Mechanically switching single-molecule fluorescenceof GFP by unfolding and refoldingZiad Ganima,b,1 and Matthias Riefb,c,1

aDepartment of Chemistry, Yale University, New Haven, CT 06520; bPhysik Department E22, Technische Universitat Munchen, 85748 Garching, Germany;and cMunich Center for Integrated Protein Science, 81377 Munchen, Germany

Edited by Steven M. Block, Stanford University, Stanford, CA, and approved September 8, 2017 (received for review March 24, 2017)

Green fluorescent protein (GFP) variants are widely used as genet-ically encoded fluorescent fusion tags, and there is an increas-ing interest in engineering their structure to develop in vivooptical sensors, such as for optogenetics and force transduction.Ensemble experiments have shown that the fluorescence of GFPis quenched upon denaturation. Here we study the dependenceof fluorescence on protein structure by driving single moleculesof GFP into different conformational states with optical tweezersand simultaneously probing the chromophore with fluorescence.Our results show that fluorescence is lost during the earliestevents in unfolding, 3.5 ms before secondary structure is dis-rupted. No fluorescence is observed from the unfolding interme-diates or the ensemble of compact and extended states popu-lated during refolding. We further demonstrate that GFP can bemechanically switched between emissive and dark states. Thesedata definitively establish that complete structural integrity isnecessary to observe single-molecule fluorescence of GFP.

optical tweezers | protein folding | fluorescent protein | mechanoswitch

Green fluorescent protein (GFP) is a 27-kDa β-barrel pro-tein with an intrinsic chromophore. (1, 2) It is widely used in

imaging applications that rely on its structural stability [to thermaland high-pressure unfolding (3), in fusion constructs (4), and tocircular permutation (5)] or its optical response to environment,such as in biosensors for force transduction (6–9), calcium con-centration (10), protease activity (11), and pH (12, 13). Under-standing the relationship between protein structure and fluores-cence is essential for these applications. Photophysical propertiesof the chromophore are sensitive to its hydrogen bonding, solva-tion, isomerization state, and binding-pocket structure. (1, 2, 14).It is known that fluorescence is quenched upon denaturation andthat several intermediates have been observed in folding experi-ments and simulations (3, 15–21), although it has not been pos-sible to definitively probe the fluorescence properties of partiallystructured intermediates in isolation from the native state. Spec-ulation exists regarding whether single-molecule photobleachingcan be reversed by unfolding and refolding the protein (22). Itis not known whether tension on the native state alters fluores-cence in similarity to the compressive, pressure-induced elasticeffect (3) and whether its emission can be reversibly mechani-cally switched. In this study, we address these questions by makingan explicit connection between structure and fluorescence, usingsingle-molecule methods.

Single-molecule experiments are widely used to reveal dis-tributions in biophysical structure and kinetics that under-lie ensemble-averaged properties. Single-molecule manipulationusing force experiments such as optical tweezers can be used toobserve states nominally at negligible concentration in a bulkdistribution (23–25). Single-molecule fluorescence experimentshave been used to probe the chemical structure and environmentof chromophores (14, 26, 27). The combination of these twotools can provide an entirely optical method to drive conforma-tional changes and probe several orthogonal coordinates; how-ever, their joint implementation is challenged by factors includ-ing rapid sample photodegradation unless the trapping and

fluorescence excitation spots are temporally or spatially dis-placed (28–31). In this work, we reveal the structural require-ments for fluorescence from the GFP chromophore by usingoptical trapping force experiments to prepare a range of pro-tein structures. The fluorescence measured here directly reportson the solvation of the chromophore and rigidity of its bindingpocket, rather than its more common use as a tag or a proxydistance measurement. With the ability to monitor fluorescencewith millisecond time resolution while independently observingprotein structure, we find that unfolding GFP disrupts the envi-ronment of the chromophore and affects a loss of fluorescence3.5 ms before any secondary structure changes are observed withforce. No fluorescence is observed from the unfolding interme-diates or the ensemble of states GFP populates during refold-ing. We show that fluorescence can be recovered by refoldingthe protein and demonstrate that GFP can be switched betweenemissive and dark states by mechanical unfolding and refolding.

ResultsSingle-Molecule Fluorescence and Force-Jump Unfolding. We de-signed a mutant of GFP, including enhanced fluorescence muta-tions (32, 33) and cysteine residues at the N- and C terminithat allowed attachment of DNA handles for dual-bead opti-cal trapping experiments (Fig. 1). The length of the DNA han-dles gave an 860-nm separation of GFP from the trapping laserbeam foci to circumvent undesirable photophysical processesknown to accompany combined optical trapping/single-moleculeforce experiments (28). Native single-molecule fluorescence wasobserved from GFP in the optical trap as characterized by unper-turbed lifetime, blinking, and photobleaching (14, 34) (Figs. S1and S2). Traces that did not show both the expected fluorescence

Significance

Green fluorescent protein (GFP) is widely used as a tag to watchotherwise invisible proteins and as a sensor of its local chemicalenvironment. Since GFP can form many partially folded states,it is critical to know how these structural changes affect itssignature fluorescence. We use optical tweezers to force sin-gle molecules of GFP into folding and unfolding intermediatestates and simultaneously probe single-molecule fluorescencefrom each state. It was found that GFP fluorescence requirescomplete structural integrity; none of the unfolding or refold-ing intermediates were observed to fluoresce, but fluorescencecould be recovered by complete refolding. This feature wasexploited to reversibly, mechanically switch GFP between onand off fluorescence states.

Author contributions: Z.G. and M.R. designed research; Z.G. and M.R. performedresearch; Z.G. and M.R. analyzed data; Z.G. prepared samples; and Z.G. and M.R. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: ziad.ganim@yale.edu or mrief@ph.tum.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1704937114/-/DCSupplemental.

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Fig. 1. Schematic of the force-jump and single-molecule fluorescence assay. Cycling the trap separation (blue trace) over an 800-nm range provides periodichigh (47 pN) and low (0 pN) force intervals to facilitate unfolding and refolding. The time resolutions were 4.8 ms for the fluorescence (green) and 33 µs forthe force (gray) measurements. The red trace shows the force smoothed to 0.66 ms. The excitation is switched on and fluorescence is detected only duringthe high-force intervals. Unfolding occurs at 0 s and refolding occurs during the low-force interval at 0.8 s, which is observed at 0.853 s. Laser off and laseron fluorescence backgrounds are visible during low-force intervals and when GFP is unfolded, respectively.

and contour length changes upon unfolding were excluded.Single-molecule fluorescence of GFP was probed throughoutunfolding and refolding events with a force-jump assay (Fig. 1and refs. 35 and 36). To investigate unfolding, a force jump (from0 pN to 30–60 pN) is applied by abruptly increasing the distancebetween the optical traps. With each such force jump, the pro-tein had a chance to unfold that increased with the magnitudeand duration of high-force application. Unfolding was evidencedby a loss of fluorescence and an increase in the end-to-end lengthof the protein as folded regions lose structure under force. Asunfolded proteins were not observed to refold at high-force con-ditions, a subsequent jump back to 0 pN allowed for the foldedstate and fluorescence to be recovered. Periodic repetition offorce jumps up and down shows that single-molecule fluores-cence cycling is possible by unfolding and refolding the proteinand allowed for optical characterization of the intermediates.

Characterization of Unfolding Intermediates. As GFP unfolds, theforce measurement probes the end-to-end length of the proteinwith sufficient time resolution (33 µs) to characterize unfold-ing intermediates (Fig. 2 A and B). Time resolution of thefluorescence experiment was dictated by the emission rate,which is proportional to the excitation laser intensity; for thebest time resolution following the force jump (4.8 ms), laserpower causing bleaching in ∼2 s was needed (Fig. S1). Thelength change upon complete unfolding (∆LF-U = 79.1± 3 nm)was consistent with that measured from force-extension exper-iments (Fig. S3) and the difference arising from unfoldingof the structured amino acids between the attachment points(LN-C = 225 aa× 0.365 nm/aa = 82.1 nm) and the distancebetween attachment points in the folded protein (dN-C = 2 nm;∆LF-U = LN-C × dN-C.) Signature force changes were ob-served for two intermediates, I1 and I2 (∆LF-I1 = 10.0± 2.2 nm,∆LF-I2 = 39.4± 3.1 nm), which are consistent with the ∆β11and ∆β11–7 previously observed from GFP unfolding with the

ClpXP mechanical protease (21). Repeated measurements withdifferent force jumps yield the force-dependent unfolding rate[fitted to a Bell model (37, 38), kU(F) = k0exp(−F*dx/kT); withk0 = 0.33 s−1 and ∆x = 0.23 nm; Fig. 2B and Fig. S4]. The valueof k0 is greatly affected by the unavailability of low-force datapoints and is likely overestimated (16). The fluorescence wasobserved to cease before any evidence of unfolding was seen inthe force signal. Due to the fluorescence intermittency of GFP,statistically significant sampling of the fluorescence intensity dur-ing unfolding was facilitated by averaging 30 trajectories. By syn-chronizing all trajectories to the F→I1 force transition, a delayof 3.5 ms was observed between loss of fluorescence and lengthchange (Fig. 2 C and D, alternative analysis in Fig. S5). Thisoffset—which is shorter than the detector integration time—was provided by the synchronization and averaging procedurewith no deconvolution. Model calculations that demonstrate thisresolution enhancement as well as independence of the offsetwith respect to GFP blinking are presented in SI Materials andMethods (and Fig. S6). The averaged force signal represents thestatistical lifetime of the two intermediate states, and the aver-aged fluorescence signal represents both the statistical blinkingand loss of fluorescence due to unfolding. These results showthat the earliest events in unfolding GFP quench its fluorescencebefore any disruption of the β-barrel structure is observable viachange in the end-to-end distance with force.

Refolding Intermediates. In a different set of measurements,real-time recovery of fluorescence with refolding was observed.Unfolding occurred at high force as described above and the flu-orescence was probed continuously at a low force of 3 pN (Fig.3A). The observed contour length fluctuations indicate that GFPsamples an ensemble of compact and extended states (35, 39)during refolding, although these low forces did not provide suffi-cient length resolution for structural assignment. (This refoldingensemble was also observed in force-extension measurements;

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Fig. 2. Characterization of unfolding intermediates. (A) Sample unfold-ing trace showing sequential transition from folded state (F) to I1, I2, andU (33-µs resolution in gray, 0.66-ms smoothed data in red). (B) Logarithmof rate of first unfolding is linearly dependent on force. (C) Fluorescenceintermittency (blinking) is observed as well as quenching upon unfoldingin single-molecule traces (green). The concomitant force data are shown inred (33-µs resolution). Synchronization defines t = 0 ms at the first unfold-ing, F→ I1. (D) Average of 30 synchronized single-molecule traces shows thatfluorescence is quenched before the force drop due to unfolding. Fit values:t0 = 0 ms, τ = 4.5 ms (force) and t0 = −3.5 ms, τ = 1.0 ms (fluorescence). Thesynchronization procedure yields better resolution than the time betweenacquisition points (4.8 ms).

Fig. S7). Fluorescence was observed abruptly upon the cessationof the contour length fluctuations when refolding was complete;as such, we conclude that the refolding ensemble did not containany sufficiently fluorescent species with an approximately mil-lisecond or longer lifetime(zoomed-in view in Fig. S8). Native-state stability was verified by a single force-extension cycle (atFig. 3A, †), during which GFP was shifted away from the exci-tation spot. The force-jump assay was used to probe the successrate of refolding as a function of time spent at 0 pN and ana-lyzed with a maximum-likelihood algorithm to yield a foldingrate of kF = 0.62 ± 0.06 s−1 (Table S1). A significant fractionof GFP was not observed to refold on the ∼5-min experimentaltimescale, which we attribute to the formation of slowly reequili-brating proline isomers (16) or nonnative disulfide linkages (40).This strict dependence of fluorescence on the native state wasfurther verified by concatenating the fluorescence from the high-force intervals across 26 unfolding/refolding force-jump cycles(Fig. 3B). The folded/unfolded state of the protein is indicatedby the average contour length during that period. These datashow that force-jump cycles can be tuned to flip GFP betweenfolded/fluorescent and unfolded/nonfluorescent states and com-plement the information obtained by synchronizing many traces.The reversibility of this process is limited by photobleachingand the propensity for GFP to enter a slowly refolding state.These results demonstrate that the fluorescence can be reversiblymechanically switched in single molecules of GFP.

DiscussionWe observe that the fluorescence of GFP requires the struc-tural rigidity afforded by the native apoprotein; fluorescence isquenched in the first unfolding event, is not recovered while sam-pling compact and extended states in the refolding ensemble, andcompletely recovers upon full refolding. GFP unfolding beginswith disruption of the chromophore fluorescence, followed byunfolding of β11 and then β(7–11) before reaching the fully

unfolded state. The nonfluorescent state with native end-to-endlength may correspond to internal rearrangements, such as theprotonated dark state (14, 22). Alternatively, fluorescence maybe quenched by external solvent penetration to the chromophorewith contacts remaining among all β strands (3). As the unfold-ing is force dependent, the 3.5-ms offset in fluorescence quench-ing is also likely to display force dependence; if the quenchedstate is a protonated dark state of GFP (41), then its lifetimewould be dictated by the sum of the rates for deprotonation andforce-dependent unfolding. The structural changes we observeare consistent with previous single-molecule unfolding studies(21), H/D exchange pointing to β7–10 as a region of increasedsolvent accessibility (17), and SAXS data indicating an interme-diate with native-like secondary structure (16). However, as thequintessential feature of GFP is its fluorescence, the ability ofcombined single-molecule force and fluorescence to make clearstatements about the fluorescent properties of intermediates iskey. In a mixture containing multiple potentially fluorescentspecies, the total fluorescence is proportional to both the concen-tration and the brightness (= extinction coefficient× quantumyield) of each species, which has previously limited the ability toquantify the fluorescence of GFP intermediates. Our data indi-cate that the weak fluorescence observed in bulk denaturationarises from residual native GFP or states not appreciably popu-lated on the approximately millisecond timescale (16, 17).

GFP folding proceeds by transitions within an ensemble of dis-ordered states, evidenced by rapid contour length fluctuationswith no fluorescence (35, 39). From this ensemble, either thenative state is reformed swiftly (0.62 s−1) and fluorescence isrecovered or GFP enters a slowly reequilibrating state. For theconstruct studied here, both proline isomerization [which reequi-librates on a 3-min timescale (16)] and formation of a nonnativeCys49-Cys71 disulfide [potentially as rapid as 1 s−1 (40)] may

Fig. 3. Fluorescence recovers upon refolding and can be switched. (A) Nofluorescence is observed as GFP samples different contour length states dur-ing refolding; fluorescence recovers immediately upon formation of thenative state at *. † indicates where force extension was used to verifynative-state formation. The time resolutions were 50 ms for the fluores-cence (green) and 33 µs for the force (gray) measurements. The red traceshows the force smoothed to 0.66 ms. (B) Driving GFP fluorescence off/on byrepeated unfolding and refolding cycles. Data such as those shown in Fig. 1were compressed to show the fluorescence counts (green) during the high-force intervals from 26 sequential force-jump cycles. For each interval, theaverage contour length is shown (red circles). The fluorescence time resolu-tion was 4.8 ms, and the laser on background fluorescence was 40 counts.

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contribute to the slow timescale. Previous measurements providebackground that the lack of fluorescence from all partially foldedGFP intermediate states observed here can be generalized toGFP variants despite structural differences (e.g., circularly per-muted GFP) and may arise due to both a reduction in fluores-cence quantum yield and molar extinction; studies on the isolatedGFP chromophore have established that the fluorescence quan-tum yield may drop by a factor of 103 in a nonrigid matrix (42,43), and the absorption of GFP is reduced by a factor of >200upon deleting as few as five N-terminal amino acids (20).

Once photobleached, fluorescence could not be recovered byunfolding and refolding, which provides evidence against the sug-gestion that secondary structure rearrangements can restore flu-orescence (Fig. S9) (22). Finally, we have shown that cycling thefluorescence by mechanical force application is possible, whichallows GFP to be calibrated as a force sensor in the 30- to 60-pNrange. The data presented here can be used to expand uponresults showing that integrin-mediated forces are sufficient todenature and quench the structurally comparable superfolderGFP (6). For sensing in the lower, ∼4- to 20-pN force regime,it may be possible to design mutants that are more mechanicallysusceptible to unfolding and to calibrate the force sensor usingthe methods presented here. These findings may provide addi-tional tools to the growing community of optical force sensing(7–9).

The combination of single-molecule manipulation and com-plementary optical probing provides a powerful tool to systemat-ically induce chemical changes and observe the effects on func-tion, which may be obscured in ensemble-averaged experiments.The methods presented here may be expanded by using flu-orescence to probe other degrees of freedom, such as redoxstate or ATP binding (26, 44). Moreover, the development ofnew single-molecule microscopies with chemical structuralresolution—using Raman scattering (45–48), direct absorption(49), photothermal contrast (50), and modulation of conductiv-ity (51)—provides exciting opportunities for combined manipu-lation and characterization of chemical structure.

Materials and MethodsThe instrument for optical trapping and single-molecule fluorescence wasadapted from that previously described (52, 53). Single-molecule fluores-cence capability was built using a 488-nm, <100-ps excitation laser (BDL-488-SMC; Becker & Hickl) operated at 20 MHz and modulated electroni-cally using the same field-programmable gate array (FPGA) used to steerthe optical trap and acquire data (NI PCI-7833R 3M; National Instruments).A polarizer and λ/2 waveplate were used to control the excitation inten-sity, followed by a λ/4 waveplate to provide circularly polarized light

[WPH10M-488 and WPQ10M-488 (ThorLabs) and G335719000 (Qioptiq)].The excitation light was combined with the 1,064-nm trapping lasers, usinga 1,064/visible dichroic mirror (R 1,064 nm>90%, T 400–800 nm>90%; Pre-cision Photonics), and provided confocal excitation. Alignment was facili-tated by a 1-mM rhodamine B sample, which allowed for two-photon fluo-rescence to visualize the two 1,064-nm trapping laser foci and one-photonfluorescence to visualize the excitation laser focus (Fig. S10); the excitationlaser was iteratively aligned to the midpoint between the two traps by cen-troid fitting of the three point-spread functions. Excitation laser power atthe sample was 10–110 nW, depending on the desired time resolution andobservation length; data shown were acquired at 110 nW (Figs. 1 and 2),10 nW (Fig. 3A), and 50 nW (Fig. 3B). Fluorescence passed through the1,064/visible dichroic mirror and a dichroic mirror to block the excitationlight (F52-477; AHF analysentechnik) and passed through two filters toremove scattered light from the trapping laser, 850 nm brightfield, and exci-tation laser (F74-750 and F33-473Z; AHF analysentechnik) before detection.An EMCCD (iXon; Andor Technology) was used to acquire all fluorescencetrajectories with a time resolution of 4.8 ms (Figs. 1 and 2) or 50 ms (Fig. 3A).To enhance sensitivity, the pixels were binned to 4×4 superpixels. Inten-sity was monitored on either the superpixel centered on the sample (Figs. 1and 3) or a sum over the five most central superpixels (Fig. 2). Tuning theseintegration conditions causes the fluorescence counts to vary across figures.For fluorescence lifetime measurements (Fig. S2), an avalanche photodiode(id100-50-STD; ID Quantique SA) and time-correlated single-photon count-ing card (TimeHarp 200 PCI; PicoQuant GmbH) were used to provide 150-pstime resolution.

In the force-jump experiments, the mobile trap was steered with a squarewave to produce a trap separation as shown in Fig. 1. The rate at which thetrap distance changed was limited by the speed of the two-axis piezoelectrictip/tilt actuator (Mad City Labs) with typical times of 5 ms required to jump1,000 nm (0.2 mm/s). The 488-nm excitation spot is fixed throughout theexperiment, but the position of GFP (precisely in the middle of the fixed andmobile optical traps) changes with applied force. Therefore, the excitationwas aligned separately to probe fluorescence at 2 pN (1,050 nm separationbetween optical trap and excitation, Fig. 3A) during refolding experimentsand between 30 pN and 60 pN (1,800–2,200 nm separation; Figs. 1, 2, and3B) during unfolding experiments. (This is evident in the fluorescence dipat Fig. 3A, †.) The fluorescence laser was turned on simultaneously with thejump up (or jump down) by a signal from the FPGA when fluorescence dur-ing unfolding (or refolding) was measured. The EMCCD recorded continu-ously throughout the experiment with 4×4 pixel binning and images weresaved as a stacked tagged image file (TIF). Each binned superpixel imagedlight from a 560- × 560-nm region in the sample, and single-molecule fluo-rescence trajectories were assembled by integrating four superpixels, whichresulted in maximal collection efficiency with minimal background from theoptically trapped microspheres.

ACKNOWLEDGMENTS. The authors thank Matthias Reisser and DianaBeyerlein for their assistance in the early stages of these experiments. Thisresearch was supported by an SFB 863 grant from Deutsche Forschungsge-meinschaft (to M.R.) (A02) and a postdoctoral fellowship from the HumboldtFoundation (to Z.G.).

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