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Noname manuscript No.(will be inserted by the editor)

An analytical workflow for the molecular dissection ofirreversibly modified fluorescent proteins

Vivien Berthelot · Vincent Steinmetz · Luis A.Alvarez · Chantal Houée-Levin · FabienneMerola · Filippo Rusconi · Marie Erard

Received: date / Accepted: date

Abstract Owing to their ability to be genetically expressed in live cells, fluores-cent proteins have become indispensable markers in cellular and biochemical studies.These proteins can undergo a number of covalent chemical modifications that may af-fect their photophysical properties. Among others, such covalent modifications maybe induced by reactive oxygen species (ROS), as generated along a variety of bio-logical pathways, or under ionizing radiations. In a previous report [1], we showedthat exposure of the cyan fluorescent protein (ECFP) to •OH amounts that mimickthe conditions of intracellular oxidative bursts (associated with intense ROS produc-tion), led to observable changes in its photophysical properties, yet in the absence ofany direct oxidation of the ECFP chromophore. In this work, we analyse in depth theassociated structural modifications occurring in the protein. Following the quantifiedproduction of •OH, we devised a complete analytical workflow, based on chromatog-raphy and mass spectrometry, that allows us to fully characterize the oxidation events.While methionine, tyrosine and phenylalanine are the only amino-acids found to beoxidized, a semi-quantitative assessment of their oxidation level shows that the pro-tein is preferentially oxidized at eight residue positions. To account for the preferredoxidation of a few, poorly accessible methionine residues, we propose a multi-stepreaction pathway supported by pulsed radiolysis experiments. The described exper-imental workflow is widely generalizable to other fluorescent proteins, and opens

F.R. and M.E are co-last authors of this work.

V. Berthelot · V. Steinmetz · C. Houée-Levin · F. Merola · F. Rusconi · M. ErardLaboratoire de chimie physique; UMR CNRS 8000; Building 350,F-91405 Orsay Cedex, France,E-mail: marie.erard @u-psud.fr;Tel. +33 1 69 15 30 14; Fax +33 1 69 15 61 88

F. Rusconi also at“Régulation et dynamique des génomes”; U INSERM 565—UMR CNRS 7196; Muséum nationald’Histoire naturelle; 57, rue Cuvier, Case Postale 26; F-75231 Paris Cedex 05, France,E-mail: [email protected];Tel. +33 1 69 15 76 04; Fax +33 1 69 15 61 88

2 Vivien Berthelot et al.

the way to the identification of crucial covalent modifications impacting their photo-physics.

Keywords protein oxidation · mass spectrometry · •OH radicals · cyan fluorescentprotein (ECFP) · γ & pulsed radiolysis

1 Introduction

Fluorescent proteins (called FPs, for short) have revolutionized the mechanistic in-vestigations of cellular processes by live cell imaging, flow cytometry and high-throughput bioassays [2]. These FPs comprise 11 β sheets that form a barrel en-closing a coaxial α-helix (Figure 1 a) which bears the chromophore resulting fromthe cyclization of three consecutive amino-acids [65-66-67] (Figure 1 b). As such,these proteins have been used to build fully gene-encoded sensors that allow one tomonitor a large set of biological chemical events in live cells [3].

Engaging in chemical sensing through the use of FPs requires a thorough knowl-edge of their photophysical responses to the cellular micro-environment. While someattention has been paid to the side-effects of either pH [4, 5, 6, 7], chloride ions [8],or refractive index [9] on the photophysical properties of FPs, the possible conse-quences of local production of reactive oxygen species (ROS: O2•−, •OH, H2O2,HOCl) have seldom been addressed [10, 11, 12, 1]. Nevertheless, ROS have cru-cial roles in physiological and pathological processes: they can be produced in largequantities in cells, be potentially harmful and cause oxidative damage, the so-calledoxidative stress [13, 14]. For example, the dynamics of pathogen phagocytosis, a phe-nomenon correlated with high levels of ROS production by enzymatic systems likeNADPH oxidase or myeloperoxidase [15], was recently investigated with the helpof FPs, [16, 17, 18, 19] while there has been a recent push to develop members ofthe FP family as specific oxidation sensors [20, 21, 10, 11, 22, 18]. In some of thelatter cases [10, 11, 22, 18] as well as in other reports [1, 12], the consequences ofROS exposure on the photophysical properties of FPs have been reported and showeddependency on either the nature of the oxidant and its quantity, or the FP variant. Onthe other hand, the characterization of ROS-induced chemical modifications of FPsmight prove fundamental in the understanding of their photobleaching mechanisms.Indeed, these photoreactions are thought to result from the uncontrolled productionof oxidants (like ROS) from the singlet or triplet fluorophore excited states [23]. Pho-tobleaching generates variations of the fluorescence signal, known as photofatigue,thus limiting the duration and the reliability of the measurements. This photophys-ical limitation becomes critical when high illumination densities are required, likein super-resolution optical microscopy or single molecule applications [24, 25]. Sofar, very few studies have been devoted to the identification of chemical modifica-tions involved in photobleaching [23, 26] and both the pathways and the targets aregenerally still unknown. In a previous study, we analyzed in detail the photophysicalperturbations induced by controlled amounts of hydroxyl (•OH) and superoxide an-ion (O2•−) free radicals on the cyan fluorescent protein (ECFP) [1] that is the mostwidely used donor in FRET-based imaging experiments in combination with a yel-low partner (like Citrine) [3, 27]. We found significant perturbations of the ECFP

Molecular dissection of modified fluorescent proteins 3

fluorescence (decreases in intensity and lifetime, without changes in its excitationand emission spectra), which did not result from direct chemical modification of thechromophore, but rather from oxidations at other sites that remain to be identified.

Understanding the causative relationships between the exposure of FPs to ROSand the resulting modification of their photophysical properties requires a thoroughstructural characterization of the oxidized proteins. Due to the lack of appropriateanalytical tools, these questions have not yet been addressed in detail up to now.Crystallography might look like an appropriate approach because fluorescent proteinsare highly compact and can be cristallized easily. However, the expected multiplicityof the chemical modifications limits the use of crystallography in this context. Thebottom-up strategy that is often used in proteomics should hold more promise. How-ever, the compactness of the FP barrel structure makes these proteins refractory toconventional enzymatic break-down, and thus previous mass spectrometric analyses(briefly reviewed by Alvarez et al. (2009) [28]) had to resort to chemically harsh pro-cedures with the risk of introducing unwanted chemical modifications to the proteins.In a previous work, we devised a new method aimed at digesting FPs in very mildconditions that opened the way to the molecular dissection of chemically-modifiedFP variants [28].

In the present report, we build on that previous work and further enhance ouranalytical workflow to achieve the full characterization of •OH-oxidized ECFP. Thisleads to the identification of ECFP oxidized residues, along with a first semi-quanti-tation of their oxidation events. We were able to spot oxidation events, mostly re-stricted to a few specific residues that might be responsible for the observed photo-physical changes. In addition, pulsed radiolysis studies provided further insights onthe •OH primary targets leading to these selective oxidations.

2 Experimental section

2.1 Cyan fluorescent protein purification and endoAspN-based digestion

ECFP refers to AvGFP F64L/S65T/Y66W/N146I/M153T/V163A/H231L (Clontech).The production and the purification of His-tagged recombinant ECFP were performedas described previously [1]. The His-tag was cleaved away, leading to a cyan fluores-cent protein in which the starting M residue is replaced by the GA dipeptide. Theresidue numbering scheme relies on the chromophore TWG triplet residues being atpositions 65-66-67 (see Figure 1 b). The ECFP solution was dialysed against a 30 mMphosphate buffer at pH 7.5 for irradiation experiments. The protein concentration wasassessed by UV absorption of the chromophore [ε (λ434nm) = 30000 cm−1.M−1].

2.2 Radical production and radiolysis

A detailed description of the radiolysis procedures is provided in Online Resources,section OR1. Briefly, the quantitative production of •OH has been performed witheither stationary γ radiolysis or high energy electron pulses. Their selection was per-formed using the well-known method of scavengers in solutions deoxygenated and


saturated with N2O [29]. Additional pulsed radiolysis experiments were performedusing •N3 radicals as oxidants. For the irradiations, the ECFP concentration wasusually 5 µM unless otherwise specified, at neutral pH. The doses applied were inthe range 20–400 Gy (stationary radiolysis) or in the range 4–10 Gy (pulsed ra-diolysis). For an easier understanding of the results, the dose was replaced by theR = [•OH]/[ECFP] ratio, which is proportional to the irradiation dose.

The pulsed radiolysis-elicited reaction was followed spectrophotometrically be-tween λ350 nm and λ750 nm and the difference absorption spectra of the reaction in-termediates were reconstructed from the maxima of the absorbance variation in theinvestigated wavelength range.

2.3 Structural modeling of residue accessibility

The ECFP structure at physiological pH is available from PDB (ID: 2WSN) [30], butdoes not comprise the C-terminal tail of the protein (sequence 230 TLGMDELYK 238).In order to correlate the mass spectrometric results with the spatial localization of allof the ECFP residues, we generated an automated model of ECFP using the SWISS-MODEL workspace described in Online Resources, section OR2.

We calculated the solvent-accessible surface percentage of each residue usingthe GETAREA service provided by the Sealy Center for Structural Biology at theUniversity of Texas Medical Branch, Galveston, USA [31].

2.4 Chromatography

Desalting of the proteins and of peptide mixtures were performed according to theprocedure described by [32], with the R2 Poros polymeric resin (Applied Biosystems)as the reversed phase.

Peptides from endoAspN-digested ECFP [28] were separated by reversed-phasehigh-performance liquid chromatography (RP-HPLC) on an Åkta Purifier setup fromGE Healthcare, using a Stability S-C23 end-capped resin (Cil Cluzeau, Sainte-Foy-la-Grande, France). The gradient was developped from 100 % buffer A [water, 0.035 % tri-fluoroacetic acid (TFA)] to 100 % buffer B [95 % acetonitrile in 0.035 % TFA] in60 min. Each fraction was manually collected as the peak shape was monitored onthe Unicorn software chromatogram view (GE Healthcare).

2.5 Mass spectrometry

Fluorescent proteins—irradiated or not—or peptides thereof were analyzed by elec-trospray ionization (using either normal, micro- or nano-spray) on three differentmass spectrometers : 1) a QStar Pulsari hybrid quadrupole–time-of-flight (AppliedBiosystems) ; 2) a 7 T Apex Qe FT-ICR with the quadrupole-hexapole interface al-lowing for m/z -based selection, ion accumulation and cooling before injection in theICR cell (Bruker Daltonics); 3) an LTQ-Orbitrap (Thermo Scientific) with a micro-spray source in line with a Dionex Ultimate 3000 HPLC setup. In each case, a typical


Fig. 1 a. Barrel structure of ECFP. b. ECFP sequence. Residues that were found oxidized are denotedusing filled or semi-filled circles (see text for details). The chromophore is typeset in bold blue characters.

ionization protocol was used, with standard settings appropriate for either a ≈ 30 kDaprotein or peptides. All the experiments were performed in the positive ionizationmode. The typical amount of protein used for one analysis was of 25 µL of a 10 µMsolution. The peptidic solutions were typically in the 2–5 µM range. When peptideanalyzes were performed by MALDI-TOF mass spectrometry, the instrument was a4800 MALDI TOF/TOF Analyzer from AB Sciex and the sample preparation wasaccording to the conventional dried droplet procedure using α-cyano-4-hydroxycin-namic acid (ACHCA) as the matrix. All gas-phase fragmentation spectra were ac-quired using collision-induced dissociation (CID). The CID mass data were analyzedto locate the oxidation events on m/z -selected ions.

Mass spectrometric data analysis was performed by first exporting the data tosimple ASCII-formatted files so as to make use of free and open source softwarefor all the analysis steps. Mass spectrum visualization and analysis was performedby using the mMass program (http://mmass.org) [33, 34]. Mass spectrometric datasimulations and detailed analyses were performed using either the massXpert pro-gram (http://massxpert.org) [35, 36] or GNU polyXmass [37]. Data handling wasperformed by setting up a relational database using the free and open source SQLitedatabase (http://sqlite.org). All in-house programming was performed using eitherC++ or Python as programming languages under a Debian GNU/Linux computingplatform (http://debian.org).


3 Results and discussion

3.1 Oxidation of the ECFP by •OH

After γ irradiation of purified ECFP with a delivered dose yielding a R= [•OH]/[ECFP]ratio of 10, the protein was desalted/concentrated and analyzed by ESI-MS. Figure 2shows the spectra obtained for non-irradiated or irradiated ECFP.

Non-oxidized protein (panel A) produced a charge envelope with peaks havingthe expected m/z ratio (e.g.: measured, m/z 841.21, z= 32; calculated, m/z 841.01).After oxidation with •OH (panel B), that same peak almost disappeared, with theconcomitant build-up of several new peaks (denoted with asterisks) separated fromeach other by m/z 0.5, corresponding to a mass increment due to the binding of oxy-gen atoms to the protein (∆M = 16 u). At least five resolved oxidation levels couldbe observed. Although in our experimental conditions, water γ radiolysis yields anamount of •H radicals that is non-negligible ([•H]/[ECFP]≈ 1), we did not detectany of their reaction products, like, for example, loss of H2S (∆M =−34 u) or loss ofCH3SH, with ∆M =−48 u [38, 39, 40, 41]. Further, at the level of the whole protein,no other •OH-induced modification was observed, like, e.g. decarboxylation, or in-termolecular Cys- or Tyr-based dimerization [for a review of ROS-induced chemicalmodifications see [42]].

A dose-response experiment was performed whereby ECFP was submitted to•OH oxidation with R ratios in the range 0–17. The correlation between an increas-ing R ratio and the appearance of ECFP oxidized variants, along with the concomi-tant decrease of the mass peak corresponding to the unoxidized ECFP, is manifest, asshown in panel C of Figure 2. With an R ratio of 17, the protein was so heavily modi-fied that the obtained mass spectrum failed to be informative: the molecular diversityassociated to the large number of modified ECFP polypeptide triggers the classicalspectral suppression phenomenon. On the other hand, for oxidation ratios of ≈ 17,we observed only a 15 % decrease in both the fluorescence quantum yield and life-time of ECFP [1]. Such moderate concomitant photophysical perturbations indicatethat many of these ECFP oxidations actually have little or not impact on the proteinphotophysical properties.

3.2 Characterization of the •OH-oxidized variants of ECFP

3.2.1 Direct analysis of the oxidized ECFP endoAspN-produced peptidic mixture

Oxidized ECFP samples were subjected to an endoAspN digestion according to theprotocol described by [28]. The peptidic mixture was analyzed by ESI mass spec-trometry, by injection in the flow, direct infusion or nano-spray. These mass spectro-metric analyses failed to afford a correct sequence coverage, which contrasted withthe excellent coverage for non-oxidized ECFP that we described in an earlier report[28]. Most evident was the complete lack of signal corresponding to the chromopep-tide (coordinates [36–75]), comprising the three 65 TWG 67 residues that undergo aseries of post-translational modifications to form the chromophore (represented using


Fig. 2 Mass spectrometry-based monitoring of the ECFP oxidation with •OH. ECFP was either non-oxidized (a) or •OH-oxidized with R = 10 (b). The inset shows a zoomed view of the framed mass peak(z = 32). The non-oxidized protein was almost homogeneous, while the •OH-oxidized protein showedseveral variants differing by the mass of one or more oxygen atoms (∆M = 16 u). c. Oxidation progressionof ECFP as observed upon increasing the R = [•OH]/[ECFP] ratio (numbers next to the traces). The mostuseful R value for our experiments was found to be in the range 4–10. Arrows point to the mass peakcorresponding to the unmodified polypeptide.


bold blue characters in Figure 1 b). This peptide is one of the largest peptides ex-pected from an ECFP digestion by endoAspN, even with partial cleavages occurringin other regions of the protein sequence. We reasoned that this insufficient sequencecoverage was due to a huge increase in the molecular complexity of the peptidic mix-ture in the case of oxidized ECFP. Indeed, the whole set of oxidation combinationsfor each oxidizable peptide could have made the sample too complex to be succcess-fully analyzed without suffering from spectral suppression. Furthermore, because theoxidation of peptides increases their hydrophilicity, the oxidized peptides could bedefavored in their desolvation/ionization with respect to their unmodified counter-parts [43], thus leading to a mass spectrometric signal loss that would mainly affectthe subpopulation of peptides that was of greatest interest to this study [44].

3.2.2 Chromatographic separation of ECFP peptides

In a first attempt to reduce mass spectral suppression, a micro-chromatography ex-periment was performed as in [32], with a step-gradient elution in two 20 % and60 % acetonitrile fractions. Analysis of the fraction contents showed that a substantialamount of mass spectrometric signal could be recovered. Indeed, the chromopeptidewas detected in the 60 % acetonitrile fraction; however, sequence coverage was stillpartial.

Fig. 3 Reversed-phase high performance liquid chromatography separation of •OH-oxidized ECFP pep-tides following endoAspN digestion. Absorbance detection was performed at λ214 nm and λ414 nm wave-lengths, for detection of the peptidic bond and of the chromophore, respectively. The amount of proteininjected was 200 pmol. Each fraction was collected in a separate tube for further analysis by mass spec-trometry.


The peptidic mixture obtained upon endoAspN digestion of ECFP was thus sub-jected to an HPLC separation using a reversed-phase resin. Figure 3 shows the ob-tained chromatogram, with absorbance detections at λ214 nm and λ414 nm. The latterwavelength corresponds to the absorption band of the chromopeptide [4], thus allow-ing us to monitor the fraction in which it eluted (at 56 min). All the fractions were col-lected separately and later analyzed by ESI or MALDI mass spectrometry to identifythe oxidized peptides. One useful observation was that oxidized peptides did almostsystematically elute in the fraction preceding the one containing their non-oxidizedpeptide counterparts. This observation was both expected (because the oxidized pep-tides are more hydrophilic) and helpful in the analysis of the mass spectrometric databecause one could predict in which fraction to search for oxidized variants of anygiven peptide.

3.2.3 Full mapping of the oxidation sites in ECFP

The ECFP primary structure could be completely covered by our mass spectrometricanalyses. In agreement with our preceding findings (Figure 2), we focused our dataanalyzes on oxygen additions (∆M= 16 u). Upon an entirely manual scrutiny of massspectrometric data obtained for oxidized ECFP-derived peptides, all the oxidationevents were mapped to a few positions in the protein sequence. The residues found tobe oxidized are all either aromatic or sulfur-containing residues [45, 46]. Two kindsof residues could be singled out: residues oxidized with R = [•OH]/[ECFP] ratios inthe range 4–10 and residues that could only be observed as oxidized species uponintense irradiation (R ≈ 20). In Table 1 and Figure 1 (panel b), we listed only thepeptides that were found oxidized for R in the range 4–10. For each listed peptide, theresidue detected as bearing the oxidation is specified, along with its abundance and itssolvent accessibility. The different residues belonging to the oxidized peptides wereclassified into four categories: those that were found not oxidized ( /0), barely oxidized(


Table 1 ECFP oxidized peptides data for R = [•OH]/[ECFP] in the range 4–10

ID Sequence Coordinates Oxid. residue / abundance SAA %1 GAVSKGEELFTGVVPILVEL [ –18] ? / -2 DVNGHKFSVSGEGEG [21–35] F27 / 0.73 DHMKQH [76–81] M78 / 0.9

4 DFFKSAMPEGYVQERTIFFK [82–101]

F83 / /0F84 / /0

M88 /Y92 / /0F99 / /0F100 / /0

0.50.200

58.30.2

5 DGNYKTRAEVKF [103–114] ? / -

6 EYNYISHNVYITA [142–154] ? /Y151 /

-59.6

7 DGPVLLPDNHYLSTQSALSK [190–209] Y200 / 33.0

8 DHMVLLEFVTAAGITLGMDELYK [216–238]

M218 /F223 / /0

M233 /Y237 /

040.258.4100

SAA: surface accessible area. The oxidized residues were classified in four categories: those that werefound not oxidized ( /0), barely oxidized (


Fig. 4 Mass spectrometric analysis of the •OH-oxidized ECFP chromopeptide (R ≈ 20). Isotopic clusterswere found to correspond to the chromopeptide, both in the non-oxidized form, at m/z 1120.83, z = 4 andin the singly-oxidized form at m/z 1124.85. The chromopeptide was found to bear a disulfide bond.

oxidized residues hinted at indirect oxidation mechanisms that we investigated nextby performing the pulsed radiolysis experiments below.

4 Pulsed radiolysis

The first steps of the reaction of •OH with ECFP were investigated by pulsed radi-olysis, where the time span for •OH creation is short compared to the reaction time.This method affords two useful data sets: the identification of reaction intermediates(by their transient absorption spectra) along with their formation and decay kinetics.

The absorbance kinetics traces recorded after the pulsed production of •OH rad-icals were different depending on the wavelength (Figure 5, panel a). At λ315 nm,the absorbance reached a maximum value a few microseconds after the pulse andthen decayed. The build-up phase is in agreement with a rate constant of (2.5± 0.5)1010 mol−1.L.s−1, which is consistent with that of the reaction of •OH with pro-teins of the same size [49]. In the range λ390−410 nm, the absorbance reached itsmaximum ca. 50 µs after the pulse and then decayed slowly in ≈ 100 ms (notshown). These kinetics, that depend on wavelength, show that several—chemicallydifferent—intermediates are produced in the first tens of microsecond after the pulsed•OH production. Moreover, the time-scale of the absorption build-up at λ390 nm didnot depend on the [ECFP] nor on the [•OH] (Figure 5, panel a), indicating an in-tramolecular process.

The time-resolved absorption spectrum of the ECFP solution recorded 40 µs afterthe •OH pulse is shown in Figure 5, panel b, trace •OH. The signal at λ320 nm might


Fig. 5 a. Absorbance kinetic traces recorded after the pulse at λ315 nm and λ390 nm. Experimental con-ditions were, for λ315 nm, [ECFP] = 30 µM, [•OH] = 7 µM; for λ390 nm, curve 1: [ECFP] = 30 µM,[•OH] = 7 µM; for λ390 nm, curve 2: [ECFP] = 12 µM, [•OH] = 4.5 µM. b. Difference absorption spectrarecorded 40 µs and 500 µs after the pulse, for •OH radicals and •N3 radicals, respectively. Experimentalconditions were [ECFP] = 12 µM, [•OH] = 4.5 µM. The experimental set-up time response was below0.1 µs.


be attributed to addition of •OH to Tyr and Phe [50, 51]. The bands in the λ390−410 nmrange could be attributed to ECFP-(TyrO•) and/or to ECFP-(MetS∴X)•+ (X = O, Nor S) [52, 46, 45, 53] (Figure 5, panel b). To confirm the nature of these radicals, wecompared this spectrum with the one obtained after an oxidation using azide radicals(Figure 5, panel b, trace •N3), which are known to make electron transfers with ty-rosine, thus yielding only the ECFP-(TyrO•) radical, and to have low reactivity withmethionine [54, 55]. The new spectrum appeared different from the former one, withshifted maxima, confirming that TyrO-centered radicals are not the only short-livedproducts of the ECFP oxidation and that methionine-centered radicals are present.

In proteins, the tyrosine residue is usually thought to be the “end point” of oxi-dation. For example, oxidations of tryptophane or methionine residues would, in ourcase, end up with the formation of ECFP-(TyrO•) by intramolecular electron transfer[56]. However, in our experiments, we found that several methionine residues are ulti-mately oxidized, despite the fact that, in some cases, these residues are deeply buriedin the protein structure. In addition, the reaction leading to the increase of absorbancein the λ390 nm region in pulsed radiolysis experiments was of the first order, indicatingan intramolecular reaction. We propose in Scheme 6 interpretations of these obser-vations. •OH radicals can react with methionine or tyrosine residues, as observed inthe transient absorption spectra shown in Figure 5 (steps a and b, respectively). The•OH adduct on the tyrosine residue might either lead to DOPA (step c) or undergoproton-catalyzed dehydration to the TyrO• radical (step d), which in turn is repairedby a methionine residue (step e), thus leading to methionine sulfoxide (step f). Inthis protein, the distances between methionine and tyrosine residues can be short(d [M78–Y200]= 8.2 Å, d [M218–Y143]= 4.6 Å and d [M233–Y237]= 5.8 Å) orlonger (d [M88–Y39]= 20 Å (see Online Resources Figure ??). In the former case,the electron transfer should be fast, while in the latter case it can proceed by hoppingthrough aromatic residues [57, 58], as illustrated in Online Resources Figure ??. Inall cases, the electron transfer requires that the reduction potential of the methionineresidue be lower than that of tyrosine, which might indeed happen because the me-thionine reduction potential is highly dependent on its immediate environment [59].Furthermore, other studies on peptides or proteins showed that many oxidations donot always end-up at tyrosine residues [60, 61, 62, 63].

5 Conclusions

In this work, the described analytical workflow showed that fluorescent protein oxi-dation was observed on methionine, tyrosine and phenylalanine residues. One inter-esting finding is that the three-dimensional modeling of the protein sets part of theoxidized residues in the inner face of the barrel, hinting at indirect oxidation path-ways involving one or more hops across the protein structure. This observation is ofparticular interest in the context of studies involving •OH-based oxidation of proteinsto determine the three-dimensional structure of proteins or the topological organiza-tion of protein assemblies [64, 65]. Our work could indeed point at one difficulty inthis field, that might arise if the ultimate oxidation state of any given residue were notthe sole result of its mere accessibility to the solvent.


Fig. 6 Reactions proposed to explain the obtained kinetics traces

Another significant result, in combination with previous results from our lab-oratory [1], is that the chemical modifications identified in this report do elicit aperturbation of the photophysical properties of the fluorescent protein, even if thechromophore was not modified. This observation is consistent with our previouslyproposed hypothesis that the oxidation of the protein increases the barrel flexibilitythus enhancing non-radiative deexcitation paths through excited-state chromophoretorsion [1]. In addition, the present work shows that these dynamical perturbationsmight be triggered only by a few critical oxidations in the ECFP structure, all locatedat least 8 Å away from the chromophore, thus excluding its direct quenching.

Acknowledgements V.B. received a doctoral fellowship from the University of Paris-Sud, Orsay, France.L.A.A. was supported by a fellowship from the French ministry of research (MESR). The authors thankDr Philippe Maı̂tre (plateforme de spectrométrie de masse du LCP, University of Orsay) for interestingdiscussions, Drs Lionel Dubost and Arul Marie of the mass spectrometry facility of the Muséum in Paris,France and Dr Jean-Pierre Le Caer of the mass spectrometry facility of the ICSN in Gif-sur-Yvette, France.We thank Dr Vincent Favaudon for the use of the pulsed radiolysis set-up (Institut Curie, Orsay, France).We are indebted to the COST CM1001 (non-enzymatic protein oxidation) action for fruitful discussions.


1 Online Resources

1.1 OR1 – Radical production and radiolysis

The quantitative production of •OH has been performed with either stationary γ radi-olysis or high energy electron pulses. In both cases, the selection of •OH or •N3 freeradicals was obtained using the well-known method of scavengers [29]. For instance,in N2O-saturated aqueous solutions, radiolysis creates •OH radicals with a radiationchemical yield (G) equal to 0.55 µmol.J−1 [29]. In N2O-saturated solutions and in thepresence of azide ions (1 mM NaN3), azide radicals, •N3, are formed with a radiationchemical yield equal to 0.55 µmol.J−1 [29].

Irradiations using γ rays were carried at room temperature using the panoramic60[Co] source IL60PL Cis-Bio International (France) at the University Paris-Sud (Or-say, France). The dose rate was determined by Fricke dosimetry for each positionused in front of the source and ranging from 2 to 4.5 Gy·min−1. The ECFP concen-tration for irradiations was 5 µM at neutral pH (30 mM phosphate buffer, pH 7.5,sample volume in the range 100–500 µL). Samples were purged gently under agi-tation without bubbling with N2O. The irradiation doses delivered were in the range20–400 Gy. For an easier presentation and understanding of the results, the dose wasreplaced by the concentration R = [radicals]/[ECFP] ratio, which is proportional tothe dose where [radicals] = dose ·G(radical).

In pulsed radiolysis experiments, free radicals were generated by delivering, intoan aqueous solution, a 800 ns pulse of high energy electrons (≈ 4 MeV) from thelinear accelerator located at the Curie Institute in Orsay, France [66]. The doses perpulse were calibrated from the absorption of the thiocyanate radical SCN•− obtainedby radiolysis of the thiocyanate ion solution in N2O-saturated phosphate. These doseswere in the range 4–10 Gy. The reaction was followed spectrophotometrically be-tween λ350 nm and λ750 nm, in a 2 cm path length cuvette designed for pulse radiolysisexperiments. The spectra of the intermediates were reconstructed from the maximaof the absorbance variation in the investigated wavelength range. The time responseof the experimental setup is below 0.1 µs.

1.2 OR2 – Structural modeling of residue accessibility

ECFP structure at physiological pH is available from PDB (ID: 2WSN [30], but doesnot contain the N-terminal tail of the protein (sequence 230TLGMDELYK238). Inorder to correlate the mass spectrometric results with the spatial localization of allof the ECFP residues, we generated an automated model of ECFP using the SWISS-MODEL workspace [67, 68, 69] and the crystal structure for the S65G, Y66G GFPvariant (PDB ID: 1QYO) [31] as a template that contains the [T230-Y237] stretch has97.9 % homology with ECFP. We then imported the resulting PDB file and alignedit with 2WSN with the VMD software package [70] for use of the [T230-Y237]modeled stretch in all the spatial analysis in this report in conjunction with 2WSN.


1.3 Plausible one-hop long-distance electron transfer between a tyrosine andmethionine residues

Fig. 1 Modelling of the three-dimensional distance between the exposed Tyr39 and the buried Met88. Theelectron transfer might happen by following two routes: through Phe71 or Phe8.

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