This article is published as part of a themed issue of Photochemical & Photobiological Sciences on Synthetic and natural photoswitches Guest edited by Dario Bassani, Johan Hofkens and Jean Luc Pozzo Published in issue 2, 2010

Other articles in this issue include:

Photochromic dithienylethenes with extended π-systems O. Tosic, K. Altenhöner and J. Mattay, Photochem. Photobiol. Sci., 2010, 9, 128 Hydrophilic and photochromic switches based on the opening and closing of [1,3]oxazine rings M. Tomasulo, E. Deniz, S. Sortino and F. M. Raymo, Photochem. Photobiol. Sci., 2010, 9, 136 Efficient carrier separation from a photochromic diarylethene layer T. Tsujioka, M. Yamamoto, K. Shoji and K. Tani, Photochem. Photobiol. Sci., 2010, 9, 157 Multiphoton-gated cycloreversion reactions of photochromic diarylethene derivatives with low reaction yields upon one-photon visible excitation Y. Ishibashi, K. Okuno, C. Ota et al., Photochem. Photobiol. Sci., 2010, 9, 172 Probing photochromic properties by correlation of UV-visible and infra-red absorption spectroscopy: a case study with cis-1,2-dicyano-1,2-bis(2,4,5-trimethyl-3-thienyl)ethene A. Spangenberg, J. A. Piedras Perez, A. Patra et al., Photochem. Photobiol. Sci., 2010, 9, 188 The DC gate in Channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156 M. Nack, I. Radu, M. Gossing et al., Photochem. Photobiol. Sci., 2010, 9, 194 Quantitative investigations of cation complexation of photochromic 8-benzothiazole-substituted benzopyran: towards metal-ion sensors M. I. Zakharova, C. Coudret, V. Pimienta et al., Photochem. Photobiol. Sci., 2010, 9, 199 Spiropyrans as molecular optical switches B. Seefeldt, R. Kasper, M. Beining et al., Photochem. Photobiol. Sci., 2010, 9, 213 Photoinduced shape changes of diarylethene single crystals: correlation between shape changes and molecular packing L. Kuroki, S. Takami, K. Yoza, M. Morimoto and M. Irie, Photochem. Photobiol. Sci., 2010, 9, 221 Functional interaction structures of the photochromic retinal protein rhodopsin K. Kirchberg, T.-Y. Kim, S. Haase and U. Alexiev, Photochem. Photobiol. Sci., 2010, 9, 226 Facile synthesis and characterization of new photochromic trans-dithienylethenes functionalized with pyridines and fluorenes Q. Luo, Y. Liu, X. Li and H. Tian, Photochem. Photobiol. Sci., 2010, 9, 234 Higher resolution in localization microscopy by slower switching of a photochromic protein H. Mizuno, P. Dedecker, R. Ando et al., Photochem. Photobiol. Sci., 2010, 9, 239 Optical control of quantum dot luminescence via photoisomerization of a surface-coordinated, cationic dithienylethene Z. Erno, I. Yildiz, B. Gorodetsky, F. M. Raymo and N. R. Branda, Photochem. Photobiol. Sci., 2010, 9, 249 Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins A. R. Faro, V. Adam, P. Carpentier et al., Photochem. Photobiol. Sci., 2010, 9, 254

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PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences

Higher resolution in localization microscopy by slower switching of aphotochromic protein†

Hideaki Mizuno,*a,b Peter Dedecker,b Ryoko Ando,a Takashi Fukano,a Johan Hofkensb and Atsushi Miyawakia

Received 6th October 2009, Accepted 11th November 2009First published as an Advance Article on the web 18th January 2010DOI: 10.1039/b9pp00124g

Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy,including techniques such as photoactivated localization microscopy (PALM). A determining factor inthe precision of the images generated by PALM measurements is the photon numbers that can bedetected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that hasbeen successfully used in PALM experiments. The number of photons per switching cycle that can beacquired for Dronpa depends on its off-switching rate, limiting the number of photons that can berecorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, showsslower switching, and develop a mutant that displays switching kinetics between those of Dronpa and22G. We show that the kinetics of the photoswitching are strongly related to self-association of theprotein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly wefind that higher-resolution PALM images can be acquired with slower-switching proteins due to theirhigher number of emitted photons per switching cycle.

Introduction

Fluorescence microscopy provides a powerful technique to visu-alize the molecules of life, while fluorescent proteins have madeit easy to label various molecules in living cells.1 However, thediffraction limit of light restricts the spatial resolution of thistechnology to ~200 nm, which is two orders of magnitude largerthan the typical size of macromolecules. Several new microscopictechnologies have now been developed to address this limitationby circumventing the diffraction limit.2–5 One of these techniques,photoactivated localization microscopy (PALM), is based on thestochastic switching of a small population of photoswitchablefluorescent molecules in combination with single-molecule detec-tion to achieve spatially separated emission and localization.2–4

The subdiffraction image is then reconstituted by rendering thecoordinates of a large number of individual fluorophore positionscollected over thousands of sequential images.

There are three types of photoswitchable fluorescent proteinsthat are applicable to PALM experiments.6 The first type are thephotoconvertible proteins, which display green fluorescence afterexpression but can be converted to red emitters by illuminationwith ultra-violet/violet light.7 The second type are the photoacti-vatable proteins, which are originally non-fluorescent but becomefluorescent by ultra-violet/violet light illumination.8 In these typesthe light-induced reactions are irreversible, and therefore onlya single switching cycle is possible.9 In contrast, the switchingprocess of the third type consists of reversible photochromism

aCell Function and Dynamics, Brain Science Institute, RIKEN, 2-1Hirosawa, Wako-city, Saitama, 351-0198, Japan; Fax: +81 48 467 5924;Tel: +81 48 467 5917bDepartment of Chemistry and INPAC, Katholieke Universiteit Leuven,Celestijnenlaan 200F, 3001, Heverlee, Belgium. E-mail: hideaki.mizuno@chem.kuleuven.be; Fax: +32 16 32 79 90; Tel: +32 16 32 73 99† This paper is part of a themed issue on synthetic and natural photo-switches.

between a fluorescent and a non-fluorescent state.10–13 The bestknown example of this type is Dronpa and its mutants.10–12 Thebright green fluorescence of Dronpa vanishes by strong blue/greenillumination and is regained very efficiently and reversibly by ultra-violet/violet illumination. This makes it possible to localize asingle molecule multiple times, thereby enabling PALM in dynamicsystems.

The effective imaging resolution in PALM experiments dependson a number of factors, including the labeling density, photo-switching contrast, and thermal stability, but one of the most deter-mining factors is the number of photons that can be detected froman individual fluorophore in a single switching cycle. In the case ofphotochromism, this is determined by the competition between theoff switching and radiative and nonradiative deactivation processof the fluorescent state absorbing at 480 nm, since a moleculecannot emit more photons once it has switched to the non-fluorescent state. In the case of Dronpa, the efficient off-switchinglimits the number of emitted photons, which presents a possibledrawback in these measurements. While faster switching mutantsof Dronpa have been reported previously,13,14,15 slower-switchingmutants have, to our knowledge, not been reported before. Here wefound the ancestor of Dronpa, 22G, to be a slower switcher. Usingthis protein, we achieved an increased imaging resolution in PALMexperiments. Pursuing mutants optimal for these measurements,we built a high-throughput imaging system aimed at characterizingthe photophysics of fluorescent protein mutants at the level ofindividual bacterial colonies. We succeeded in developing a mutantwith slower off-switching kinetics compared to Dronpa, providingus with insight into the structural aspects of the switching kinetics.

Experimental

Analysis of switching kinetics by fluorescence microscopy

Recombinant proteins were expressed in Escherichia coli andpurified as described previously.10 For the droplet screening,

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mineral oil (Sigma Aldrich, St. Louis, MO) was deposited ona glass bottom dish and placed on a fluorescence microscope(IX71; Olympus, Tokyo, Japan). A tiny droplet of protein solutionembedded in mineral oil (ª0.5 nl, 10 or 100 mM) was createdusing direct injection by a microinjector (Femtojet, Eppendorf,Hamburg, Germany) equipped with a glass capillary (Femtotip;Eppendorf). The fluorescence microscope consists of a 75 W xenonlamp, a motorized filter wheel (Lambda 10-2; Sutter Instrument,Novato, CA), an objective lens (UPlanSApo 20¥/0.75, Olympus)a cooled charge coupled device camera (CoolSnapHQ; Pho-tometorics, Tucson, AZ), and an image acquisition/processingsoftware package (MetaFluor Ver. 5.0r7; Molecular Devices,Downingtown, PA). To analyze the data we used Igor Pro version4.09A (Wavemetrics, Portland, OR).

Mammalian expression of b-actin fused to fluorescent proteins

Plasmid DNA for mammalian expression of b-actin fused toDronpa (Dronpa-b-actin/pMC1) and 22G (22G-b-actin/pMC1)were made by inserting the gene encoding human b-actin to theBamHI/NotI site of phKikGR1-MC1 vector (Amalgaam, Tokyo,Japan) followed by Dronpa and 22G substitution for hKikGR atthe NheI/AgeI site. The plasmid DNA was transfected into HeLacells on a glass base dish (Asahi Techno Glass Corp., Tokyo,Japan) with FuGENE 6 (Roche, Basel, Switzerland) accordingto the manufacturer’s protocol. Two days after transfection, thecells were washed three times using Hank’s balanced salt solution,fixed with 4% formaldehyde (Thermo Scientific, Rockford, IL) inphosphate buffered saline (PBS) for 20 min at room temperature,and finally washed three times in PBS before being subjected tothe PALM imaging.

PALM imaging

We used a total internal reflection fluorescence microscope with aspecial software package for PALM (Carl Zeiss, Jena, Germany).A laser beam at 489 nm emitted from a diode-pumped solidstate laser was attenuated with an acousto-optic tunable filter(AOTF) and focused on the back-focal plane near the edge ofa high numerical aperture objective lens (aPlan-APOCHROMAT100¥/1.46, Carl Zeiss) to achieve illumination in total internalrefraction (TIRF) mode. In previous reports of PALM withDronpa, 405 nm laser illumination or a two-photon absorptionprocess was used to recruit a small fluorescent population ofDronpa for the single molecule imaging.4,16 In our system, however,we could acquire the images without using the 405 nm laser, whichwe attribute to spontaneous on-switching of the protein molecules.Indeed we have observed a similar behavior in previous single-molecule measurements.4,12 The PALM images were generatedusing the software package installed on the microscope, whichcontains a processing protocol similar to that reported by Betziget al.2 PALM images were reconstituted from 4000 frames ofsequential images acquired with a frame rate of 25 Hz. Thehistogram analysis was performed with Igor Pro.

Random mutagenesis

Dronpa mutants were generated by random nucleotide replace-ment through error-prone polymerase chain reaction with TaqDNA polymerase in the presence of 100 mM MnCl2 (ref. 13).

The PCR product was inserted into the BamHI/EcoRI site ofthe pRSET bacterial expression vector (Invitrogen, Carlsbad,CA), and used for transformation of Escherichia coli, strainJM109(DE3) (Promega, Madison, WI). The bacteria were spreadon an LB plate supplemented with 100 mg ml-1 ampicillin,incubated at 37 ◦C overnight and at room temperature for anadditional one to three days before the colony imaging.

Colony imaging system

The colony imaging system is composed of a 300 W xenonlamp unit with a motorized filter exchanger (MAX-301; AsahiSpectra, Tokyo, Japan), a cooled CCD camera (CoolSnapHQ)equipped with a c-mount lens (Xenon 0.95/25; Jos. SchneiderOptische Werke, Bad Kreuznach, Germany), and an imageacquisition/processing software package (MetaFluor Ver. 5.0r1).Light from the xenon lamp illuminates the bacterial plate througha band-pass filter (BPF490/40 for off-switching and image acqui-sition, BPF390/20 for on-switching; Asahi Spectra). Time-lapseimages were acquired with the CCD camera through a band-passfilter (540AF30; Omega Optical, Brattlebolo, VT). The conceptof observation of fluorescence from colonies on a bacterial plateis the same as reported previously,17 but time-lapse analysis ofrepeated off/on cycle makes it possible to apply the system to thescreening of the photochromic proteins.

Analytical equivalent centrifugation

We used a ProteomeLab XL-A (Beckman Coulter, Fullerton, CA)for the analytical equivalent centrifugation. The concentration ofthe sample was adjusted to an optical density of 0.125 (22G andDronpa) or 0.25 (PDM1-4) at 503 nm in a 1 cm cuvette beforecentrifugation at 18.1 ¥ 103 g for 22 h. The profile of the absorbanceat 503 nm was monitored.

Results and discussion

Photochromic property of 22G

Dronpa is a monomeric mutant made from 22G, a tetramericfluorescent protein isolated from a coral of Pectiniidae sp.10

Both proteins display a comparable brightness, but 22G isan obligate tetramer and therefore seemed less interesting forapplications compared to monomeric Dronpa. The efficient,complete, and reversible photoswitching of Dronpa has beenintensively investigated,12,14 while the properties of 22G have notbeen paid much attention since it was tetrameric and apparentlynon-photochromic. We reassessed the photochromic behavior of22G using a fluorescence microscope and a small droplet of proteinsolution in mineral oil as a specimen, where the high power-densityillumination achieved by focusing the light with an objective lens,makes it possible to observe photochromism characterized by slowswitching rates. The specimen was repeatedly illuminated with 485nm light (using a 485DF15 bandpass filter), at a power density of55 mW cm-2, for 2 s followed by a 1 s interval to analyze the off-switching kinetics. A fluorescence image was acquired during theinterval using weak (3.3 mW cm-2) and short (20 ms) illuminationat 475 nm (475AF20). After 70 cycles (one off-switching event,210 s), the sample was illuminated with 380 nm light (380HT15) at7.4 mW cm-2, for 1 s followed by a 2 s interval. This was repeated for

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Fig. 1 Photochromic property of 22G revealed by fluorescent microscopy.Small droplets of 22G and Dronpa solutions in mineral oil were placed sideby side. (A) Time trace of the fluorescence intensity emitted from 22G (red)and Dronpa (green). The droplets were repeatedly illuminated using lightfrom a xenon lamp passed through either a 485 nm (indicated with cyanbars at the top) or a 380 nm (violet bars) bandpass filter. Fluorescent imagesof the droplets were continuously acquired during these irradiation phases.(B) Off-switching curves of 22G (red) and Dronpa (green) during the firstswitching step fitted to double and single exponential models, respectively(blue). (C) Acceleration of the off-switching rate of 22G. The off-switchingcurves of 22G at all iterations were normalized and overlaid. From topto bottom, the first to the tenth iteration. (D) Iteration dependency ofthe composition of the slow off-switching (Aslow, closed circle) and the fastoff-switching (Afast, open circle) components of 22G (red) and Dronpa(green, Afast only). (E) Iteration dependency of the t slow (closed circle) andt fast (open circle) for the off-switching of 22G (red) and Dronpa (green,t fast only). The t slow values might be imprecise at a first few iteration, sincethose were too large to determine within the time window used (210 s).

15 cycles to revert the sample to the bright state (one on-switchingevent, 45 s).

This first off/on step caused 97.9% of the Dronpa moleculesto convert to the non-fluorescent state, of which 96.6% wassubsequently restored to the bright state (Fig. 1A). Using thesame illumination protocol, 59.1% of 22G was rendered non-fluorescent followed by a 73.3% recovery. After ten iterations of theoff/on steps, 28.4% and 59.8% of the fluorescence of Dronpa and

Table 1 Off-switching kinetics of the proteins

Conc./mM I 0 Aslow t slow/s Afast t fast/s

22G 100 0.229 0.735 154.4a 0.038 23.510 0.191 0.729 119.0a 0.080 31.5

Dronpa 100 0.021 ND ND 1.02 10.810 0.059 ND ND 0.92 10.6

PDM1-4 100 0.113 0.749 66.0 0.147 11.010 0.086 0.518 43.3 0.387 8.6

a Approximate values are shown, since the t slow values for 22G were toolarge to determine precisely within the time window used (210 s).

22G, respectively, was lost by irreversible photobleaching. Takentogether these results showed that 22G is photochromic, but thatits switching rate and fatigue resistance are lower than Dronpa.

We next analyzed the off-switching rate in detail. The fluores-cence decay curves of Dronpa could be fit to a single exponentialfunction [eqn (1)], whereas those of 22G could be fit to a doubleexponential function [eqn (2)] (Fig. 1B, D, E).

(1)

(2)

where I(t) is the fluorescence intensity at time t. For 22G, the slowdecay component was always dominant. At the first iteration, theslow decay component was 19.9 fold larger than the fast decaycomponent. The time constant of the slow decay componentof 22G was 154.4 s, 14.3-fold slower than the time constantof Dronpa (10.8 s), whereas the fast decay component of 22Gwas comparable with that of Dronpa (23.5 s) (Table 1). TheI 0 value of 22G was 0.229, indicating that 22.9% [I 0/(I 0 + Aslow +Afast)] of the protein could not be converted to the non-fluorescentstate, whereas the non-photochromic component of Dronpa wasnegligible (2.0%). This apparent non-photochromic componentlikely consists of an equilibration between the light-inducedoff-switching and spontaneous recovery rates. Spontaneous on-switching in the dark has been reported for Dronpa and itsmutants.4,12 Reducing the illumination power, which slows thelight-induced off-switching but not the spontaneous recovery,increased the non-photochromic component (data not shown).Interestingly, the off-switching rate of 22G gradually acceleratedafter repetitive off/on cycles (Fig. 1C). This was mainly caused bydecrease in the time constant and irreversible bleaching of the slowdecay component, whereas the fast decay component was ratherstable. Dronpa, on the other hand, could always be described usinga single fast off-switching component.

PALM imaging with 22GThe precision with which an individual fluorophore can be

localized is roughly in inverse proportion to the square root ofthe photon numbers acquired from that particular fluorophore.2

In view of the comparable ensemble fluorescence brightnessbut slower off-switching rate of 22G compared to Dronpa, wereasoned that 22G fluorophores could be localized with a higherprecision than Dronpa. To investigate this hypothesis, we subjectedHeLa cells expressing b-actin fused to 22G or Dronpa to PALM

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measurements (Fig. 2A, B). High resolution images could beacquired using both 22G or Dronpa, though detailed investigationrevealed that the image acquired using 22G showed an increasedclarity. To evaluate this point quantitatively, we performed ahistogram analysis on the precision of the fitted coordinatesreported by the PALM software (Fig. 2C, D). The resultingdistributions were fit to a normal log Gaussian model [eqn (3)].

(3)

The median value of the precision, q, was calculated to be 11.1 ±1.7 nm for 22G. This was significantly smaller (p < 0.0005) than thevalue calculated for Dronpa, 26.0 ± 10.2 nm. From these results,we concluded that PALM measurements using fluorophores withslower off-switching can yield images with a significantly increasedprecision/resolution.

One possible drawback of the slower switching fluorophoreis reduction of the achievable time resolution. To evaluate thisissue, we compared the duration of the fluorescence emission fromrespective molecules on the microscope. More than 80% of the22G molecules emitted fluorescence during less than 9 frames ofthe sequential images, which corresponds to 360 ms (Fig. 2E).This was slightly longer than Dronpa (5 frames, 200 ms), butthe difference was small and the difference of achievable timeresolution should also be small.

Bacterial colony imaging system

A major drawback of 22G is its self-association to form stabletetramers,10 which sometimes interferes with the normal behaviorof proteins labeled with 22G, and highlights the need for furtherdevelopment of improved photochromic fluorescent proteins forPALM experiments. Given the large number of protein variantsthat can be designed, high-throughput screening of prospectivecandidates is one of the most essential tasks. To this end, we builtan imaging system aimed at the screening of Dronpa mutantsat the level of bacterial colonies expressing these mutants. Lightfrom a 300 W xenon lamp was passed through a 490 nm bandpassfilter (BPF490/40) and illuminated the bacterial colonies on anagar plate continuously for 150 min. During this illumination,fluorescence images of the plate were acquired every 5 minto evaluate the off-switching. Next, fluorescence recovery wasstimulated by switching to a 390 nm bandpass filter (BPF390/20)to evaluate the recovery for 30 min, while fluorescence imageswhere acquired every 5 min by transiently switching back to theBPF490/40 filter. This off/on switching cycle was automaticallyrepeated several times.

Dronpa mutants were created through error-prone PCR andsubjected to the screening. Fig. 3A shows an example of timeseries images acquired using this system, where the color scaleindicates the ratios of the fluorescence intensity at the selectedtime point to that of the initial image. Colonies with variouscolors, i.e. different off-switching rates, could be observed. Usingthe fluorescence images acquired during the switching, timetrajectories of individual colonies could be constructed (Fig. 3B),allowing the switching properties to be analyzed in detail. Controlbacteria expressing Dronpa and 22G were prepared on anotherplate and subjected to the same illumination procedure (Fig. 3C).Based on this information, we could quantitatively determine theoff-switching rate as being faster than Dronpa (blue colonies in

the bottom right of Fig. 3A), equivalent with Dronpa (greenishyellow), intermediate between Dronpa and 22G (yellow andorange), and equivalent to or slower than 22G (red). We pickedup a colony with intermediate off-switching rate, PDM1-4, andprepared a plasmid encoding the protein. For direct comparison,bacteria transformed with this plasmid were spread on the sameplate as Dronpa and 22G (Fig. 3C).

Time trajectories of Dronpa, 22G, and PDM1-4 on the bacterialplate are shown in Fig. 3D. The first illumination step at 490 nmswitched 31.2% of Dronpa to a non-fluorescent state, from which85.6% regained its fluorescence following illumination at 390 nm.Note that power density of the illumination on the bacterial plateswere 2.7 mW cm-2 at 490 nm and 0.17 mW cm-2 at 390 nm, whichare only 4.9% and 2.3%, respectively, of those for the droplet onthe microscope mentioned above. After 13 off/on cycles, 34.9% ofDronpa was lost through irreversible photobleaching. 22G showedmuch slower switching; only 7.9% was turned off during the firstcycle with 48.1% of the molecules regaining fluorescence, while27.6% was lost after 13 cycles. The off-switching rate of PDM1-4was intermediate between Dronpa and 22G, as expected; 20.0%was turned off, 95.1% regained fluorescence in the first cycle, and35.3% was lost after 13 cycles.

Photochromic property of PDM1-4

To analyze the photochromic kinetics of PDM1-4, we subjectedthe purified protein to the droplet imaging by fluorescencemicroscopy detailed above (Fig. 4A). Using the same schemeapplied to Dronpa and 22G in Fig. 1A, illumination at 480 nmturned off 85.9% of PDM1-4 followed by an 84.9% recovery uponillumination at 380 nm. After 10 cycles, 52.0% of the fluorescencewas lost to irreversible bleaching.

The off-switching rate of PDM1-4 was intermediate betweenthose of Dronpa and 22G (Fig. 4B), and could be analyzed usinga double exponential model. During the first switching cycle, thet slow and t fast values of PDM1-4 at 100 mM were 66.0 s and 11.0s, respectively (Table 1). The off-switching rate was observed toincrease at later iterations (Fig. 4C), which was apparently causedby conversion from the slow to the fast off-switching componentrather than by a change in the t values (Fig. 4D,E).

Interestingly, the off-switching rate of PDM1-4 was increasedby diluting the solution to 10 mM (Fig. 4B, Table 1). This wasagain caused by conversion of the slow off-switching componentto the fast off-switching component; the relative contribution ofthe fast off-switching component [Afast/(Aslow + Afast)] increasedfrom 16.4% at 100 mM to 42.8% at 10 mM, whereas the change in tvalues was small (from 66.0 s to 43.3 s for t slow, and from 11.0 s to8.6 s for t fast). The off-switching rate of 22G was also increased bydilution, but this was caused by a fall in the time constant not bythe conversion of the component (Fig. 4B, Table 1). The effect ofdilution on the off-switching rate of Dronpa was small (Fig. 4B,Table 1).

Quarternary structure of PDM1-4

We performed analytical equivalent centrifugation on 22G,Dronpa and PDM1-4. The concentration gradient curve of 22Gcould be fitted to a single ideal species model [eqn (4)] with the

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Fig. 2 Higher precision PALM images of actin tagged with photoswitchable proteins in HeLa cells. (A, B) Fixed cells expressing 22G-b-actin(A) or Dronpa-b-actin (B). Leftmost: TIRF images. Middle left to rightmost: PALM images with different cutoffs, in which only those positionswith localization precision estimated to be below the cutoff are retained. The scale bars indicate 1 mm in all images. (C) Representative histograms of theestimated localization precision for 22G (left) and Dronpa (right). Both were fitted to a normal log Gaussian model. We analyzed multiple images (n =20 for 22G and n = 14 for Dronpa) and got essentially the same results. (D) Statistical analysis of the median value of the precision, q. (E) Representativehistogram of the number of frames during which respective molecules emit fluorescence for 22G (left) and Dronpa (right).

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Fig. 3 Screening of Dronpa mutants using the bacterial imaging system. (A, C) Agarose plates with bacteria transformed with plasmids resulting fromrandom mutagenesis (A), or 22G, Dronpa, and PDM1-4 (C). Ratios of respective images over the initial image are shown in 24 bit mode intensity-modifiedpseudo-color display (IMD). The ratio ranges from 1.0 to 0.0 (magenta to blue). (B, D) Time course of the fluorescence intensities measured from colonies#1 and #2 (B) and Dronpa, 22G, and PDM1-4 (D). The bars in the upper plot indicate the irradiation sequence at 490 nm (cyan) or 390 nm (violet).

molecular weight of the tetramer, indicating formation of obligatetetramer, as reported previously10 (Fig. 5A).

(4)

with Ctotal(r) the weight concentration of protein at position r,Cm(r0) the concentration of monomeric protein at the meniscus,w the angular velocity, R the universal gas constant, T thethermodynamic temperature, M the molecular weight of protein,v the partial specific volume (vbar), r the density of solvent.

It has been reported that Dronpa is a monomeric mutant of22G,10 but the concentration gradient curve of Dronpa couldbe described with a self-association model in which the proteinassociates to a dimer [eqn (5)], rather than the single ideal speciesmodel (Fig. 5B).

(5)

where K ¢12 is the association coefficients for dimerization in weightconcentration. The association coefficient K ¢12 was determined tobe 1.50 (g l-1)-1. This indicates that Dronpa was in monomeric

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Fig. 4 Two component off-switching of 22G. (A) Time trace of thefluorescent intensity emitted from PDM1-4. The experimental protocolwas same as in Fig. 1. (B) Comparison of the off-switching curves of100 mM (solid line) or 10 mM (dashed line) of PDM1-4 (orange), 22G(red), and Dronpa (green) at the first iteration. (C) Acceleration of theoff-switching rate of PDM1-4. From top to bottom, the first to tenthiteration. (D) Iteration-dependent conversion from the slow off-switching(Aslow, closed circle) to the fast off-switching (Afast, open circle) componentof PDM1-4. (E) Iteration-dependent gradual fall in t slow (closed circle) andt fast (open circle) for the off-switching of PDM1-4.

form in diluted solutions (99.6% at 0.1 mM and 90.0% at 1 mM),as reported, but dimerized in concentrated solutions (24.9%,62.4%, and 86.0% were dimerized at 10 mM, 100 mM, and 1 mM,respectively) (Fig. 5C).

The concentration gradient curve of PDM1-4 could be de-scribed with a self-association model [eqn (6)] in which the proteinassociates to a dimer and tetramer (Fig. 5D).

(6)

where K ¢14 is the association coefficients for tetramerization inweight concentration. The association coefficients K ¢12 and K ¢14

were determined to be 1.84 (g l-1)-1 and 1.35 ¥ 103 (g l-1)-3,respectively. Based on the association coefficients, the contributionof the monomeric, dimeric, and tetrameric forms were estimatedat various concentration of PDM1-4 (Fig. 5E). The monomer wasdominant at 1 mM or less, but diminished at higher concentrationin favor of the tetramer, which became dominant at 100 mM ormore. The contribution of the dimeric form reached a maximum(8%) at around 4 mM, but remained a minority on the whole.

Intriguingly, the contributions of the monomeric form were7.1% and 35.4% at 100 mM and 10 mM, respectively, comparablewith the relative contributions of the fast off-switching com-ponents observed in the kinetic experiments mentioned above,16.4% and 42.8%, respectively. These results led us to concludethat the slow off-switching component of PDM1-4 correspondedto protein in the tetrameric form, while in diluted solutions,the protein dissociated to a monomer with correspondingly fastoff-switching rate. This is consistent with the fast off-switchingobserved for monomeric Dronpa and the slower switching ratedisplayed by 22G, which forms a stable homo-tetramer. Thisfinding strongly suggest that the structural interaction and/or thephotophysical coupling between protomers is critical to determinethe kinetics of reversible photochromism. Interestingly, repeatedoff/on switching cycles caused the conversion of both 22G andPDM1-4 from slow to fast off-switching components, which couldbe triggered by photo-damage to a portion of the protomersbreaking down the structural interaction and/or disordering thephotophysical coupling between the protomers.

Sequence analysis revealed that PDM1-4 differs from Dronpain only a single substitution of aspartate for lysine145 (Table 2).This residue is not located in the vicinity of the chromophore, butrather on the interface between protomers A and C (Fig. 5F, cyan).This substitution likely increases the tendency of PDM1-4 to self-associate along the A/C interface, with dimers self-associatingalong the A/B interface to form tetramers even though there isno amino acid substitution along the A/B interface. Cooperativeassociation along the A/B and A/C interfaces has been shownpreviously while monomerizing 22G, for which two substitutions,I102N on the A/C interface and G218E on the A/B interfacewere required (Fig. 5F, orange and yellow). A single substitutionof one of these amino acids was insufficient to break each interfacecompletely.11

Our previous NMR experiments indicated that the mechanismof the photoswitching is based on dynamic flexibility of thechromophore and b-barrel (Fig. 5F, magenta),11 and cannotbe explained by a discussion of the chromophore configura-tion/conformation alone. The fact that PDM1-4 contains just asingle mutation located on the exterior of the b-barrel rather thannear the chromophore strongly supports this viewpoint.

Table 2 Comparison of amino acid residues. All the differential residuesare shown

Residue number

102 114 145 162 194 205 218

22G I F K L R N GDronpa N Y K S H S EPDM1-4 N Y N S H S E

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Fig. 5 Quaternary structure of 22G, Dronpa, and PDM1-4. (A, B, D) Equilibrium radial absorbance profile of 22G (A), Dronpa (B), and PDM1-4 (D)by analytical equivalent centrifugation (blue circles). The profile of the absorbance at 503 nm was successfully fitted to the single ideal species model (22G)or the self-association model (Dronpa and PDM1-4) (red line). (C, E) Composition of monomer, dimer, and tetramer Dronpa (C; without tetramer)and PDM1-4 (E) estimated from the association constants. (F) Mapping on a crystal structure of Dronpa (PDB ID 2Z6Y). Magenta indicates a flexibleregion only in the dark state.11 PDM1-4 was made by asparagine substitution of Dronpa lysine 145 (cyan). Replacement of isoleucine 102 to asparagine(orange) on the A/B interface and glutamine 218 to glycine (yellow) on the A/C interface were critical to dissociate the tetrameric structure of 22G tomonomer.

Conclusions

In this work we have revealed that 22G, the parental clone ofDronpa, is photochromic and can be used as a fluorescent tag forPALM. The slower off-switching rate of 22G allows an increased

number of photons to be detected for individual fluorophores,which makes it possible to improve the resolution of PALMimages. Using a home-built bacterial colony imaging system, wesucceeded in developing a Dronpa mutant, PDM1-4, which has anintermediate switching speed between Dronpa and 22G. Kinetic

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and structural analyses of PDM1-4 revealed an intricate linkbetween self-association of the protein and the kinetics of theswitching process. The plate imaging system is expected to be apowerful approach in pursuing effective fluorescent proteins as atag for PALM experiments.

Acknowledgements

We thank Carl Zeiss MicroImaging GmbH for technical assis-tance. The authors acknowledge Molecular Ensemble Programat RIKEN, Japan MEXT Grant-in Aid for Scientific Researchon priority areas, the Research Foundation – Flanders (FWO)(Grant G.0366.06), the KULeuven Research Fund (ConcertedResearch Action GOA 2006/2, Center of Exellence INPAC,CREA2007), and the Federal Science Policy of Belgium (IAP-VI/27) for financial support. Peter Dedecker is a postdoctoralfellow of the Research Foundation – Flanders (FWO).

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