Selective Double-Labeling of Cell-Free Synthesized Proteins for More

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Selective double-labeling of cell-free synthesized proteins for more accurate smFRET studies Mayuri Sadoine, Michele Cerminara, Noémie Kempf, Michael Gerrits, Joerg Fitter, and Alexandros Katranidis Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01639 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Analytical Chemistry

Selective double-labeling of cell-free synthesized proteins for more accurate smFRET studies

Mayuri Sadoine†, Michele Cerminara†, Noémie Kempf†,¶, Michael Gerrits‡, Jörg Fitter *, †, § and Alexandros Katranidis*, † †

Forschungszentrum Jülich, Institute of Complex Systems ICS-5, Jülich, Germany

‡

biotechrabbit GmbH, Berlin, Germany

§

RWTH Aachen, 1. Physikalisches Institut (IA), Aachen, Germany

* Correspondence should be addressed to [email protected] Tel.: +49 2461 612036 Fax: +49 2461 611448 or [email protected] Tel.: +49 241 80 27209 Fax: +49 241 80 22331

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ABSTRACT: Förster Resonance Energy Transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) which provides proteins with two different functional groups for posttranslational labeling at the specific amino acid positions. Besides the sulfhydryl-group of a cysteine we make use of an azido-group of a pazido-L-phenylalanine to achieve chemical orthogonality. Herewith, we achieve not only a site-specific, but most importantly also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.

Single-molecule fluorescence methods and in particular smFRET constitute a powerful tool for the study of protein structural and conformational dynamics, as well as the characterization of protein interactions with their counterparts both in vivo and in vitro1-3. As a requirement, the protein needs to be site-specifically double-labeled with a pair of donor and acceptor fluorophores. The commonly used and well-established methods take advantage of the naturally occurring side-chains of the protein’s lysine and cysteine residues, which can react with N-hydroxy-succinimide (NHS) esters and maleimides, respectively4. Due to the high abundance of lysines, site-specific labeling is practically limited to cysteine residues. Typically wild-type cysteines, if present, are removed and new cysteines are introduced at desired positions by site-directed mutagenesis. Even though double-labeling is sitespecific at the two cysteine positions, it lacks selectivity due to the same functionality of the two residues. The fact that each of both cysteine positions has the ability to be connected to the donor dye as well as to the acceptor dye gives rise to heterogeneously labeled populations and prevents a fine analysis of the smFRET data. The development of new strategies includes the site-specific incorporation of unnatural amino acids (UAAs) at desired positions via cell-free protein synthesis (CFPS)5. The open nature of the CFPS systems facilitates protein synthesis, manipulation and subsequent analysis, while protein yields were shown to be sufficient for some fluorescence-based methods6. The successful production of a double-labeled protein using CFPS was already reported7,8. Chemically aminoacylated suppressor tRNAs were used in order to incorporate co-translationally two different fluorescent UAAs via combination of either amber and four-base codon or two different four-base codons. Although this approach directly inserts both dyes already during the protein synthesis, a major disadvantage of co-translational incorporation of fluorescent UAAs is the inevitable use of small dyes, mostly belonging to the BODIPY family, which are not optimized for single-molecule experiments due to their weak photostability9,10. Moreover, protein synthesis yields are comparatively low, since co-translationally incorporation of the dyes suffers from the weak

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binding of the corresponding pre-charged tRNAs to elongation factor Tu (EF-Tu) that is strongly dependent on size, charge and

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structure of the attached amino acid side chain11,12. A different approach is based on the site-specific incorporation of UAAs containing bio-orthogonal functional groups, which can be post-translationally labeled with chemoselective reactive reagents13. So-called orthogonal systems were originally established in vivo 14-17, but nowadays they are also available for the incorporation of single chemoselective reactive amino acids using CFPS18,19. The post-translational labeling allows the choice of more photostable dyes, suitable for single-molecule studies. Orthogonal systems achieve higher protein yields (several hundreds µg/mL) than systems with co-translationally incorporated dyes (ng/mL up to few µg/mL). In this work we report a new approach, in which we combine the strength of smFRET to resolve populations and conformational dynamics in equilibrium with the flexibility of CFPS for sample production. For this purpose we synthesize a protein, sitespecifically mutated with a single cysteine residue and a single UAA, namely the chemoselective reactive p-azido-L-phenylalanine (AzF). We subsequently prove that the selective labeling, enabled by the orthogonal reactivity of the sulfhydryl- and the azidogroup, allows for more accurate quantitative analysis of the smFRET data.

EXPERIMENTAL SECTION Construct. The gene of human CaM was cloned into a pRSET vector (Thermo Fischer Scientific) and single (CaM T34C, CaM T110C) or double mutants (CaM T34C T110amb, CaM T34amb T110C) were generated via site-directed mutagenesis.

Cell-free expression and incorporation of p-azido-phenylalanine. Protein synthesis was performed using an E. coli derived cell-free transcription/translation system depleted from termination factor RF1 and supplemented with enriched fractions of orthogonal amber suppressor tRNA and p-azido-phenylalanyl-tRNA synthetase specific for p-azido-phenylalanine18,20. For the full composition of the system see Table S1. The system is now available at biotechrabbit GmbH. Protein synthesis was performed at 32°C either in vials for 120 min (batch mode) or in RTS 100 dialysis devices for 24 h (continuous exchange mode). Proteins were purified via Ni-NTA magnetic agarose beads (Qiagen). The product was desalted with either PBS (pH 7.5) or PBS-TCEP (pH 7.5) by using size exclusion Zeba™ desalting columns (Thermo Fisher Scientific).

Labeling with fluorescent dyes. Single mutants containing a cysteine were labeled after purification with AF488-maleimide (Invitrogen). At least a 10-fold excess of Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to avoid the presence of disulfide bonds and the PBS-TCEP (pH 7.5) buffer was degassed for at least 120 min. A 30-fold excess of dye was used for the labeling, which was performed at 4°C for 15 h after degassing. In the case of double-labeled samples, the AzF was labeled first with AF647-DIBO alkyne (Thermo Fisher Scientific) in PBS (pH 7.5), using a 25-fold excess of dye for 15 h at 25°C. For SDS-

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PAGE, only a 3-fold excess of AF488-maleimide and a 2.5-fold excess of AF647-DIBO were used, in order to avoid saturation of

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the measured fluorescence signal. The single-labeled CaM was purified via Ni-NTA magnetic agarose beads (Qiagen). The cysteine residue was labeled subsequently with AF488-maleimide using the same conditions as for the labeling of the single mutants. The double-labeled protein was finally purified using the magnetic beads as described in the previous paragraph. The occurrence of the labeling was verified using SDS-PAGE: 20 µl of sample were separated by 15% SDS-PAGE and the gel was read using a fluorescence scanner (Typhoon FLA 9500, GE Healthcare Life Sciences) before being stained with Coomassie Brilliant Blue for at least 15 h. The gel was then washed twice with milliQ water before being visualized using a gel imager (Biorad ChemiDoc MP imaging system).

Samples for circular dichroism. For circular dichroism non-labeled proteins were used to avoid saturation of the signal by the UV absorption of the dye. Expression was performed using the CFPS system in a continuous exchange mode to reach an optimal concentration. Samples were prepared in 25 mM phosphate buffer at pH 7.5 with a nominal concentration of hundreds of µg/ml.

Samples for fluorescence microscopy. The proteins were synthesized in batch mode and diluted in the final buffer to adjust the optimal concentration for each experiment. For Fluorescence Correlation Spectroscopy (FCS) measurements the final concentration was in the nM range, for smFRET around 50 pM and for single-molecule two-color coincidence detection (TCCD) below 10 pM. All samples were then incubated for at least 1 h at room temperature to reach equilibrium conditions. Experiments with apoCaM were performed in apo-buffer (10 mM EGTA, 50 mM MOPS). Binding experiments with calcium ions were performed in holo-buffer (10 mM CaCl2, 50 mM MOPS) and the sample was incubated overnight to reach equilibrium. Binding experiments with partners were performed in holo-binding-buffer (10 mM CaCl2, 50 mM MOPS, 150 mM KCl) and in presence of a high excess of either CaMKII (150 µM) or M13 (100 µM) binding peptide to perform measurements in saturated condition (KD from subpicomolar to nanomolar). Inhibition of the calcium-dependent binding of CaMKII and M13 was achieved by chelation of calcium ions by addition of an equivalent volume of apo-binding-buffer (10 mM EGTA, 50 mM MOPS, 150 mM KCl).

Circular Dichroism. CD measurements were performed by using a Jasco J-1100-Spectropolarimeter (JASCO International Co. Ltd.) with a quartz cell of path length of 1 mm at room temperature. CD spectra were recorded over the range 190-260 nm and the background spectrum of the corresponding buffer was subtracted. Since CaM lacks tryptophan residues, it has a very small extinction coefficient (ε280). As a result the concentration of the unlabeled protein cannot be determined with high precision, making the calculation of absolute molar ellipticity unreliable. To avoid this problem, the relative amount of secondary structural elements was evaluated by using the Secondary Structure Estimation software included in the Jasco package.

Slide preparation. All the measurements performed with the confocal fluorescence microscope were done using high precision coverslides with a thickness of 170 ± 5 µm (No. 1.5H, Marienfeld Superior, Germany). In order to prevent the adhesion of

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the labeled protein on the slides and consequently a loss of sample concentration during the measurement, the slides were

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passivated. Furthermore, to avoid sample evaporation during the course of the experiments, a closed chamber was constructed by gluing a half-cut low-binding Eppendorf tube to the slide. The glass slides were initially cleaned with piranha solution (H2SO4 and 30% H2O2, 2:1 v/v). Then they were functionalized with amino-groups by incubating for 10 min in 2% solution of 3-aminopropyltriethoxysilane (APTES) (Sigma-Aldrich) in acetone. The slides were finally passivated with polyethylene glycol (PEG) by reaction with a solution of 200 mg/ml PEG-NHS ester in 1 M NaHCO3 (pH 8.3) at 4°C for at least 12 h. Before usage, the excess of PEG was washed with milliQ water, acetone and methanol and dried with nitrogen.

Confocal fluorescence detection. FCS, TCCD and smFRET measurements of freely diffusing fluorescent species were performed with a MicroTime 200 (PicoQuant GmbH, Berlin, Germany) confocal inverted microscope. AF488 and AF647 were excited by using a 485 nm and 640 nm laser (LDH-D-C 640B and LDH-D-C 485B from PicoQuant), respectively. The excitation light was focused on the sample by using a high numerical aperture water immersion objective (UPLSAPO 60x; Olympus); the fluorescence emitted was collected through the same objective and spatially filtered by using a 75 µm pinhole in confocal configuration. Emission signal was separated by a dichroic mirror (T600lpxr, Chroma Technology) and filtered by using a 535 nm band pass filter (535/55, Semrock) for the donor channel and a 635 nm long-pass filter (635 LP, Semrock) for the acceptor channel. Finally, photons were detected by single-photon avalanche diodes (τ-SPAD, PicoQuant, Berlin, Germany for the donor channel and SPCM-CD3077-H and SPCM-AQR-14, Perkin-Elmer Inc., Waltham, USA for the acceptor channel). The arrival time of each photon was recorded with a time-correlated single-photon counting module (HydraHarp400, PicoQuant, Berlin, Germany). For smFRET and TCCD measurements, a Pulsed Interleaved Excitation (PIE) scheme was applied, in which excitation of the donor and acceptor are alternated in order to verify the presence of the acceptor by direct excitation and minimize the artifacts due to the presence of donor-only molecules.

Data analysis. Analysis of the FCS data (i.e. calculation of the correlation function and fit with model functions for diffusion) was performed using the SymPhoTime64 software (PicoQuant, Berlin, Germany). For TCCD measurements, data analysis was done with self-written Matlab routines (Mathworks, Natick, USA) using an approach similar to the one used by Li et al.21. Analysis of smFRET data was performed by using self-written Matlab routines (Mathworks, Natick, USA) whose details are described in Gabba et.al.22. Briefly, the bursts (i.e. the ensemble of photons emitted by the dye pair when a labeled protein crosses the confocal volume) were identified from the raw data according to the inter-photon lag-time between two adjacent photons, which has to be below a certain threshold. Furthermore only bursts having a total number of photons above a threshold value were considered for further analysis. According to established standard procedures, for each burst the numbers of photons emitted from the donor and from the acceptor were counted and the corresponding fluorescence intensities were calculated by correcting for the background

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photon counts, the crosstalk of the donor fluorescence into the acceptor channel, the detection efficiencies for the two channels and

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for the different quantum yields of the dyes23. The FRET efficiencies were then calculated and the resulting smFRET histograms were fitted with multiple Gaussian peaks to identify the different populations present in equilibrium. As a comparison, Gaussian peaks were calculated by using the intrinsic theoretical variance values expected from experimental photon statistics (shot noise)24 to highlight the extent of extra broadening that could be due to conformational dynamics or conformational heterogeneity.

Quantum yield (QY) determination. The QYs of the dyes bound to CaM were determined by a comparative method previously described by Kempe et al.25. In brief, FCS was used to calculate the molecular brightness (MB) of the single-labeled proteins at different values of excitation intensity. By comparing the slope of the MB vs. excitation intensity it is possible to calculate the QY of the sample with respect to the one of a reference with a known QY. We used as reference AF647-NHS and AF488-NHS for the acceptor and donor calculations, respectively.

Accessible Volume (AV) calculations. The AVs were determined by using an algorithm that was previously described by Höfig et al.26. In brief, this algorithm generates for each dye bound to the protein an AV cloud, where each point is sterically accessible for the fluorophore. This algorithm takes into account the length of the linker as well as the shape of the dyes and the fact that the dye is free to move around the anchor position. By averaging all the possible positions for both dyes, a mean donoracceptor distance is calculated.

RESULTS AND DISCUSSION Cell-free synthesis and selective double labeling of CaM. In order to demonstrate the potential of our approach we chose the human calmodulin (CaM), a small two-domain protein with two calcium-binding EF-hand motifs in each domain. When the calcium-free CaM (apoCaM) binds four calcium ions (holoCaM), it is able to interact with different partners27, leading to a wide range of conformational changes28. CaM is an ideal model protein, since it is intensively studied and many three-dimensional structures and functional mutants are reported in the literature. To observe and analyze the conformational changes using smFRET, two mutations were introduced within the N- and C-terminal domains at positions 34 and 110, respectively. These positions were previously used to successfully label CaM, keeping the structure and functionality of the protein intact29-31. The two threonine residues at these positions were substituted by a cysteine and an amber codon, to generate the two double-mutant constructs CaM T34C T110amb and CaM T34amb T110C (Figure S1). The UAA AzF was previously incorporated into proteins via amber suppression both in vivo and in CFPS systems15,18,32,33. Here, cell-free synthesis and purification of both CaM mutants CaM T34C T110AzF and CaM T34AzF T110C, was performed using a modified coupled transcription/translation system for efficient incorporation of AzF (Figure 1A, top panel). Termination factor

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RF1 that competes with AzF incorporation at the amber codon was partially depleted from the CFPS system20 in order to enable

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efficient incorporation of AzF. The incorporated azido group can be converted with alkynes in a copper-dependent azide-alkyne cycloaddition (CuAAC) reaction or using the milder copper-free click chemistry, namely the Staudinger ligation with phosphines or the “strain-promoted alkyne-azide cycloaddition” (SPAAC) with cyclooctynes13. We took advantage of the differing orthogonal groups, namely the sulfhydryl- and the azido-group and combined maleimide-cysteine and alkyne-azide ligations in order to selectively label the CaM mutants with the FRET dye pair Alexa Fluor 488 (AF488) and Alexa Fluor 647 (AF647) (Figure 1A, middle and bottom panels). The selective labeling of CaM with both donor and acceptor fluorophores was visualized on SDS PAGE by excitation at 473 nm and 635 nm. As expected, only CaM with both functional groups gave fluorescent signals for the two dyes. On the contrary, the single-mutant CaM T110C containing only a cysteine gave a signal only for the donor dye and the single-mutant CaM T34AzF containing only the azido-group gave a signal only for the acceptor dye, while the CaM wild type (CaM wt) showed no fluorescence at all, even though all protein constructs were incubated with both fluorophores at the same time (Figure 1B). The percentage of CaM that is indeed labeled with both dyes was measured with single-molecule two-color coincidence detection (TCCD) technique and found to be ~30%. Thus, we concluded that CaM was labeled selectively with both donor and acceptor dyes and, most importantly, without any cross-reactivity.

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Figure 1: Selective double-labeling of CaM at residues 34 and 110. (A) Site-specific co-translational incorporation of cysteine and AzF into CaM (top). This approach allowed selective double-labeling using an orthogonal chemistry via maleimide-cysteine and azide-alkyne ligations (middle). CaM labeled at residues 34 and 110 with AF488 (cyan sphere) and AF647 (red sphere) respectively is illustrated as a cartoon (bottom). (B) Each of four different CaM constructs was post-translationally incubated with both fluorophores and fluorescent signals are shown on SDS PAGE.

Structure and functionality of cell-free synthesized CaM. To initially determine whether the structure of CaM remained unaffected by the incorporation of cysteine and AzF, circular dichroism (CD) spectra were acquired for CaM T34AzF T110C, CaM T34C T110AzF and CaM wt. A secondary structure analysis software was used to analyze the CD data in terms of secondary structure composition. The obtained compositions (i.e. α-helix, β-sheet, turn and random coil content) were rather similar for all measured constructs (Figure 2A). These results are furthermore consistent with previous reports concerning the secondary structure content of CaM34. Although CD spectroscopy is a low resolution technique that does not allow to resolve the three-dimensional structure at atomic level, it indicates that the structure of cell-free synthesized CaM is not strongly affected by the introduction of the two functional groups.

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Figure 2: Structure and functionality of mutant CaM. (A) CD spectra of CaM wt (black solid line), CaM T34AzF T110C (blue solid line) and CaM T34C T110AzF (red solid line). In all cases the corresponding dotted lines represent a fit to the experimental data. (B) FRET histograms of the CaM T34AzFAF647 T110CAF488 mutant in apo- (top) and holo-conditions (bottom). The apoCaM structure with the accessible volumes for the two dyes is illustrated, as well as two extreme structures of holoCaM. (C) FRET histograms of holoCaM in holo-binding buffer in the absence (left) or presence of CaMKII (middle) and after addition of EGTA (right). Gaussian fits were applied to all histograms and a mean FRET efficiency value was calculated for each peak. Figure 2B bottom and 2C are related to two different experiments done in similar but not identical conditions (see Experimental section). The relative weight of the observed subpopulations is strongly sensitive to minor changes in the experimental conditions. The position of the high FRET peak is not altered, while the low FRET populations exhibit some minor variations35, also because of slight differences in the experimental conditions (mainly the ionic strength of the buffer used for the binding experiment that is well known to stabilize more compact structures36, as well as temperature, stock concentrations, pipetting errors etc. can have an effect).

As a further step, we determined the effect of the labeling procedure on the functionality of CaM by performing smFRET experiments either in the presence of the calcium chelator EGTA to obtain the apo-condition or in saturation of CaCl2 to achieve the

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holo-condition (Figure 2B). In the presence of EGTA, calcium is not bound to CaM (apoCaM). A structure of the apoCaM was

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previously determined and is available in the Protein Data Bank (PDB) (PDB ID: 1DMO)37. Based on this three-dimensional structure, we calculated the expected FRET efficiency, by determining the average donor-acceptor distance (RDA) whereby we considered the entire accessible volume (AV) that can be occupied by both fluorophores26 (Figure 2B, top). Following this approach we obtained a calculated RDA for the double-labeled CaM T34AzFAF647 T110CAF488 mutant of 53.78 Å, which gave an expected FRET efficiency of 49.2%. This value is very close to the 48% of FRET efficiency obtained experimentally. Similar results were also observed for the second double-labeled mutant (CaM T34CAF488 T110AzFAF647) (Figure S2A). In the presence of 10 mM CaCl2, four calcium ions are bound to each CaM (holoCaM). smFRET experiments were performed under calcium-saturating conditions and related FRET histograms indicated the presence of three apparent populations (Figure 2B, bottom). These results are consistent with the fact that holoCaM is conformationally highly heterogeneous and many different structures are available in the PDB. Interestingly, we have seen that the low and high FRET populations are consistent with two of them, one extended (PDB ID: 1CLL) and one compact form (PDB ID: 1PRW) (Figure 2B, bottom)38,39. In the compact form the donor/acceptor distance is ~20 Å, thus with the AF488/AF647 pair exhibiting a Förster radius of ~53 Å we were not able to resolve such a short distance between the two labeling positions accurately. As a consequence the peak corresponding to this population is quite narrow and shifted to high transfer efficiencies compared to the other population peaks in the FRET histogram. To our knowledge, the peak in the center of the histogram is not consistent with any specific structure resolved so far, but most probably represents an ensemble of multiple conformations that can also interchange during the time the molecule crosses the observation volume. However, similar results describing three populations for the holoCaM were previously reported40,41. Due to the high heterogeneity and dynamical behavior of holoCaM, this state of the protein is particularly prone to be affected by subtle changes in the actual experimental conditions; minor differences (e.g. in salt concentration, temperature, stock concentrations etc.) can alter the relative weight of the three major populations, as well as the position and size of the central peak that is not representative of a single, well defined structure but a convolution of multiple conformations. In summary, our smFRET results show that the cell-free synthesized and selectively labeled CaM behaves as expected in apo- and holo-conditions. This suggests that the labeling procedure neither disturbs the structure of the protein nor affects the conformational changes that CaM undergoes upon calcium binding. In order to investigate the functionality of the double-labeled CaM more deeply, we performed binding experiments with two CaM partners, the calmodulin-binding domain of the calcium/calmodulin-dependent protein kinase II (CaMKII) (Figure 2C and Figure S2B) and the calmodulin-binding domain of skeletal muscle myosin light chain kinase (M13) (Figure S3). Upon addition of the partner (in high excess to achieve saturation) the population distribution shifts towards the one that is stabilized by the interaction forming the complex. The three-dimensional structures of CaM in complex with these two partners are available in the

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PDB (PDB IDs: 2BBM and 1CDM)42,43. In both complexes CaM is bound to four calcium ions and is in a compact form. In this

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conformation, the obtained calculated RDA value is again rather small (~20 Å) and as expected, it gives a narrow high FRET population in the related histogram (Figure 2C, middle). Consistent with the fact that the complex formation is calcium dependent, the binding interaction is inhibited by addition of EGTA. This becomes visible in our results by a FRET histogram, which is almost identical to the one we already observed for the apo-form of CaM (Figure 2C, right and Figure 2B, top). The reversible binding of interaction partners strongly indicates that the functionality of the double-labeled CaM remained unaffected.

Effect of selective labeling on FRET histograms. Finally, we also examined the effect of selective labeling on the accuracy of the distance determination calculated by the FRET histograms. To this aim, we performed smFRET measurements with both double-labeled constructs, which differed for the positions of the fluorophores. The first construct had AF647 at position 34 and AF488 at position 110 (CaM T34AzFAF647 T110CAF488), while the second construct had the dyes the opposite way (CaM T34CAF488 T110AzFAF647). (Figure 3A, top and middle). After measuring smFRET for both constructs, the FRET efficiencies were calculated by applying two different strategies to consider the obtained quantum yields (QYs) of the dyes when bound at a specific position (Figure 3B and 3C). Indeed, the two different constructs (see above) allowed us to determine the respective QYs of both fluorophores at each position independently. Altogether we obtained four independent QY values (Table S2). The reliable determination of QY values was possible using our method range)25, which requires only very small sample amounts and low dye concentration (nM range). In the first approach to consider the QYs for calculating FRET efficiencies, we took the arithmetical average of the QY of each dye obtained at both positions (34 and 110). We used these two averaged values (for the donor and the acceptor) to calculate the FRET histograms of data measured with both constructs (Figure 3B, top and middle, “averaged correction”). This method to correct for the QY is identical to the one used when a sample contains a mixed population of both CaM constructs (Figure 3A, bottom). Exactly such kind of sample is obtained when the commonly used non-selective method of labeling based on double cysteine mutagenesis is used44. After the above described correction, we observed that the FRET histograms of the two constructs were shifted with respect to each other (Figure 3B, top and middle). To mimic the double cysteine approach we then calculated a new FRET histogram by averaging those of each construct (Figure 3B, bottom), since in the non-selective approach there is no preference for any of the two positions. It becomes evident that as a consequence of averaging two peaks shifted with respect to each other, the resulting position is the average of those of the two independent constructs and that a further broadening is introduced.

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Figure 3: Increased accuracy of the FRET histograms due to selective labeling. (A) The constructs with corresponding AVs are shown for each case, in which experimental data were obtained, see (B) and (C). (B) FRET efficiency histograms of CaM T34AzFAF647 T110CAF488 (top), CaM T34CAF488 T110AzFAF647 (middle) and both constructs together (bottom) using the averaged correction for the QYs of the fluorophores in both positions. (C) FRET efficiency histograms of the same constructs using the specific correction for the QYs of the fluorophores at their specific positions. A vertical dashed red line centered on the mean efficiency of CaM T34AzFAF647 T110CAF488 (top) is drawn to illustrate relative shifts between corresponding peaks in each of the three cases. Dotted lines represent the shot-noise limited width expected for each peak. While the widths of the histograms in the top and middle lane are almost identical within the limits of error, we observe an additional broadening only for the averaged correction (bottom lane) caused by a relative shift of two peaks (top and middle lane) in this case.

In order to correct for the actual QY of each dye at its specific position in the construct, we developed a second approach, that we call “specific correction”. For this, we used the QYs of donor and acceptor, previously measured at the exact positions within the respective construct (Table S2). This kind of correction is only possible in case of selective labeling but would not be applicable for a double cysteine mutant. We observed that the peaks in the two FRET histograms were not anymore significantly shifted, in line with the fact that the RDA is almost identical for both constructs (Figure 3C, top and middle). After averaging both corrected histograms and analyzing the overall data set, we can see that for the specific correction no shift nor further broadening is introduced. It is therefore evident that the double cysteine approach introduces a source of error in the FRET histograms (shift of

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the peak position and further broadening) that is not present when selective labeling is used. (Figure 3B and 3C, bottom). In this

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specific case the broadening is not dramatic because we chose labeling positions that were previously characterized and do not alter the photophysics of the dyes significantly. Nevertheless with our approach we are intrinsically sensitive to any kind of local effect and we can still appreciate also a small effect that otherwise would have been neglected. An additional advantage of our approach is that even if the QY of a dye is relevantly changed due to the specific position of the labeling, it is not necessary to discard that position. This advantage can also be very beneficial in smFRET studies where not only single distances within a protein are in the focus of the study, but where the investigation of distance networks is targeted in order to implement an integrated structural modeling based on FRET data45. On the contrary, the actual correction can be performed and information that would otherwise be neglected can be obtained. These results demonstrate that selective labeling of proteins can significantly improve the precision in estimating molecular distances by increasing the accuracy of the smFRET histograms.

CONCLUSION In the present study, we describe a new CFPS-based approach for the synthesis of selectively double-labeled proteins for more accurate smFRET studies. Selective double-labeling was achieved by combining the well-established procedure of cysteine mutagenesis and labeling via maleimide reagents with an orthogonal strategy, the site-directed labeling of a site-specifically incorporated azido-group. We used an improved RF1-depleted orthogonal CFPS system for azido protein synthesis. Here we demonstrate for the first time, to our knowledge, that such a system is well suited for the synthesis of proteins that can be sitedirected and selectively double-labeled with commercially available photostable dyes, suitable for smFRET experiments. In addition, the perfect suitability of protein samples produced by CFPS for smFRET studies implies at the same time that techniques like smFRET expand the scope of CFPS to applications which up to now were not exploitable due to the higher amount of proteins required. Selective labeling results in a homogeneous double-labeled population, rendering any additional purification steps unnecessary. Hence, the protein yield is sufficient for performing smFRET experiments. Furthermore, in our method, design of additional singlemutant constructs is not needed, since the orthogonality of the labeling chemistry makes it easier and more straightforward to obtain from the same construct the single-labeled proteins to evaluate the effect of the local environment on the photophysics of the attached fluorophores, without the need of time-consuming and expensive mutagenesis. Our approach is a clear improvement to previous cell-free methods that are based on the use of pre-charged suppressor tRNAs carrying fluorescently-modified amino acids7,8 that are not well suited for smFRET studies. With this work we achieved to replace the analogue in vivo method15,17 by a more convenient cell-free approach. Even proteins that are toxic to living cells or for other reasons difficult to express (proteases, membrane proteins, etc.) can be produced, since

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synthesis takes place independently from recombinant organisms, especially in combination with - commercially available - de

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novo gene synthesis and PCR-based template generation. Moreover, our cell-free method is an ideal tool for labs focusing on spectroscopic techniques like smFRET, since cell cultures and safety regulations for recombinant organisms are not necessary. The presented CFPS approach is suitable to investigate biologically relevant processes, as demonstrated by the proper folding and functionality of the selectively double-labeled CaM. Selective labeling was shown to be crucial for the increased accuracy of the smFRET data analysis, due to the improved correction of the smFRET histograms. As mentioned throughout this study, our E.coli CFPS-based method is an advance to single-molecule studies. On the contrary, in vivo established protein synthesis is still the most suitable for ensemble measurements. One limitation of using a bacterial system is the restriction in post-translational modifications for functional proteins; the use of a eukaryotic system could solve this problem, but at the expense of lower protein yields. Another limitation comes from the use of a cysteine codon that allows the study of proteins with no or limited number of non-essential cysteines. This problem can be tackled by introducing alternative codons (4base or 5-base codons) to incorporate functional groups with different chemistries. In summary we report a fast, general and simple method for selective double-labeling of proteins, increasing smFRET precision by taking advantage of the strength of CFPS. More specifically we demonstrated (i) that the combination of sulfhydryl- and azidereactive reagents is easy to handle and allows selective labeling, (ii) that the orthogonal labeling scheme in combination with CFPS is fast and allows for a straightforward production of protein mutants suitable for smFRET measurements and (iii) that the selective labeling allows for a more accurate quantitative analysis of smFRET data, a prerequisite for more precise single-molecule distance determination. Our study emphasized that CFPS and smFRET represent a perfect combination to achieve the full potential of analyzing protein structures with fluorescence based techniques.

ASSOCIATED CONTENT Supporting Information Figures S1-S3 showing the amino acid sequence of the constructs and FRET histograms of the second double-labeled mutant, as well as the binding of the second CaM partner (M13). Also Tables S1 and S2 listing the components of the CFPS system and the quantum yields of the used fluorophores (PDF).

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AUTHOR INFORMATION Corresponding Authors

* [email protected] Tel.: +49 2461 612036 * [email protected] Tel.: +49 241 8027209 Present Address ¶ Laboratoire de Biologie Moléculaire des Eucaryote (LBME), CNRS UMR 5099, Toulouse, France Author Contributions The manuscript was written through contributions of all authors and all authors have given approval to the final version of the manuscript. Notes The authors declare competing financial interests. M.G. was employed during the research project by biotechrabbit GmbH.

ACKNOWLEDGMENTS The authors would like to thank Dr. Matteo Gabba for developing and establishing the smFRET data analysis programs in our institute; Nadine Jordan for helping with the Typhoon fluorescence scanner; Ilona Ritter and Ramona Choy for preparing the CaM constructs; Prof. Wolf B. Frommer for critical reading of the manuscript.

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