Cotranslational Incorporation into Proteins of a Fluorophore Suitable

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Co-translational incorporation into proteins of a fluorophore suitable for smFRET studies Mayuri Sadoine, Michele Cerminara, Michael Gerrits, Joerg Fitter, and Alexandros Katranidis ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00433 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

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ACS Synthetic Biology

Co-translational incorporation into proteins of a fluorophore suitable for smFRET studies

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

†

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

‡

Biocatalysis Group, Department of Chemistry, Technische Universität Berlin, Berlin, Germany

§

RWTH Aachen University, I.Physikalisches Institut (IA), Aachen, Germany

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids co-translationally using cell-free protein synthesis systems. Here, we demonstrate the co-translational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that co-translational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies. Keywords: unnatural amino acids, single-molecule FRET, double labeling, cell-free protein synthesis, precharged tRNA

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Single‐molecule Förster resonance energy transfer (smFRET) is a powerful fluorescence technique able to study structural and conformational dynamics of double‐labeled proteins1-3. The most common approach to obtain a protein with a FRET dye pair is to label cysteine residues introduced at the desired positions by site‐directed mutagenesis. Nevertheless, a better approach is to introduce at these positions at least one unnatural amino acid (UAA) with a unique reactive group to allow a selective labeling of the protein4-6. However, the above mentioned strategies cannot be used when studying the synthesis and folding of nascent polypeptide chains on the ribosome in real time. For this kind of studies it is necessary to co‐translationally incorporate two different fluorescent UAAs into cell‐free synthesized proteins, a quite elegant approach of double labeling that also ensures site‐specificity and selectivity. Small fluorophores belonging to the BODIPY family of dyes were already shown to incorporate efficiently into proteins7, 8. However, BODIPY dyes are not well suited for single‐molecule studies due to their poor photophysical properties and weak photostability9, 10. Although unbound BODIPY dyes exhibit rather large quantum yields (QY) in aqueous buffers, corresponding dye‐protein conjugates often suffer from a drastically reduced fluorophore brightness restraining single molecule sensitivity11. On the other hand, larger fluorophores with much better photophysical properties were shown to incorporate only at the N‐terminus of a protein12, but so far not used for co‐translational double labeling in combination with a second dye. However, an important prerequisite of employing FRET for elucidating structural and dynamical properties is a high flexibility in choosing two eligible label positions. Despite these difficulties there is undoubtedly a strong motivation to achieve co‐translational incorporation of dyes into nascent chains suitable for single molecule studies, because this provides for example unique prospects for studying co‐translational protein folding13, 14. smFRET studies on nascent polypeptide chains is the key to obtain detailed insight into protein folding on the ribosome, including time resolved information about transient conformational states and dynamics from individual proteins15.


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Therefore, the primary goal of this study is to identify other fluorophores than BODIPY dyes, which can be incorporated into protein structures (i) showing better photophysical properties and (ii) allowing the correct folding of the respective polypeptide chain into its native structure. It is well known that co‐ translational incorporation of fluorescent dyes reduces significantly the protein yield in cell free protein synthesis16, 17. However, a further strength of the smFRET approach is that in principle only small sample amounts, both in terms of volume and concentration, are required for the measurements. The commercially available dye Atto633 has a rigid structure with a high fluorescence QY and excellent photostability, rendering it highly suitable for single‐molecule detection applications18. In this study we demonstrate that Atto633 incorporates co‐translationally into a protein without altering the structure and functionality, as revealed by smFRET measurements. We also present for the first time the co‐translational incorporation of Atto633 together with a second fluorophore, the efficiently incorporated BODIPY‐FL, at two defined positions into the same protein. Combining the sense cysteine codon UGC19 and the nonsense amber codon UAG, we offer a new possibility to introduce co‐ translationally a FRET pair of fluorophores into a protein and to measure conformational states by smFRET. In our view, the combination of amber and cysteine codon is highly advantageous compared to the previously reported combinations of either amber and quadruplet codons8 or two different quadruplets7, because the latter are limited by the low incorporation efficiency and inflexible use of the quadruplet codon. Even though the use of the sense cysteine codon is not suitable for all proteins, thus lacking generality as an approach, it can be applied to numerous proteins, since cysteine is still an underrepresented amino acid. Moreover, engineering of proteins to reduce the number of cysteines in order to allow a site‐directed modification with sulfhydryl‐reactive dyes is a common method to tailor proteins. As a model protein we chose the intensively studied human calmodulin (CaM). The calcium‐free form of CaM (apoCaM) binds four calcium ions, which induce conformational changes. These changes 3 ACS Paragon Plus Environment


enable the calcium‐bound CaM (holoCaM) to recognize and interact with different partners20, leading to further conformational diversity21. In order to be sensitive for analyzing functional conformational changes within this protein, positions 34 and 110 were identified in previous studies as good for dye labeling. Therefore, we introduced either an amber codon at the N‐terminal domain at position 34 or a cysteine codon at the C‐terminal domain at position 110 (Figure S1), both previously reported to be “tolerant” to point mutations22, 23. We started our study by exploring the possibility to incorporate co‐translationally different fluorophores using commercially available precharged tRNAs. To this aim, a number of coupled transcription/translation cell‐free protein synthesis (CFPS) reactions were performed, in which a single‐ mutant DNA construct (CaM T34amb or CaM T110C) and the respective suppressor tRNA (tRNAChemCUA or tRNACys) carrying a single fluorophore was added. Depending on the DNA template the fluorophores were successfully incorporated in a site‐directed manner into the N‐ or C‐terminal domain of CaM either via amber codon suppression or the sense cysteine codon (Figure 1). In the latter case free cysteine was omitted in the CFPS reaction in order to ensure the efficient incorporation of the UAA by the dye‐ charged tRNACys. In order to improve the efficiency of amber suppression, a release factor 1 (RF1) depleted system24 was used. Among the fluorophores that were successfully incorporated, Atto633 displays the best spectroscopic characteristics and was used for the rest of the study.


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Figure 1. Single fluorophore incorporation. (A) Site‐directed co‐translational incorporation of a fluorophore into CaM via amber suppression at position 34 (top) or the sense cysteine codon at position 110 (bottom). Labeled CaM at the N‐ or C‐terminal domain is illustrated as a cartoon. (B) Fluorescent signals of single fluorophores incorporated into CaM taken from different gels.



To investigate whether the structure as well as the functionality of CaM remained unchanged upon insertion of Atto633, we performed smFRET measurements with a construct containing mutations both in its N‐ and C‐terminal domains. An amber and a cysteine codon were introduced at positions 34 and 110 of CaM, respectively, generating the double‐mutant DNA construct CaM T34amb T110C (Figure S1). Atto633 was linked either to p‐aminophenylalanine (AF) or to lysine (K) and it was successfully incorporated at position 34 in both cases. In addition, to perform smFRET measurements a donor dye is also needed. To this end, the acceptor‐labeled cell‐free synthesized CaM was labeled post‐ translationally at position 110 with Alexa Fluor 488 (AF488) as donor, using the well‐established maleimide‐cysteine ligation25. Independently on the used amino acid (AF or K) of the acceptor the FRET histogram showed a peak at the same position (Figure S2). For further smFRET experiments the Atto633‐AF was used as the acceptor dye. CaM can experience a wide range of conformations depending on the actual experimental conditions26, 27. Many structures are available in the Protein Data Bank (PDB) (for example PDB IDs: 1DMO, 1CLL, 1PRW, 1CDM). smFRET experiments were performed according to Sadoine et al.6 in calcium‐saturating conditions (holo‐condition)(Figure 2A), in the presence of the calmodulin‐binding domain of the calcium/calmodulin‐dependent protein kinase II (CaMKII)28 (Figure 2C) and following addition of the calcium chelator EGTA (apo‐condition) that inhibits the binding of CaMKII (Figure 2D). In holo‐conditions CaM exhibits a quite broad range of configurations and a very dynamic behavior; therefore the smFRET histograms show numerous subpopulations. The obtained histograms were fitted with two Gaussians, one at high FRET values, corresponding to a subpopulation that adopts a compact conformation, and the other at intermediate values that do not correspond to a specific structure, but to an ensemble of structures rapidly interchanging between each other. Upon addition of CaMKII the equilibrium shifts towards the compact state since the structure of CaM interacting with this ligand is a compact one. The compact structures reported for CaM have


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distances between the labeling positions quite below the Förster radius of the dye pair, thus giving values of FRET efficiency close to 100% that cannot be used to determine the exact distance between the dyes. Nevertheless, it is still shown that the functionality of the protein, i.e. the interaction with its partners, is maintained. Upon addition of EGTA the calcium ions are chelated and the interaction of CaM with the partners is switched off. Therefore, the histogram shows a single peak corresponding to the apo‐structure, further confirming the functionality of the expressed protein. The structure of the produced protein cannot be investigated with the usual biophysical methods (i.e. circular dichroism, activity tests) because the concentration of the sample is far too low to enable this kind of measurements. However, with these rather small sample amounts, smFRET measurements can also be employed directly to prove the structural and functional integrity of the synthesized proteins. The apoCaM has a well‐defined conformation and based on the three‐dimensional structure available in the PDB (PDB ID: 1DMO) we calculated the expected FRET efficiency, taking into account the entire accessible volume (AV) that can be occupied by the two fluorophores29. Following this approach we obtained a calculated average donor‐acceptor distance RDA for the double‐labeled apoCaM of 53.8 Å, which gave an expected FRET efficiency of 55%. This value is very close to the 57% of FRET efficiency obtained experimentally (see also Figure S2). The results are consistent with the expected calculated values and also similar to previously reported FRET histograms of CaM6, 26, 27. Therefore, we have strong indications that the co‐translational incorporation of the fluorophore maintains the structure and functionality of CaM unchanged.



Figure 2. Structure and functionality of mutant CaM. (A) smFRET histogram of holoCaM labeled co‐translationally with Atto633 and post‐translationally with AF488. (B) Stoichiometry vs FRET Efficiency graph of the same sample using the data from (A). The graph is constituted by about 1200 bursts, accumulated over three hours. An extended discussion about the statistics of the bursts forming the histograms is given in the Supporting Information. (C) smFRET histogram of the holoCaM in the presence of CaMKII and (D) after addition of EGTA. Gaussian fits were applied to all histograms and a mean FRET efficiency value was calculated for each peak. The area at FRET values below 0.15 is shaded because in that range the main contribution to the histogram comes from donor‐only molecules, giving rise to the zero‐peak artifact.


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Finally, we investigated the possibility to achieve co‐translational incorporation of two different fluorophores into the double mutant CaM T34amb T110C using once again commercially available precharged tRNAChemCUA in combination with tRNACys. Hereby, the acceptor dye Atto633‐AF was introduced at position 34 via amber suppression and the donor dye BODIPY‐FL at position 110 via the sense cysteine codon (Figure S3). smFRET experiments were performed in the presence of calcium to achieve the holo‐conformation. As in the previous experiment (Figure 2A), the FRET histograms of the holoCaM revealed two distinct populations, one corresponding to the compact state and a broad peak resulting from the presence of many different structures in fast exchange (Figure 3). In this case, checking the activity of the protein was not possible, because in the presence of EGTA (apo‐conditions) the acquisition of the corresponding FRET histogram for the calcium depleted CaM structure was not feasible, due to the fact that the BODIPY‐FL dye at position 110 suffered in this structure from strong local quenching (Table S1). However, Figure 3 demonstrates that the insertion of one fluorophore with improved photophysical properties makes it possible to actually perform smFRET experiments. Nevertheless, because of the poor molecular brightness of the donor dye BODIPY‐FL, any further worsening of its properties due to local quenching (e.g. due to conformational changes upon addition of EGTA) results in a complete loss of the signal. It is then foreseeable that the insertion of a donor with better photophysical properties will allow to obtain smFRET data with better statistics in a broader range of conditions.



Figure 3. smFRET of co-translationally double-labeled CaM. (A) smFRET histograms of CaM labeled with co‐ translationally incorporated Atto633 (AF) at the position 34 and BODIPY‐FL (C) at the position 110 in the presence of calcium ions. In the samples, in which both dyes are co‐translationally incorporated, the possibility to have a molecule with only one dye (due to read‐through of the amber codon) is higher with respect to samples obtained with post‐translational labeling, thus the weight of the zero‐peak is higher (grey‐shaded area). (B) Stoichiometry vs FRET Efficiency graph of the same sample using the data from (A). The graph is formed by about 500 bursts, resulting from 5 hours of acquisition. An extended discussion about the statistics of the bursts forming the histograms is given in the Supporting Information. (C) SDS‐gel showing fluorescent signals of single and double incorporation of BODIPY‐FL and Atto633 into CaM.


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In this study, we demonstrated using commercially available precharged suppressor tRNAs the co‐ translational incorporation of different dyes, among them some with superior characteristics suitable for single molecule measurements. The incorporation could take place in combination with the incorporation of a second fluorophore producing sufficient amounts of double‐labeled protein to perform smFRET experiments. For the success of our approach two aspects appeared to be of relevance. (i) The incorporation of only one single‐molecule suitable dye (Atto633) seems already to be sufficient in order to achieve single molecule sensitivity, at least if the second dye (BODIPY‐FL) is attached at a position where local dye quenching is not excessively strong (see Table S1). Since we already incorporated successfully one dye suitable for smFRET studies, we have an excellent starting point to achieve also a simultaneous incorporation of two dyes of this kind. With the utmost probability, such a sample would give even better results. (ii) Rather low amounts of double‐labeled protein from the CFPS are already sufficient if state‐of‐the‐art single‐molecule data acquisition and data analysis procedures are employed. In particular, pulsed interleaved excitation (PIE) and a burst selection procedure based on inter‐photon distance determination30 allowed for sufficiently high numbers of selected events that finally enter the FRET histograms. Furthermore, the application of AV calculations for the attached dyes29 allowed us to determine reliable inter‐dye distances (RDA), which can be validated by existing model structures of the investigated protein. The feasibility of this measure is of particular importance for our approach since the low yield in CFPS typically does not allow the application of other techniques. In this respect, smFRET and CFPS represent a perfect match since smFRET allows not only to monitor structural and dynamical properties of the proteins but inherently also allows to verify the structural and functional integrity of the proteins, which is not possible by other techniques, due to the low sample amounts. So far we applied our approach only in the case of CaM at two selected positions in the structure. Further studies with other proteins attempting to incorporate dyes at various different positions can give 11 ACS Paragon Plus Environment


valuable indications about the generality of our approach. However, our achievements constitute already a first step forward, since until now the inferior properties of the co‐translationally incorporated fluorophores forbid the use of this powerful technique in smFRET studies. In this respect, our approach represents a valuable component for emerging technologies to probe co‐translational protein folding, enabling a whole field of research that now is limited due to lack of experimental methods.

METHODS Construct. The gene of human CaM containing a C‐terminal His‐tag was cloned into a pRSET vector (Thermo Fischer Scientific) and two single mutants (CaM T34amb, CaM T110C), as well as a double mutant (CaM T34amb T110C) were generated via site‐directed mutagenesis. Synthesis of precharged tRNAs. Fluorescent tRNAs that recognize the sense cysteine codon were prepared and purchased from Biotechrabbit GmbH (Berlin, Germany). Shortly, to prepare E. coli tRNACys charged with BODIPY‐FL‐Cys (or Cy5‐Cys, Atto633‐Cys and BODIPY576‐Cys) the tRNA was generated by in vitro transcription with T7 RNA polymerase and charged in a first working step enzymatically with cysteine. The sulfhydryl group of Cys‐tRNACys was then reacted with BODIPY‐FL‐N‐(2‐aminoethyl)‐ maleimide (Molecular Probes, USA) to obtain BODIPY‐FL‐Cys‐tRNACys, Cy5‐maleimide (GE Healthcare, USA) to obtain Cy5‐Cys‐tRNACys, Atto633‐maleimide (AttoTec, Siegen, Germany) to obtain Atto633‐Cys‐ tRNACys or BODIPY576‐maleimide (Molecular Probes, USA) to obtain BODIPY576‐Cys‐tRNACys. Amber suppressor tRNA with Atto633 coupled via p‐amino‐phenylalanine was obtained from ProteinExpress (Japan). Amber suppressor tRNA precharged with Atto633‐lysine (Biotechrabbit GmbH) was prepared using a modified route for chemical aminoacylation according to Ellman et al. and Hohsaka et al.31-33. In brief, a truncated amber suppressor tRNA with enhanced suppression efficiency lacking two nucleotides at the 3’‐end (tRNAChemCUA[CA]) was generated by in vitro transcription with T7 RNA polymerase. The tRNA was ligated with dinucleotide pdCpA carrying Nα‐NVOC and Nε‐pentenoyl 12 ACS Paragon Plus Environment

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protected lysine resulting in (Nα‐NVOC)‐Lys(Nε‐pentenoyl)‐tRNAChemCUA. Following deprotection of the ε‐ amino group of lysine, (Nα‐NVOC)‐Lys‐tRNACUA was reacted with the NHS‐ester of Atto633 resulting in (Nα‐NVOC)‐Lys(Atto633)‐tRNACUA. Functional Atto633‐Lys‐tRNACUA was finally obtained after cleavage of the NVOC protection group by irradiation with a pulsed 360 nm laser beam. Hereby, the dye was protected by passing the beam through a special glass window with a transmission range selective for UV light. Amber suppressor tRNAs charged with BODIPY‐FL‐lysine or BODIPY576‐lysine were prepared in a similar way using the respective NHS esters (Molecular Probes, USA). The synthesis of precharged tRNAs is described in detail in the supporting information. Cell-free protein synthesis and incorporation of fluorescent UAAs. Protein synthesis was performed using an E. coli derived cell‐free transcription/translation system depleted from termination factor RF124. The reaction solution contained all amino acids except cysteine (Biotechrabbit GmbH). Fluorophore‐ charged tRNAChemCUA and tRNACys were added in a final concentration of 10 μM each. For control reactions in the absence of fluorophore‐charged tRNACys, 1.2 mM of free cysteine was added. Reaction mixtures were incubated in Eppendorf tubes at 32°C for 2 h. The synthesized protein was purified via Ni‐ NTA magnetic agarose beads (Qiagen) and desalted with PBS (pH 7.5) using size exclusion Zeba™ desalting columns (Thermo Fisher Scientific). The incorporation of the fluorescent UAAs was verified by loading 20 μl of the sample in presence of EGTA onto a 15% SDS‐PAGE and visualizing the gel using a fluorescence scanner (Typhoon FLA 9500, GE Healthcare Life Sciences). Samples for fluorescence microscopy. The protein was synthesized using a CFPS reaction and diluted in the final buffer to adjust the optimal concentration for each experiment. For FCS measurements the final concentration was in the nM range and for smFRET around 50 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, 150mM KCl). Binding experiments with calcium ions were performed in holo‐buffer (10 mM CaCl2, 50 mM MOPS, 150mM KCl) and the sample was 13 ACS Paragon Plus Environment


incubated overnight to reach equilibrium. Binding experiments with CaMKII were performed in the same holo‐buffer and in presence of a high excess of CaMKII (150 μM) to perform measurements in saturated condition. Inhibition of the calcium‐dependent binding of CaMKII was achieved by addition of an equivalent volume of apo‐buffer, which chelates calcium ions. 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 the labeled protein on the slides and consequently a loss of sample concentration during the measurement, the slides were 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. smFRET measurements and data analysis. smFRET measurements were performed with molecules freely diffusing in solution on an inverted confocal microscope employing pulsed excitation and time resolved photon detection (MicroTime 200, PicoQuant GmbH, Berlin, Germany). Analysis of FCS data was performed using the SymPhoTime64 software (PicoQuant, Berlin, Germany). Analysis of smFRET data was performed by using self‐written Matlab routines (Mathworks, Natick, USA). A detailed description of the microscope setup and the employed procedures for data treatment and analysis is given in the Supporting Information.


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ASSOCIATED CONTENT Supporting Information

Figures S1-S3 showing the primary sequence of the constructs, the incorporation of Atto633-AF and Atto633-K, as well as the double incorporation of dyes into CaM. Also, Supporting Methods referring to confocal fluorescence detection, QY determination and AV calculations, as well as discussing the effect of the local environment on the properties of the bound dyes, including Table S1 mentioning the change in the QY of dyes bound to CaM. Data analysis procedure and burst statistics including Table S2 and Figure S4 showing S / E graphs. Finally, explaining in more detail the preparation steps of precharged tRNAs including Tables S3-S4 showing the gradients used for the tRNA purification. (PDF).

AUTHOR INFORMATION Corresponding Authors

* [email protected].: +49 2461 612036 * [email protected].: +49 241 8027209 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 no competing financial interests.

ACKNOWLEDGEMENTS 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. We thank Kerstin Beyer and Iris Claußnitzer (RiNA GmbH, now biotechrabbit GmbH) for the synthesis of precharged tRNAs. This work was supported by Forschungszentrum Jülich resources.



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Co-translational incorporation into proteins of a fluorophore suitable for smFRET studies Mayuri Sadoine, Michele Cerminara, Michael Gerrits, Jörg Fitter and Alexandros Katranidis


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Cotranslational Incorporation into Proteins of a Fluorophore Suitable

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