Letter pubs.acs.org/JPCL
A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation Joshua D. Slocum and Lauren J. Webb* Department of Chemistry, Center for Nano and Molecular Science and Technology, and Institute for Cell and Molecular Biology, The University of Texas at Austin, 105 East 24th Street STOP A5300, Austin, Texas 78712-1224, United States S Supporting Information *
ABSTRACT: A photoactivatable variant of superfolder green fluorescent protein (GFP) was created by replacing the threonine at position 203 with aspartic acid. Photoactivation by exposure of this mutant to UV light resulted in conversion of the fluorophore from the neutral to the negatively charged form, accompanied by a ∼95-fold increase in fluorescence under 488 nm excitation. Mass spectrometry before and after exposure to UV light revealed a change in mass of 88 Da, attributed to the double decarboxylation of Glu 222 and Asp 203. Kinetics studies and nonlinear power-dependence of the initial rate of photoconversion indicated that the double decarboxylation occurred via a multiphoton absorption process at 254 nm. In addition to providing a photoactivatable GFP with robust folding properties, a detailed mechanistic understanding of this double decarboxylation in GFP will lead to a better understanding of charge transfer in fluorescent proteins.
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quantum yield (∼0.6), either directly from the excited B state or from a deprotonated form of the excited A state after excited state proton transfer.10,12 Although fluorescence is the dominant decay pathway of these excited states, several infrequent CT pathways exist that lead to interesting fluorophore chemistry, including bleaching, oxidative reddening, and decarboxylation of Glu 222. Upon repeated illumination, these pathways can lead to the accumulation of various photoproducts,13,14 and has been the subject of a recent review regarding photoinduced chemistry in FPs.15 For example, the accumulation of decarboxylated Glu 222 in GFP causes an increase in the B state absorption at the expense of the A state. This is ultimately due to the change in fluorophore pKa that accompanies the decarboxylation of Glu 222. Optimization of the residues surrounding the fluorophore led to the development of PA-GFP, which exhibits a high fluorescence contrast (under 488 nm excitation) upon accumulation of decarboxylated Glu 222 and the concomitant population shift from neutral to charged fluorophore.3 In contrast to the SYG fluorophore, the one formed from Thr 65, Tyr 66, and Gly 67 (Figures 1A and 1B; hereafter TYG) has several interesting properties. First, mutants containing the TYG fluorophore typically have a dominant B state absorption at physiological pH. This largely has to do with the extra methyl group on Thr 65, which forces Glu 222 to donate a hydrogen bond to the −OH side chain of Thr 65, ultimately allowing Glu 222 to be neutral even at high pH.16,17 The neutral Glu 222 allows the negatively charged form of the fluorophore to be favored, compared to the mutants with Ser
he discovery and subsequent manipulation of naturally occurring fluorescent proteins (FPs) has revolutionized the field of biological microscopy by enabling the visualization of biomolecules in vivo.1,2 In addition to emission spectra that span much of the visible region of the spectrum, many FPs have photoconversion functionalities that make them useful for tracking biomolecule dynamics in live cells and have been used for superresolution imaging techniques. Perhaps the most wellknown FP with photoconversion functionality is photoactivatable green fluorescent protein (PA-GFP), which can be irreversibly photoactivated from a dark to a brightly emissive state with high contrast upon exposure to near-UV light.3 This photoactivation is thought to be initiated by an excited state electron transfer process, which has been proposed to occur in many FPs, and has been exploited in the development of other photoconvertable FPs including PA-GFP,3,4 DsRed,5 PAmCherry,6 and LSS-mOrange.7 Here, we show that this charge transfer (CT) pathway can be altered by a single amino acid substitution to impart the photoactivatable functionality onto superfolder GFP (sfGFP), which is otherwise not photoactivatable. In addition to providing a basis for the modulation of photoactivation in other FPs, we think that this photoactivatable mutant will provide a convenient model for understanding the mechanisms of CT in FPs. Photoconversion in FPs was first observed in the wild type form of GFP,1,8−10 whose embedded fluorophore is formed from the residues Ser 65, Tyr 66, and Gly 67 (hereafter SYG). In the ground state, the GFP fluorophore can be either neutral or negatively charged depending on the protonation state of Tyr 66. The neutral form (A state) absorbs maximally at 400 nm and is present in a ∼ 6:1 ratio relative to the negative form (B state), which absorbs maximally at 490 nm.11 Excitation of either of these states leads to green fluorescence with a high © XXXX American Chemical Society
Received: May 5, 2017 Accepted: June 9, 2017 Published: June 9, 2017 2862
DOI: 10.1021/acs.jpclett.7b01101 J. Phys. Chem. Lett. 2017, 8, 2862−2868
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Figure 1. (A) Top-down view of the crystal structure of sfGFP (2b3p) showing the embedded TYG fluorophore and residues Glu 222 and Thr 203. (B) A closer view of the fluorophore in sfGFP. Interactions between Thr 203 and the phenolate of Tyr 66 are shown, as well as the interaction between Glu 222 and Thr 65. (C) The absorption spectrum of sfGFP before and after 10 min of exposure to UV light. (D) The absorption spectrum of sfGFP T203D before and after 10 min of exposure to UV light.
residue near TYG would be necessary to force the equilibrium toward the A state such that its absorption spectrum resembled that of SYG. To do this, we replaced Thr 203 with Asp 203 because of the proximity of position 203 to Tyr 66 (Figures 1A and 1B), and because of the well-known influence of the position 203 side chain on the fluorophore pKa.19,20 We found that this T203D (which abbreviates the replacement of Thr at position 203 with Asp) mutation was enough to force the TYG equilibrium entirely to the A state (Figure 1D; black). Titration of the T203D mutant revealed that the B state of the fluorophore was only present above pH 9 (Figure 2, black circles), with the protein unfolding before the end of the titration. This suggests that the TYG fluorophore in the T203D mutant has a pKa of at least 10, which is at least 3 pH units higher than the pKa of wild-type sfGFP (Figure 2, green circles; pKa = 6.7) and almost 2 pH units higher than the highest
65, where Glu 222 is typically negative and destabilizes the B state. Additionally, the TYG fluorophore matures more quickly, has a higher extinction coefficient, and is more photostable, which has earned mutants containing the TYG fluorophore the title of “enhanced” GFPs due to their improved use as fluorescence tags for microscopy experiments.8 While the TYG fluorophore offers spectroscopic benefits, it is inherently incompatible with the high contrast photoactivation that is observed in PA-GFP due to its increased B state absorbance. Additionally, if Glu 222 is indeed neutral in GFP mutants with Thr 65, then decarboxylation of this group would not represent as drastic of an electrostatic change (compared to the decarboxylation of negative Glu 222) and would not be expected to shift the fluorophore equilibrium significantly. Furthermore, the reduction potential of neutral glutamic acid is much lower than that of the negatively charged carboxylate, which means that neutral Glu 222 will be less likely to undergo the initial CT step that is necessary for decarboxylation. To illustrate these points, we attempted to photoactivate super folder GFP (sfGFP), which has desirable folding properties and contains the TYG fluorophore.18 Figure 1C shows the absorption spectrum of sfGFP before (black) and after (red) 10 min of exposure to 254 nm light with a power density of ∼7 mW/cm2. As expected, because of the dominant B state absorption before UV exposure, we observed only a minimal increase in the B state absorption (∼10%) after 10 min of UV exposure. We then became interested in making mutations that would allow the TYG fluorophore in sfGFP to be photoactivated with a similar contrast to that of PA-GFP, which contains the SYG fluorophore. Because the SYG and TYG fluorophores generally have different hydrogen bonding networks that drastically affect the pKa Glu 222,16,17 we reasoned that a negatively charged
Figure 2. pH titrations of the TYG fluorophore. B state absorption relative to total protein concentration plotted against the solution pH. T203D titrates with a pKa of at least 10 before UV exposure (black circles), and decreases to 7.2 after UV exposure (red circles). For comparison, sfGFP (green circles) titrates with a pKa of 6.7. 2863
DOI: 10.1021/acs.jpclett.7b01101 J. Phys. Chem. Lett. 2017, 8, 2862−2868
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The Journal of Physical Chemistry Letters Scheme 1. Kolbe-Type Decarboxylation of Glu 222 As It Occurs in Fluorescent Proteins with the SYG Fluorophorea
a Upon the generation of an excited A state (represented by the asterisk), Glu 222 (green) transfers an electron (red) to the fluorophore, which results in the generation of a radical intermediate and the release of CO2 (green). The alkyl radical is quickly quenched, presumably by back transfer of an electron and a proton of the fluorophore, resulting in the stable decarboxylated Glu 222 and a deprotonation of the phenolic oxygen of the fluorophore. Scheme adapted from ref 9.
reported pKa in a FP.21 Clearly, the negative charge on Asp 203 strongly inhibits the deprotonation of the fluorophore. To test whether this T203D mutant could be photoconverted, we exposed it to UV light using the same conditions as before. The red circles in Figure 1D show the T203D mutant spectrum after 10 min of UV exposure. Interestingly, we observed a very high contrast in the B state absorption, similar to that of PA-GFP. Additionally, after UV exposure the pKa of the T203D mutant decreased to 7.2 (Figure 2, red circles), which is similar to the wild-type sfGFP pKa. We also looked for differences in infrared absorption in the carbonyl stretching region before and after the UV exposure and observed only a slight bleaching of signal in the 1650 cm−1 region (not shown), consistent with the loss of deprotonated carboxylic acid carbonyl stretching. This, coupled with the large increase in pKa in the T203D mutant, suggests that both Glu 222 and the newly inserted Asp 203 are both negatively charged. We note that Patterson and Lippincott-Schwartz made the T203D mutation in their development of PA-GFP, but observed no absorption of the resulting protein between 350 and 550 nm for undetermined reasons.22 We hypothesize that this was due to improper protein folding or the absence of fluorophore formation, which appears to be overcome here by the use of sfGFP, which has robust folding properties and a high tolerance for random mutations.18 Because of its potential use as a robustly folding photolabel in live cells, we wanted to compare the cause of photoactivation in this T203D mutant to the well-known Kolbe-type decarboxylation of Glu 222 depicted in Scheme 1.9 Following the generation of an excited state in GFP, Glu 222 can donate its electron to the fluorophore, which leads to the release of CO2 and the formation of an alkyl radical at position 222. The radical is then quenched by back electron transfer from the fluorophore and extraction of a proton, presumably from a water or nearby titratable residue. This process results in a methylated alanine at position 222 and a change in the electrostatic environment around the fluorophore that now favors the B state. While no intermediate species predicted by this reaction scheme have been directly observed, considerable experimental and theoretical evidence supports the Kolbe-type decarboxylation. van Thor et al. reported crystal structures and mass spectra of wild-type GFP before and after exposure to UV light and observed the loss of electron density in the carboxylate side chain of Glu 222, as well as a mass reduction of 44 Da, which indicated the formation of one CO2 equivalent according to Scheme 1.9 Bell et al. showed that the efficiency of the decarboxylation decreases with increasing illumination
wavelength in the order of 254 nm >280 nm >476 nm, which they attributed to the population of higher excited states that are stronger oxidizers and thus can accept an electron from Glu 222 more efficiently. In both studies by van Thor et al. and Bell et al., the authors observed that the rate of photoconversion followed first order kinetics, and the independent studies generally agreed upon the observed rate constant. Grigorenko et al. performed electronic structure calculations that showed that CT states between Glu 222 and the excited fluorophore could feasibly be populated by several pathways.14 Additionally, the authors showed that one of the key residues in PA-GFP (His 203) serves to lower the energy of one possible CT state, which supports the hypothesis that the decarboxylation process is initiated by CT between Glu 222 and the fluorophore. To confirm that photoconversion of the TYG fluorophore in the T203D mutant occurred following a similar process to the CT-initiated decarboxylation outlined in Scheme 1, we performed electrospray ionization mass spectrometry to determine the difference in mass before and after the exposure to UV light. Figure 3A shows the resulting mass spectrum of the T203D mutant before UV exposure. The most abundant peak, with a mass of 27887.5 Da, corresponds to the full protein chain including a hexa-histidine affinity tag. The smaller mass corresponds to the same chain minus the N-terminal methionine residue, which is commonly lost during ionization. Figure 3B shows the resulting mass spectrum of the T203D mutant after it was exposed to UV light as described above. Interestingly, the most abundant mass in this spectrum (27800.0 Da) corresponds to the pre-exposure mass from Figure 3A minus 87.5 Da. There is also a mass loss of 87.3 Da for the protein chain that lacks the N-terminal methionine. These mass changes are strongly indicative of the loss of two CO2 groups, which can be explained by the decarboxylation of both Glu 222 and the newly inserted Asp 203. Further inspection of Figure 3B shows several other masses that were not present in Figure 3A, which correspond to the singly decarboxylated species and a small amount of protein that did not decarboxylate at all. The observation that both Glu 222 and Asp 203 became decarboxylated is further support that both residues are negatively charged, considering that anionic carboxylate is a better electron donor than neutral carboxylic acid. This finding suggests that Asp 203 can also donate an electron to the excited fluorophore to initiate a decarboxylation similar to the one outlined in Scheme 1. If Asp 203 can also initiate the CT, then the site of initial CT could be a tunable 2864
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more relevant factor in determining the efficiency of CT should simply be the distance between the donor and acceptor. Stark spectroscopy has revealed that the imidazolinone ring is electron deficient in the excited A state,25 which supports its role as the initial electron acceptor as drawn in Scheme 1. If this holds true in the T203D mutant, then it should be the case that CT from Asp 203 is less efficient than CT from Glu 222 due to its increased distance from the imidazolinone ring of the fluorophore (0.8 vs 0.6 nm, estimated from crystal structure 2b3p; Figure 1A). As such, it seems likely that the kinetics of this double decarboxylation process might not obey a firstorder rate law if there are indeed two electrons that transfer with different efficiencies. Because the mechanism for this double decarboxylation reaction, and thus the kinetics, will depend on the relative CT efficiencies between Asp 203 and Glu 222 to the fluorophore, we made preliminary steady-state kinetics measurements of the T203D mutant. We irradiated the T203D mutant with the same power of UV light as before and recorded the absorption spectrum of the TYG fluorophore at regular intervals over the course of 15 min. The resulting spectra are shown in Figure 4B. We observed a clean isosbestic point at 432 nm, which indicated that the UV exposure induced a photoconversion between only two states. Fifteen minutes of exposure at this irradiation power caused conversion from a fluorophore population that existed almost entirely in the A state to a fluorophore population with a ∼ 2:3 ratio of A to B state. We note that this does not imply an incomplete photoconversion. Rather, the photoactivated fluorophore has a pKa of 7.2 (Figure 2), which means there is still an A state population, even after the double decarboxylation of Glu 222 and Asp 203. Plotting the B state absorption as a function of the irradiation time (Figure 4C, green data points) for this photoconversion yielded a curve that was not well-described by first-order kinetics (see Figures S1 and S2), which is in contrast to the kinetics that have been observed for photoconversion reactions in GFP and PA-GFP.4,9 However, as mentioned above, the presence of two CT processes in the same system occurring with different efficiencies, either in parallel or in series, might lead to a complex rate equation for this double decarboxylation. Preliminary attempts to quantify the kinetics suggest that the reaction might obey second-order kinetics. Further experiments are underway to build a fully descriptive kinetic model for this process, which will lead to an understanding of the determinants of CT efficiencies to the GFP fluorophore. In addition to the kinetics of decarboxylation, we were also interested in the power-dependence of the photoconversion rate, because of the information it contains about the reactive excited state (denoted by the asterisk in Scheme 1). In Figure 4C, we show the kinetics of the photoconversion at different irradiation powers between 0.6−57.7 mW/cm2. In all cases, the growth of the B state absorption as a function of time could not be well-described by a first-order kinetic process (Figures S1 and S2), which further supports the complex kinetics of the double decarboxylation. The best-fit lines drawn in Figure 4C are linear fits over the initial linear range of each trace, which we used to estimate the initial rates of the reaction at different powers. Figure 4D shows the initial rates plotted against the corresponding irradiation powers. We clearly observed a nonlinear power-dependence of the rates over this range, which suggests that the reactive form of the excited A state is not generated by a single photon absorption. This is in contrast to previously observed single decarboxylation events, which
Figure 3. Exposure to UV light leads to a mass reduction of 87.5 Da. (A) Electrospray ionisation mass spectrometry (ESI-MS) of sfGFP T203D before UV exposure shows two main species, which correspond to the full protein chain (T203D) and a chain missing the N-terminal methionine (T203D−Met1). The dominant species is 27887.5 Da. (B) After UV exposure, both species from panel A shift in mass by ∼88 Da. Additional peaks appear due to the loss of ∼44 Da by both species from (A). Additionally, there is still protein present that does not undergo decarboxylation (indicated by −0 Da).
parameter in the design of fluorescent proteins with increased photoconversion efficiencies. To assess the utility of the Asp 203 mutant as a high contrast fluorescence agent, we recorded the fluorescence spectrum under 488 nm excitation before and after the UV exposure. This is shown in Figure 4A, where we observed a ∼95-fold increase in the integrated fluorescence after 10 min of UV exposure, which is similar to the activation efficiency that occurs with PA-GFP.3 In this regard, we have constructed a photoactivatable variant of sfGFP, which could offer improvements over PA-GFP in studies where the robust folding properties of sfGFP are desired. Notably, Berlin et al. reported a construct of sfGFP with the mutations of T203H, T65S, and F64L in sfGFP to convert it to an analogue of PAGFP.23 However, to our knowledge, the mutant in the present study is the first mutant to contain the desirable TYG fluorophore and exhibit high contrast photoactivation. In addition to its potential use as a fluorescence label in live cells, this T203D mutant of sfGFP also offers significant insight into photoinduced chemistry in FPs. If CT can occur from either Glu 222 or Asp 203 to the excited state of the fluorophore, then it might be expected that the CT processes occur with different efficiencies. It has been proposed that CT efficiencies between Glu 222 and the fluorophore could be altered by mutating residues near the fluorophore cavity that alter the redox coupling between Glu 222 and the fluorophore.24 However, the possibility of increasing CT efficiency by moving the charge donor site closer to the acceptor has so far not been suggested. Grigorenko et al. showed that the residue at position 203 can modulate the energies of the proposed CT states, which makes populating them more efficient.14 However, in the case of the T203D mutant, where CT might actually occur from position 203, the 2865
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Figure 4. Spectroscopic and kinetic properties of photoconversion. (A) Fluorescence of the T203D mutant before (black) and after (red) 10 min of UV exposure indicating a ∼95-fold increase in the fluorescence under 488 nm excitation due to the photoconversion. (Inset) Enhanced view of the black spectrum that is partially masked by the x-axis. The area under the red spectrum is ∼95 times more than that under the black spectrum. (B) Absorption spectra of the TYG fluorophore in the T203D mutant as a function of UV irradiation time, plotted at 1 min intervals for a total of 15 min. The black spectrum was taken before irradiation and the greener spectra correspond to later time points. (C) The B state absorption as a function of time for several different UV irradiation powers. Best-fit lines are drawn over the time course that corresponds to the linear portion of the curve to estimate the initial rates. (D) The initial rates of photoconversion from panel C plotted against irradiation power.
because those values ultimately determine the concentrations of negative carboxylates, and thus the reactive species. High resolution crystal structures of the T203D mutant could provide information about the complex hydrogen bonding network surrounding the fluorophore in the ground state. Moreover, the nature of the excited state that acts as the initial electron acceptor could be probed using ultrafast, pulsed excitations and electronic structure calculations. Specific questions of interest are: (1) Is the reactive excited state generated by excited state absorption? (2) What is the nature of the reactive excited state? Is the reactive excited state in this T203D mutant the same as it is in PA-GFP? (3) How do the pKa values of Glu 222 and Asp 203 depend on either group being decarboxylated? For instance, would the decarboxylation of Glu 222 change the pKa of Asp 203, and thus the concentration of reactive acid? A detailed mechanistic view of the double decarboxylation reported here will allow for a better understanding of CT reactions that occur in a wide range of FPs. Mutations of this nature could provide a tunable parameter in the design of FPs with new properties, which has so far been unexplored.
display linear power-dependence with 254 nm irradiation over a similar range of irradiation power.9,24 Rather, the logarithmic power dependence that we observed in Figure 4D suggests that the reactive form of the excited A state is generated by an excited state absorption process. We note that while here we have only analyzed the initial rates, we observed the same logarithmic power dependence when analyzing the rate constants obtained from higher-order exponential fits (data not shown). However, the nature of the reactive excited state is currently unknown, and undoubtedly plays a major role in the CT efficiencies between the fluorophore and Asp 203 and Glu 222 (and thus the observed kinetics). Until we have information from experiments that can probe the dynamics of the excited fluorophore, we can only speculate about the mechanism of this photoconversion and the observed kinetics. Altogether, these observations on the photoconversion properties of sfGFP mutant T203D lead to several questions regarding the Kolbe-type decarboxylation that has been proposed to occur throughout the FP family.5−7 First and foremost, does the decarboxylation of Asp 203 occur via a similar process to the one outlined in Scheme 1? If so, then it might be the case that CT to the excited state of the fluorophore can be tuned to achieve more efficient photoconversion. Because of the generality of this Kolbe-type decarboxylation among the FP family, the tunability of CT distance could be exploited to improve other photoactivatable FPs that function similarly to PA-GFP. Future studies of the kinetics of this decarboxylation will focus on elucidating the pKa of both Glu 222 and Asp 203,
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EXPERIMENTAL METHODS The gene for sfGFP was generously provided by Ryan Mehl and was mutated at position 203 using a QuickChange Mutagenesis kit from Stratagene following the manufacturer recommended procedure.26 The mutated sequences were verified by Sanger sequencing, and all sfGFP genes were expressed and purified as detailed elsewhere.27 Purified proteins 2866
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in the Dark and Fluorescent States. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (50), 21097−21102. (7) Fron, E.; De Keersmaecker, H.; Rocha, S.; Baeten, Y.; Lu, G.; UjiI, H.; Van Der Auweraer, M.; Hofkens, J.; Mizuno, H. Mechanism behind the Apparent Large Stokes Shift in LSSmOrange Investigated by Time-Resolved Spectroscopy. J. Phys. Chem. B 2015, 119 (47), 14880−14891. (8) Heim, R.; Cubitt, A. B.; Tsien, R. Y. Improved Green Fluorescence. Nature 1995, 373 (6516), 663−664. (9) van Thor, J. J.; Gensch, T.; Hellingwerf, K. J.; Johnson, L. N. Phototransformation of Green Fluorescent Protein with UV and Visible Light Leads to Decarboxylation of Glutamate 222. Nat. Struct. Biol. 2002, 9 (1), 37−41. (10) Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (16), 8362−8367. (11) Tsien, R. Y. The Green Fluorescent Protein. Annu. Rev. Biochem. 1998, 67, 509−544. (12) Stoner-Ma, D.; Jaye, A. A.; Matousek, P.; Towrie, M.; Meech, S. R.; Tonge, P. J. Observation of Excited-State Proton Transfer in Green Fluorescent Protein Using Ultrafast Vibrational Spectroscopy. J. Am. Chem. Soc. 2005, 127 (9), 2864−2865. (13) Bogdanov, A. M.; Acharya, A.; Titelmayer, A. V.; Mamontova, A. V.; Bravaya, K. B.; Kolomeisky, A. B.; Lukyanov, K. A.; Krylov, A. I. Turning on and off Photoinduced Electron Transfer in Fluorescent Proteins by Pi-Stacking, Halide Binding, and Tyr145 Mutations. J. Am. Chem. Soc. 2016, 138 (14), 4807−4817. (14) Grigorenko, B. L.; Nemukhin, A. V.; Morozov, D. I.; Polyakov, I. V.; Bravaya, K. B.; Krylov, A. I. Toward Molecular-Level Characterization of Photoinduced Decarboxylation of the Green Fluorescent Protein: Accessibility of the Charge-Transfer States. J. Chem. Theory Comput. 2012, 8 (6), 1912−1920. (15) Acharya, A.; Bogdanov, A. M.; Grigorenko, B. L.; Bravaya, K. B.; Nemukhin, A. V.; Lukyanov, K. A.; Krylov, A. I. Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. 2017, 117 (2), 758−795. (16) Brejc, K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien, R. Y.; Ormo, M.; Remington, S. J. Structural Basis for Dual Excitation and Photoisomerization of the Aequorea Victoria Green Fluorescent Protein. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (6), 2306−2311. (17) Wachter, R. M.; Hanson, G. T.; Kallio, K.; Remington, S. J. Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH. Biochemistry 1999, 38, 5296−5301. (18) Pédelacq, J.-D.; Cabantous, S.; Tran, T.; Terwilliger, T. C.; Waldo, G. S. Engineering and Characterization of a Superfolder Green Fluorescent Protein. Nat. Biotechnol. 2006, 24 (1), 79−88. (19) Wachter, R. M.; Elsliger, M. A.; Kallio, K.; Hanson, G. T.; Remington, S. J. Structural Basis of Spectral Shifts in the YellowEmission Variants of Green Fluorescent Protein. Structure 1998, 6 (10), 1267−1277. (20) Jung, G.; Wiehler, J.; Zumbusch, A. The Photophysics of Green Fluorescent Protein: Influence of the Key Amino Acids at Positions 65, 203, and 222. Biophys. J. 2005, 88 (3), 1932−1947. (21) Hanson, G. T.; Mcananey, T. B.; Park, E. S.; Rendell, M. E. P.; Yarbrough, D. K.; Chu, S.; Xi, L.; Boxer, S. G.; Montrose, M. H.; Remington, S. J. Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 1. Biochemistry 2002, 41, 15477−15488. (22) Patterson, G. H.; Lippincott-Schwartz, J. Development of a Photoactivatable Fluorescent Protein from Aequoria Victoria GFP. Proc. SPIE 2004, 5329, 13−22. (23) Berlin, S.; Carroll, E. C.; Newman, Z. L.; Okada, H. O.; Quinn, C. M.; Kallman, B.; Rockwell, N. C.; Martin, S. S.; Lagarias, J. C.; Isacoff, E. Y. Photoactivatable Genetically Encoded Calcium Indicators for Targeted Neuronal Imaging. Nat. Methods 2015, 12 (9), 852−858. (24) Bell, A. F.; Stoner-Ma, D.; Wachter, R. M.; Tonge, P. J. Light Driven Decarboxylation of Wild-Type Green Fluorescent Protein. J. Am. Chem. Soc. 2003, 125 (5), 6919−6926.
were exposed to UV light (254 nm peak wavelength) from a Spectroline XX-15G lamp at distances of 42.2, 22.1, 12.1, 7.6, and 4.1 cm to achieve power densities of 0.6 mW/cm2, 1.9 mW/cm2, 6.9 mW/cm2, 17.2 mW/cm2, and 57.7 mW/cm2, respectively. Titrations were carried out by buffer exchanging the purified protein into a master buffer of 50 mM phosphate, 50 mM citrate, and 100 mM NaCl at pH 7.5. The protein was then concentrated to ∼1 mM and diluted by 150x with the master buffer (adjusted to the desired pH with NaOH or HCl) into a transparent 96-well plate. Visible absorbance was measured on a Biotek Epoch 2 plate reader. Fluorescence spectra were recorded with a Horiba Fluorolog 3 spectrometer.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b01101. Single and double exponential fits to time-dependent spectral changes of sfGFP T203D (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Joshua D. Slocum: 0000-0002-8705-3054 Lauren J. Webb: 0000-0001-9999-5500 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Welch Foundation (F-1722). We thank the Institute for Cell and Molecular Biology core facility at the University of Texas at Austin for providing DNA sequencing and protein mass analysis. We would like to thank the Institute for Biomaterials, Drug Delivery, and Regenerative Medicine at the University of Texas at Austin for allowing us to use their plate reader for the experimental pKa measurements. We would also like to thank Ryan Mehl for providing the sfGFP gene.
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REFERENCES
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DOI: 10.1021/acs.jpclett.7b01101 J. Phys. Chem. Lett. 2017, 8, 2862−2868
Letter
The Journal of Physical Chemistry Letters (25) Bublitz, G.; King, B. A.; Boxer, S. G. Electronic Structure of the Chromophore in Green Fluorescent Protein (GFP). J. Am. Chem. Soc. 1998, 120 (36), 9370−9371. (26) Hammill, J. T.; Miyake-Stoner, S.; Hazen, J. L.; Jackson, J. C.; Mehl, R. A. Preparation of Site-Specifically Labeled Fluorinated Proteins for 19F-NMR Structural Characterization. Nat. Protoc. 2007, 2 (10), 2601−2607. (27) Slocum, J. D.; Webb, L. J. Nitrile Probes of Electric Field Agree with Independently Measured Fields in Green Fluorescent Protein Even in the Presence of Hydrogen Bonding. J. Am. Chem. Soc. 2016, 138 (20), 6561−6570.
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DOI: 10.1021/acs.jpclett.7b01101 J. Phys. Chem. Lett. 2017, 8, 2862−2868
