli
Shaner et al., 2004
106880
tdTomato
60
E. coli
Shaner et al., 2004
106876
mPlum
100
H. sapiens, B cell line
Wang wt al., 2004
106878