Precursor-based selective methyl labelling of cell-free synthesized

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Precursor-based selective methyl labelling of cell-free synthesized proteins Mariya Lazarova, Frank L#hr, Ralf-Bernhardt Rues, Robin Kleebach, Volker Dötsch, and Frank Bernhard ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00338 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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Precursor-based selective methyl labelling of cell-free synthesized proteins Mariya Lazarova, Frank Löhr, Ralf-Bernhardt Rues, Robin Kleebach, Volker Dötsch, Frank Bernhard* Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt am Main, Germany

ABSTRACT: NMR studies of large proteins are complicated by pronounced spectral overlap and large line width. Reducing complexity by [13C, 1H] selective labelling of L-Val, L-Leu and/or L-Ile residues in combination with optional perdeuteration is therefore commonly approached by supplying labelled amino acid precursors into bacterial expression cultures, although often compromised by high label costs, precursor instability and label scrambling. Cell-free expression combines efficient production of membrane proteins with significant advantages for protein labelling such as small reaction volumes, defined amino acid pools, and reliable label incorporation. While amino acid specific isotopic labelling of proteins is routine application, the amino acid methyl side-chain labelling was so far difficult as appropriately labelled amino acids are hardly available. Based on recent proteome analyses of cell-free lysates, we have developed a competitive strategy for efficient methyl labelling of proteins based on conversion of supplied precursors. Pathway complexity of methyl side-chain labelling was reduced by implementing the promiscuous aminotransferase IlvE catalysing the selective LLeu, L-Val or L-isoleucine biosynthesis from specific ketoacid precursors. Precursor-based L-Leu and L-Val synthesis was demonstrated with the cell-free labelling of peptidyl-prolyl cis/trans isomerase cyclophilin D and of the proton pump proteorhodopsin. The strategy is fast and cost-effective and enables the straight forward methyl side-chain labelling of individual amino acid types. It can easily be applied to any cell-free synthesized protein.

membrane proteins and larger soluble proteins.1, 4-7 Further applications are proton-detected MAS solid-state NMR spectroscopy8 and the use of 1H, 13C labelled methyl groups for ligand mapping and as reporters for protein/ligand interactions.9, 10 This strategy is complementary to the use of 1H, 15N amide group labelled probes and attractive as highly sensitive 2D 1H, 13C chemical shift correlation spectra can be recorded within few minutes and with low sample concentrations. It furthermore allows analyzing the binding of small molecules into hydrophobic pockets or cores of soluble proteins or into membrane embedded domains of membrane proteins. Specifically labelled methyl groups can further be used as probes for protein side-chain dynamics.11

INTRODUCTION The characterization of structure, interaction and dynamics of biological macromolecules requires high spectral resolution and reduced signal complexity. Fast decay of NMR signals due to the overall slow reorientation of larger molecules reduces sensitivity and resolution. However, methyl groups are partly decoupled from slow tumbling and thus provide excellent properties for recording 1 H-13C correlation spectra due to rapid rotation even in large molecules as well as to signal averaging by the three-fold proton multiplicity.1 Site-specific protonation of methyl groups in combination with perdeuteration can further confer sensitivity enhancements in transverse relaxation-optimized spectroscopy (TROSY) through elimination of intra-methyl proton-proton and proton-13C dipole-dipole relaxation mechanisms.2 The aliphatic amino acids L-Ile, L-Leu and L-Val roughly represent 30% of the amino acids in α-helical membrane proteins.3 Their clustering in protein cores and often close vicinity give rise to intra- and intermolecular nuclear Overhauser effects (NOE’s) providing valuable restraints of structure, dynamics and interactions. Methyl side-chain labelling strategy thus extends structural NMR studies towards

Conventionally, methyl side-chain labelled amino acids are synthesized in bacterial cells overexpressing recombinant proteins by supplying the appropriately labelled precursors 2-ketobutyric acid (KBY) for L-Ile synthesis and 2-ketoisovaleric acid (KIV) for combined L-Val/L-Leu synthesis.12, 13 In case of deuteration, cells are grown in D2O minimal medium. As the methyl groups of L-Leu and L-Val are not stereoselectively 13C, 1H labelled, signals will be obtained from the γ1/δ1 methyl group and the γ2/δ2 1


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methyl group at 50% intensity each. Bottlenecks of the established cell-based protein methyl side-chain labelling procedure are often reduced growth and protein production efficiencies in D2O minimal medium, in particular for membrane proteins. The relatively high volumes require substantial amounts of expensive labelled precursor.12-14 Precursor instability and inefficient transport into the cells can further increase the required amounts. Furthermore, using the precursor KIV will result in the combined labelling of L-Val and L-Leu residues, while suppression of nondesired L-Leu labelling can be attempted by including large amounts of either unlabelled L-Leu or of unlabelled 4-methyl-2-oxovalerate (MOV), a specific ketoacid precursor for L-Leu biosynthesis.15, 16 Alternatively, appropriately labelled MOV precursor may be prepared by customized chemical synthesis and supplied to the medium for specific L-Leu labelling.14

branched chain aminotransferase IlvE for the exclusive synthesis of L-Leu, L-Val or L-Ile based on the supply of appropriate ketoacid precursors. We demonstrate the precursor-based preparative scale protein production in standard S30 lysates and we could drastically reduce precursor requirements to sub-millimolar concentrations with further optimized S30 lysates. The precursor-based CF labelling and NMR analysis of L-Leu or L-Val residues was exemplified with the 20.5 kDa chaperone cyclophilin D (CypD) and with the 27.6 kDa membrane protein proteorhodopsin (PR) containing seven transmembrane segments. RESULTS S30 and S12 lysate proteome analysis of branched chain amino acid metabolic pathways. Two lysate variations, S30 lysate fractionated by centrifugation at 30.000 xg and S12 lysates fractionated by centrifugation at 12.000 xg are commonly used for CF expression approaches and their proteomes have recently been analysed.21, 22 Starting with the commercially available standard precursors KBY for L-Ile biosynthesis and KIV for L-Leu and L-Val biosynthesis, pathways comprising several enzymatic steps are necessary (Table S1). However, the key enzyme necessary for biosynthesis of all three amino acids is the promiscuous branched chain aminotransferase IlvE catalyzing the final amino acid conversion from three specific precursors by using L-Glu as amino group donor (Fig. 1). With the precursor KIV, only IlvE is sufficient for the conversion to L-Val (Table S1). However, for the synthesis of the two other amino acids, IlvE requires the precursor MOV for conversion to L-Leu and the precursor (S)-3-methyl-oxopentanoate (MOP) for conversion to LIle (Fig. 1). Therefore, two possible strategies for the CF methyl side-chain labelling of branched chain amino acids exist: (I) Using the three appropriately labelled precursors KIV, MOV and MOP and only implementing the enzyme IlvE, or (II) recruiting the endogenous biosynthetic pathways implementing in addition to IlvE the enzymes IlvC, D, G and M for L-Ile synthesis and LeuA-D for L-Leu synthesis (Table S1).

Cell-free (CF) expression offers numerous advantages for the labelling of proteins. Most obvious are low reaction volumes of just few mL, reliable and fast protein production and reduced and controllable metabolic processes. Working with defined precursor pools ensures 100% label incorporation. Finally but not least important, the reduced system complexity of CF reactions allows fast access to even formerly very difficult proteins such as membrane proteins.17, 18 Aliphatic amino acid residues are more abundant in membrane proteins and averaged frequencies are roughly 9% for L-Ile, 10% for L-Val and 12% for L-Leu.3 The anticipated methyl side-chain labelling is therefore of particular value for this class of proteins. Methyl labelling and stereospecific isotope labelling of CF synthesized proteins has previously been accomplished by supplying appropriately site-specific labelled amino acids into the reaction.17, 19 In particular the incorporation of supplied stereo-specifically methyl labelled amino acids into CF or cell-based synthesized proteins has been documented.19, 20 However, these amino acids are difficult to obtain and must be isolated either from hydrolyzed labelled proteins or synthesized by elaborated chemical or enzymatic reactions. The limited availability and high costs of site-specifically labelled amino acids has therefore widely restricted the application of CF side-chain labelling so far.

However, from all relevant enzymes listed in Table S1, the core proteome of S30 lysates contains only IlvE, IlvD and the non-essential enzyme AvtA, thus making strategy II very unlikely. Taking the emPAI values as a preliminary measure of protein abundance, IlvE and LeuD are even present in relatively low amounts. Alternatively, the analysed S12 proteome did not reveal the key enzyme IlvE, while the L-Leu specific biosynthetic enzymes LeuA-D were occasionally detected in individual measurements.22

Recent proteome analyses of the S30 and S12 lysates has transferred CF expression to a better defined synthetic system.21, 22 The S30 lysate or alternatively the more complex S12 lysate are core components of CF reactions and produced by standardized procedures from E. coli cells. Processing of the S30 lysate results into fractionation and removal of approximately two third of the E. coli proteome.21 Analysis of the residual amino acid biosynthetic portfolio in the S30 lysate revealed elimination of most enzymes necessary for L-Val, L-Leu and L-Ile biosynthesis according to the Kyoto encyclopedia of genes and genomes pathway (http://www.genome.jp/kegg/pathway.html, KEGG). The reduced metabolic complexity allows to implement the

Determining branched chain amino acid synthesis in S30 and S12 lysates. Despite the obvious absence of most enzymes required for strategy II, we nevertheless tested both approaches for their feasibility. For strategy I, the standard precursor KIV is available as unlabelled compound as well as suitable isotopically labelled deriva2



tive. Unfortunately, the precursor MOV is only commercially available as 13C labelled derivative at the C1 position, while the precursor (S)-3-methyl-oxopentanoate (MOP) can currently only be obtained by custom synthesis. In order to test for IlvE function in the S30 lysate, a GFP expression with a 19 amino acid mixture either lacking LLeu but containing 1 mM MOV, or lacking L-Val but containing 1 mM KIV was performed (Fig. S1). The alternative conversion of KIV to L-Leu by implementing the addi-

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tional enzymes LeuA-D for strategy II was analysed by expressing GFP with 1 mM KIV in a L-Leu deficient 19 amino acid mixture (Fig. S1). This approach should verify the absence of the enzymes LeuA-D in the CF lysate, as already indicated by the previous proteomics analysis.21 Negative controls without supplements indicated very low background GFP expression and alternative biosynthetic pathways in S30 lysates can thus be excluded.

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Figure 1. Structure and conversion of precursors necessary for L-Val, L-Leu and L-Ile biosynthesis. (a) Reaction scheme indicating two alternative strategies. Vertical arrows indicate the IlvE-based conversion of the three precursors KIV, MOV and MOP to L-Val, L-Leu and L-Ile (= strategy I). Horizontal arrows indicate the multi-step enzymatic synthesis (= strategy II) of L-Leu and L-Ile by the enzymes LeuA-D + IlvE and IlvC, D, G, M + IlvE, respectively. Cofactors required by the enzymes are indicated. (b) Isotope labels of the KIV and MOV precursor used in this study for NMR sample preparation. The position of the labels in the corresponding amino acids after conversion by IlvE is indicated.

Already considerable amounts of GFP of 2.4 0.1 mg/mL with MOV, and of 2.0 0.2 mg/mL with KIV/L-Leu were produced by the endogenous S30 IlvE activity, while GFP production based on L-Leu synthesis in the KIV/L-Val reaction implementing the LeuA-D pathway for strategy II was not observed (Fig. S1). Modified reactions containing in addition extra amounts of the cofactors acetyl-CoA and NAD+ were also negative (Fig. S1). This result correlates to the absence of LeuA-D in the S30 lysate proteome and it demonstrates that in contrast to in vivo conditions, the conversion of KIV to L-Leu does not occur in CF reactions based on standard S30 lysates. The CF L-Ile synthesis by strategy II was analysed with

the commercially available precursor 2-ketobutyrate (KBY) requiring the enzymes IlvC, D, G and M as well as IlvE for amino acid conversion (Table S1). CF reactions with KBY with and without supplied cofactors did not result into GFP expression and the result correlates with the previously reported absence of IlvC, D, G and M in the S30 proteome (Fig. S2 and Table S1). Strategy I for CF L-Ile synthesis could not be tested as the required precursor MOP is currently not available. Alternative to S30 lysates, we prepared S12 lysates according to published procedures from strain A19 and as indicated in the methods section. Furthermore, we prepared a modified S12 lysate with an additional heat/salt 3


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ACS Chemical Biology Optimizing L-Val and L-Leu biosynthesis in S30 lysates based on IlvE activity. The initial KIV and MOV based GFP expression indicated some limitations of precursor conversion if compared with the positive controls (Fig. S1). Further increased MOV or KIV concentrations correlated with improved GFP production (Fig. 2a, b). Alternatively, increasing S30 lysate concentrations from the standard 30% concentration up to 50% improved GFP expression with 2 mM KIV in presence of a 19 amino acid mixture lacking L-Val (Fig. 2c). Increased concentrations of the amino group donor L-Glu up to 5 mM did not improve GFP expression efficiency (data not shown). Likewise, increasing L-Ala concentrations up to 5 mM to improve the conversion of L-Ala + KIV to L-Val + pyruvate by the detected enzyme AvtA did not improve GFP expression.

treatment we routinely apply for S30 lysate preparation and that considerably improved the efficiency of subsequent CF expression reactions (Fig. S3). The only difference between this S12-H and the S30 lysate is thus the gforce used for centrifugation, whereas the S12 lysate in addition lacks the routine heat/salt treatment. The results of precursor conversion were similar to that with S30 lysate, showing that strategy I for methyl side-chain labelling implementing only IlvE and different precursors is also feasible with S12 lysate. KIV is converted to L-Val although the IlvE enzyme was not detected in the previous S12 proteome study.22 Like with the S30 lysate, the application of strategy II by conversion of KIV to L-Leu over the LeuA-D pathway as well as the KBY to L-Ile conversion is not feasible as well with S12 lysate (Fig. S3).

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Figure 2. KIV and MOV based protein synthesis in S30 lysates (n = 3). S30 lysate concentrations in (a) and (b) are 30%. (a) Concentration screen for MOV. GFP was CF expressed with an L-Leu deficient 19 amino acids mix and increasing MOV concentrations. Controls are with 1 mM L-Leu and without supplement (-). (b) Concentration screen for KIV. GFP was expressed with an L-Val deficient 19 amino acids mix and increasing KIV concentrations. Controls are with 1 mM L-Val and without supplement (-). (c) GFP expression with KIV and different S30 lysate concentrations. GFP was expressed with an L-Val deficient amino acid mix with 2 mM KIV and different concentrations of S30 extract (30%, 40% and 50%) in the reaction mixture. As control a sample with 40% lysate and 1 mM L-Val and a sample without supplement (-) were prepared.

ing concentrations of MOV (Fig. 3). The addition of extra IlvE enzyme had a clear beneficial effect on the expression efficiency with both precursors and GFP yields comparable to the control with supplied L-Val and L-Leu were already obtained with 0.1 mM KIV and 0.3 mM MOV (Fig. 3). Taken together, the results indicate IlvE activity as limiting factor for precursor-based methyl labelling in standard S30 lysates, while this limitation could be addressed by either increased precursor or S30 lysate concentrations.

To analyze increased IlvE concentrations in CF reactions, an E. coli codon optimized synthetic IlvE gene containing a C-terminal StrepII-tag was cloned into pET21 and expressed in BL21 (DE3) Star (Fig. S4). The enzyme could be purified by affinity chromatography to apparent homogeneity (Fig. S5). The purified IlvE was supplied into CF reactions at final concentrations of 1 mg/mL. GFP was synthesized with a 19 amino acid mixture lacking L-Val but containing varying concentrations of KIV and with a 19 amino acid mixture lacking L-Leu but containing vary-

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Labelling of the soluble protein CypD. CypD is a mitochondrial peptidyl-prolyl isomerase and its CF preparation was already established for NMR investigation.23 For precursor-based L-Leu labelling, a commercially available MOV derivative containing a 13C label at the carboxyl-

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group and thus theoretically resulting in selective L-Leu labelling at the CypD backbone CO position was used (Fig. 1). Unfortunately, this labelling scheme does not allow acquisition of a 2D experiment and could be monitored only by a 1D 13C spectrum (Fig. 4).

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Figure 3. KIV and MOV based protein synthesis in IlvE enriched S30 lysates (n = 3). S30 lysate concentration is 30%. (a) KIV concentration screen. (b) MOV concentration screen. GFP was expressed with a 19 amino acid mix lacking L-Val (KIV) or L-Leu (MOV) and the reaction was supplemented with 1 mg/mL IlvE. Controls are with 1 mM L-Val or L-Leu, or without supplement ().

specific 13C methyl labelling of L-Val residues based on the KIV precursor without significant metabolic scrambling.

In the 1D spectrum a set of peaks with chemical shifts between 173-180 ppm were detected. In this region resonances of backbone carbonyl carbons are typically found. Integration of the peaks suggests that the signals are evoked by 11-12 13C nuclei, which corresponds to the expectation since the CypD construct contains 11 L-Leu residues. Although the spectrum does not unambiguously show the L-Leu labelling, it strongly suggest that the CF expression with MOV results in incorporation of the 13C nuclei at the L-Leu backbone carbonyl position.

Labelling of the membrane protein PR. In order to investigate whether the proposed precursor labelling strategy can also be applied for a more challenging membrane protein target, KIV was used for the CF preparation of a L-Val 13C/2H labelled PR sample. The sample preparation of PR in DH7PC followed an established procedure25, 26 with substitution of L-Val for KIV in the CF reaction. Labelling of the protein was monitored by a [13C,1H]SOFAST-HMQC (Fig. 5b). Besides the protein signals, only natural abundance signals of acetate buffer and the detergent were detected. Although the number of methyl cross peaks is difficult to determine exactly, it is clearly in the expected range for the 20 L-Val residues in the sequence of PR. The spectrum was compared to a previously acquired SOFAST-HMQC of a PR sample with u-13C labelling of all methyl containing residues and L-Trp.

The L-Val labelling of CypD expressed in presence of [13C/2H] labelled KIV could be evaluated by [13C, 1H]HSQC measurements, since the precursor harbours a labelled carbon at the methyl side-chain. The 2D spectrum (Fig. 5a) displays a set of peaks at the region where resonances for L-Val methyl side-chains are expected. Apart from the methyl resonances only a couple of weak low field signals were detected. These are most probably induced by supplied low molecular weight protease inhibitors at 13C natural abundance. The region of methyl sidechain resonances reveals some 20 resolved peaks together with a set of overlapping signals. CypD contains 15 L-Val residues, and since the precursor is not stereo-specific labelled a maximum of 30 signals is expected. Approximately half of the signals could be assigned to CypD L-Val residues using previously published chemical shifts list of the protein.24 As the reference spectrum was acquired with modified conditions and with a different labelling scheme, only well-resolved cross peaks were unambiguously assigned. The [13C, 1H]-HSQC therefore validates the

All peaks of the sample prepared with KIV display twobond and three-bond isotope shifts due to the deuteration of the precursor and the lack of a corresponding 2Hlabelling of the u-13C labelled reference sample. Nevertheless, the overlay of the spectra validates the signal detection of the KIV sample in the region of the PR methyl side-chain resonances. Furthermore, the comparison demonstrates the tremendous gain in resolution by selective 1H/13C labelling of one methyl group in otherwise deuterated L-Val residues owing to the lack of 13C-13C and 1H-1H scalar couplings as well as reduced transverse relaxation rates in isolated 13CH3 groups. In accordance to 5


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the results for CypD, the PR sample verifies the successful L-Val labelling by CF expression with KIV without signifi-

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scrambling.

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Figure 4. 1D C spectrum of CypD expressed with C MOV. 1D C was acquired for CypD expressed without L-Leu in presence 13 of C MOV at a 500 MHz Bruker Avance II spectrometer at 298 K. The measurement was performed with 250 µM sample in 50 mM sodium phosphate pH 7.0, 1 mM DTT, 5% (v/v) D2O, 0.15 mM DSS, 0.05% (w/v) NaN3 and 1x complete protease inhibitor. The spectrum results from accumulation of 64k scans using an acquisition time of 0.15 s relaxation delay of 2 s. To enable signal counting, integrals are normalised to the resolved peak at 173.25 ppm. The integral values are indicated.scans using an acquisition time of 0.15 s relaxation delay of 2 s. To enable signal counting, integrals are normalised to the resolved peak at 173.25 ppm. The integral values are indicated.

DISCUSSION CF expression provides unique advantages for the production of membrane proteins as problems with toxicity, inefficient membrane insertion, template instability or low yields are eliminated.18 Further benefits are rapid purification strategies and multiple options to support the co-translational folding of synthesized membrane proteins by supplying appropriate hydrophobic environments.18, 26 Branched-chain amino acids are enriched in membrane proteins and are excellent reporters for internal molecular motion and intermolecular interactions.7, 27, 28 Selective methyl side-chain-labelling using cell-based protein expression was already successfully applied for structural studies of challenging membrane proteins 6, 29, 30 and investigation of substrate interactions in high molecular weight complexes.31, 32 However, the difficult preparation of accordingly labelled amino acids limited this application for CF expression approaches so far. Amino acids obtained from hydrolysed labelled carrier proteins previously synthesized in E. coli by the conventional precursorbased strategy have been used for CF methyl labelling.17 However, this procedure still relies on cellular expression, and yields and purity of the isolated amino acids depend on hydrolysis. The strategy to incorporate chemically

synthesized stereospecifically labelled amino acids into CF synthesized proteins is restricted by high costs and limited availability.19, 20 We have developed a straight-forward one step strategy for the direct methyl labelling in CF reactions by precursor-based synthesis of the corresponding amino acids. The strategy uses the promiscuous activity of the branched chain aminotransferase IlvE to specifically convert the precursors KIV, MOV and MOP into the amino acids L-Val, L-Leu and L-Ile, respectively. Already in standard S30 and S12 lysates, preparative scale protein production can be obtained with low mM concentrations of KIV and MOV precursors. Precursors could be further reduced to sub-millimolar concentrations by enrichment of lysates with 1 mg/mL recombinant IlvE enzyme. The amino acid synthesis was strictly selective without any apparent scrambling activities e.g. by converting KIV to MOV by IlvE for subsequent L-Leu synthesis. The 27.5 kDa GFP contains 16 L-Val and 22 L-Leu residues. 1 mg/mL GFP expression therefore corresponds to a 36 µM protein concentration in the CF reaction mixture. The CF reactions were performed in the two-compartment dialysis configuration containing a reaction mixture (RM) and a feeding mixture (FM).18 Taking into account the used 6



RM to FM ratio of 1:18, a total of 19 mL per reaction has to be considered. The required minimal L-Val concentration per mL for the production of 1 mg GFP in this reaction would be 36 µM x 16 (# of L-Val residues) = 576 µM divided by 19 (# of total reaction mL) = 30 µM. For L-Leu labelling, according to this calculation a minimal concentration of 42 µM per mL for production of 1 mg/mL GFP would be necessary. In the exemplified CF reactions supplied with IlvE (Fig. 3), GFP was synthesised with yields of 3 mg/mL with 100 µM KIV and 3.5 mg/mL with 300 µM MOV. Considering that the minimally required precursor concentrations were then approximately 90 µM for KIV and 147 µM for MOV, an amino acid conversion and incorporation efficiency of 90 % for KIV and of 50% for MOV has been achieved. The almost complete KIV to LVal conversion indicates a high precursor stability in the

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CF reaction and breakdown metabolisms can be neglected, whereas the MOV to L-Leu conversion is somehow less efficient but still at a high level. For 100 µM KIV concentration, the required amount of precursor would be 14.3 µg/mL = 272 µg for a 1 mL (=1 mL reaction mixture + 18 mL feeding mixture) CF reaction. The L-Val methyl side-chain labelled PR sample was isolated from 3 mL CF reaction and would thus theoretically require a total amount of approximately 820 µg labelled KIV precursor. The current costs for the precursor would be less than 2 € (Sigma # 691887, 1.7 €/mg). For comparison, for the cellbased methyl labelling of a target protein, approximately 100-200 mg of labelled precursor has to be supplied per liter bacterial culture.13 Certainly, it should be considered that protein expression efficiencies in CF systems are target specific and costs will thus vary accordingly.

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Figure 5. NMR Spectra of CypD and PR expressed without L-Val and in presence of [ C, H] KIV. (a) [ C, H]-HSQC spectrum of CypD acquired at a 500 MHz Bruker Avance II spectrometer at a temperature of 298 K. The measurement was performed with 300 µM sample in 50 mM sodium phosphate buffer, pH 7.0, containing 1 mM DTT, 5% (v/v) D2O, 0.15 mM DSS, 0.05% (w/v) 24 NaN3 and 1x complete protease inhibitor. Individual assignments of L-Val methyl resonances are given in the inset. (b) HMQC spectrum of PR acquired at a 950 MHz Bruker Avance III spectrometer at 323 K. The measurement was performed with 250 µM sample in 25 mM sodium acetate pH 5.0, 2 mM DTT, 2% (w/v) DH7PC, 5% (v/v) D2O, 0.15 mM DSS, 0.05% (w/v) NaN3. The inset 13 1 shows an overlay with a [ C, H]-SOFAST HMQC spectrum of a non-deuterated sample in which all methyl containing residues 13 15 (Ala, Ile, Leu, Met, Thr, Val) as well as Trp were u-[ C, N]-labelled (green contours), recorded under the same experimental conditions. Notation: d – DH7PC signals; a – acetate signal.

The presented approach is new and still has limitations. Due to the lack of the MOP precursor, we could only demonstrate the L-Val and L-Leu synthesis by IlvE based on the KIV and MOV precursor, although the acceptance of MOP by IlvE is very likely. The required appropriately labelled precursors of MOV and MOP are currently not standard chemicals, but can be chemically synthesized.14 Furthermore, a quoting for the custom synthesis of the two labelled precursors would be approximately 20 €/mg

(Sigma, personal communication). However, the ease of CF methyl labelling of either individual or combinatorial branched-chain amino acid types in proteins might increase future demands of the precursors and result into their inclusion into standard chemical product portfolios. Depending on the intended application, membrane proteins often require perdeuteration for NMR investigations due to the high molecular weight of the protein in combination with the artificial hydrophobic environment 7


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such as detergent micelles or nanodiscs. Deuteration in vivo requires growth of bacterial cultures in D2O media, which additionally increases the costs for sample preparation (approximately 500 € per liter D2O). Deuteration in CF systems requires supply of [2H]-labelled amino acids to the CF reaction. These are commercially available (average costs are 1 €/mg) and are efficiently incorporated in the protein without affecting expression yields. However, residual transaminase activities in the CF reactions can cause loss of deuteration at Cα and Cβ positions.33 Transaminase inhibitors could then not be used as they would also inhibit IlvE. In cases where such deuteration scrambling is not acceptable, either CF expression in D2O or a two-step procedure with the pre-synthesis of methyl sidechain labelled amino acids by IlvE in a separate reaction might be considered.

containing a total volume of 19 mL. The costs could most likely be reduced by using the one-compartment CF batch configuration.35 Batch CF systems consist only of the RM and have thus drastically reduced volumes. As a consequence, lower product yields are obtained, while still expression efficiencies of approximately 1 mg/mL have been reported.35 A balance in between costs, workload and time investment might therefore be considered when selecting either of the two CF configurations. In conclusion, the demonstrated approach of precursor based methyl side-chain labelling in CF systems allows straightforward and time-efficient protein labelling with minimal optimisation of standard CF expression protocols. Specifically labelled NMR samples of membrane proteins, toxins or any other CF synthesized protein can be obtained within 24 hours. Moreover, the strategy allows the selective labelling of L-Val or L-Leu residues. Depending on the intended applications and the required labelled precursors, significant cost savings could be obtained as well. Future refinements will help to expand the process as a standard tool for the structural and functional characterization of even challenging membrane protein targets.

In general, additional costs for operating the CF expression reaction have to be considered as well. For S30 lysate preparation, efficient and low cost procedures have been described that can generate > 100 mL of S30 lysate within two days.34 For the standard CF reaction components such as energy precursors or NTPs, approximately 50 € can be calculated for the described two-compartment dialysis configuration with a RM to FM ratio of 1:18 and

EXPERIMENTAL SECTION DNA and templates. Standard superfolder-GFP cloned in the pET21 vector was used as reporter for CF protein expression. The cypD and PR constructs used for NMR sample preparation were described previously.23, 25 Briefly, the CypD construct contains amino acid 43–207 of the human CypD and an N-terminal His6-tag with a TEV protease cleavage site and is cloned in the pIVex2.3-MCS vector. The PR construct contains an N-terminal strepIItag and is cloned in the pIVEX2.3d vector. E. coli IlvE was cloned in the pET21a vector with a C-terminal strepII-tag (see also Fig. S3).

12.7 mg/mL. Purified IlvE was supplied to CF reactions without additional pyridoxal phosphate cofactor. Extract preparation. All lysates for CF expression were prepared with the A19 E. coli strain. The S30 lysate was prepared as described previously.34 Briefly, the bacterial cells were grown in a fermenter at 37 °C until an OD600 of 4.0-4.5 was reached. The cell culture was cooled down to approximately 20 °C and harvested by centrifugation at 6800 xg for 15 min. The cells were washed with S30 buffer A (10 mM Tris-acetate, 14 mM Mg(OAc)2, 60 mM KCl, 6 mM 2-mercaptoethanol, pH 8.2) and resuspended in 110% (v/w) S30 buffer B (10 mM Tris-acetate, 14 mM Mg(OAc)2, 60 mM KCl, 1 mM DTT, 1 mM PMSF, pH 8.2) for lysis. The cells were disrupted with a French press and the lysate was centrifuged two times at 30,000 xg for 30 min. NaCl was added to the supernatant to the final concentration of 400 mM and the solution was incubated in a water bath at 42 °C for 45 min. The solution was dialysed against S30 buffer C (10 mM Tris-acetate, 14 mM Mg(OAc)2, 60 mM KOAc, 0.5 mM DTT, pH 8.2) with one buffer exchange and finally centrifuged two times at 30,000 xg for 30 min. The supernatant was collected, shock frozen in liquid nitrogen and stored at -80 °C. The S12-H lysate was prepared similar to the S30 lysate except that all centrifugation steps at 30,000 xg were performed at 12,000 xg instead. For preparation of the S12 lysate, in addition the incubation step at 42oC with 400 mM NaCl was omitted.34

IlvE expression. IlvE was expressed in BL21 (DE3) Star express cells. LB medium was inoculated with an overnight pre-culture and the cells were grown at 37 °C and 180 rpm until an OD600 of 0.8 was reached. The IlvE expression was induced by addition of 1 mM IPTG. The cells were incubated at 37 °C for another 4 h and harvested at 4500 xg and 4 °C for 20 min. The pellet was resuspended in wash buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1x complete protease inhibitor) and cell lysis was achieved by sonification. The lysate was centrifuged for 30 min at 30 000 xg and 4 °C in order to remove cell debris. The supernatant was filtered through a sterile 0.45 μm filter and purified with strep-tactin resin (IBA) according the manufacturers protocol. The eluate was dialysed against 10 mM K2HPO4 pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 50% (v/v) glycerol. The purified protein was concentrated using Amicon centrifugal filters with a MWCO of 30 kDa to the final concentration of

Analytical CF expression. For protein expression the two compartment continuous exchange CF system consisting out of a reaction mixture (RM) and a feeding mix8



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ture (FM) in a ratio of 1:18 (v/v) and separated by a membrane was used. Reaction set-up and protein production was performed as described previously.24 Analytical CF expressions were performed with 55 µL RM and 1 mL FM. Amino acids as well as all tested cofactors were supplemented to the reaction with the final concentration of 1 mM. The precursors KIV, MOV and KBY were added with varying concentrations from a 100 mM stock in water. GFP expression was used as a reporter for the efficiency of protein production. The amount of expressed protein per mL RM was evaluated by measurement of the specific GFP fluorescence at λex = 485 nm and λem = 510 nm directly in the RM. The results of three independent CF reactions were averaged.

enhanced pulse sequence with 300 µM sample. PR spectra were acquired on a Bruker Avance III 950 MHz spectrometer equipped with a cryogenic probe at 323 K. The KIV labeled sample (200 µM protein concentration) had a volume of 380 µl in a 4 mm tube. The u-13C/15N-Ala, Ile, Leu, Met, Thr, Trp, Val labelled sample (400 µM) had a volume of 300 µl in a 5 mm shigemi tube. [13C, 1H]SOFAST-HMQC was acquired with 250 µM sample with a 1H excitation bandwidth of 3.5, centered at 0.5 ppm using a pulse sequence adapted to incorporate gradient coherence selection to improve suppression of the strong detergent and acetate signals.36

NMR sample preparation. CypD was expressed with 3-4 mL RM and a RM:FM ratio of 1:15. For L-Leu labelling, a L-Leu deficient amino acids mix was used and MOV (2-keto-4-methylpentanoic-1-13C acid, Sigma) was added to the final concentration of 2 mM. For L-Val labelling, a L-Val deficient amino acids mix was used. KIV (2-keto-3methyl-d3-butyric acid-4-13C,d, Sigma) was added to the final concentration of 2 mM. CypD was purified with IMAC as described previously (Hein 2017). For acquisition of NMR spectra the sample buffer was exchanged to 50 mM sodium phosphate pH 7.0, 1 mM DTT, 5% (v/v) D2O, 0.15 mM DSS, 0.05% (w/v) NaN3 and 1x complete protease inhibitor through dialysis. The sample was concentrated to the final concentration of 250-300 µM with Amicon centrifugal filters with a MWCO of 10 kDa.

Supporting Information. Further material including details about the residual biosynthetic pathways in E. coli S30 and S12 lysates, data for L-Ile biosynthesis with KBY and details about the utilized IlvE construct. This material is available free of charge via the Internet at http://pubs.acs.org.

PR was expressed with 3 mL RM and a RM:FM ratio of 1:15. The PR bound cofactor all-trans-retinal was added to the RM with final concentration of 0.6 mM. For cotranslational solubilisation GDN and DH7PC were added to the reaction with final concentrations of 0.4% (w/v) and 0.04% (w/v) respectively as described previously.25, 26 For L-Val labelling KIV was supplemented to the reaction as described for CypD. For labelling of all methylcontaining amino acids an amino acid mix containing u[13C, 15N]-labelled L-Ala, L-Ile, L-Leu, L-Met, L-Thr, L-Trp and L-Val was used. The protein was purified with streptactin resin (IBA) according the manufacturers protocol. 0.1% (w/v) DH7PC was added to all buffers. For NMR spectra acquisition the sample buffer was exchanged to 25 mM NaOAc pH 5.0, 2 mM DTT, 0,1% (w/v) DH7PC, 5% (v/v) D2O, 0.15 mM DSS, 0.05% (w/v) NaN3 with PD10 desalting columns. L-Val methyl selective and u-13C/15N labeled samples were concentrated to 200 and 400 µM, respectively.

This work was funded by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG). This work was supported by the state of Hessen (Center for Biomolecular Magnetic Resonance) and the German Research Foundation (DO545/11, SFB 807).

ASSOCIATED CONTENT

AUTHOR INFORMATION Corresponding Author *correspondence should be addressed to [email protected]

Author Contributions The manuscript was written through contributions of all authors.

Funding Sources

ACKNOWLEDGEMENTS This work was supported by the state of Hessen (Center for Biomolecular Magnetic Resonance) and the German Research Foundation (DO545/11, SFB 807). ABBREVIATIONS SEC, size exclusion chromatography; IMAC, immobilized metal-ion affinity chromatography; CV, column volume; CF; cell-free; RM, reaction mixture; FM, feeding mixture; DTT, dithiothreitol; GFP, green fluorescent protein; PR, proteorhodopsin; CypD, cyclophilin D; L-Val, L-valine; L-Leu, Lleucine; L-Ile, L-isoleucine; L-Ala, L-alanine; L-Met, Lmethionine; L-Thr, L-threonine; L-Trp, L-tryptophan; KIV, 2ketoisovalerate (3-methyl-2-oxobutanoate); KBY, 2ketobutyrate, KEGG, Kyoto encyclopedia of genes and genomes; MOV, 4-methyl-2-oxopentanoate; MOP, (S)-3methyl-2-oxopentanoate; DSS, 4,4-dimethyl-4-silapentane-1sulfonic acid.

NMR measurements. CypD spectra were acquired on a Bruker Avance II 500 MHz spectrometer equipped with a room-temperature triple-resonance probe at 298 K with 300 µl sample volume in 5 mm shigemi tubes. A 0.4-ms 1D spin-echo 13C spectrum was acquired for the MOV labeled protein with 250 µM sample to prevent a baseline-roll otherwise occurring as a result of probe ringing. [13C, 1H]HSQC was acquired for the KIV labeled sample using a standard gradient-coherence selected, non-sensitivity

REFERENCES 1. Tugarinov, V., Kay, L. E. (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new

9


Page 11 of 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


labeling strategy and novel NMR methods, J Am Chem Soc 125, 13868–13878. 2. Tugarinov, V., Kanelis, V., Kay, L. E. (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy, Nat Protoc 1, 749–754. 3. Deber, C. M., Brandl, C. J., Deber, R. B., Hsu, L. C., Young, X. K. (1986) Amino acid composition of the membrane and aqueous domains of integral membrane proteins, Arch Biochem Biophys 251, 68–76. 4. Hiller, S., Garces, R. G., Malia, T. J., Orekhov, V. Y., Colombini, M., Wagner, G. (2008) Solution structure of the integral human membrane protein VDAC-1 in detergent micelles, Science 321, 1206–1210. 5. Imai, S., Osawa, M., Takeuchi, K., Shimada, I. (2010) Structural basis underlying the dual gate properties of KcsA, Proc Natl Acad Sci U S A 107, 6216–6221. 6. Gautier, A., Mott, H. R., Bostock, M. J., Kirkpatrick, J. P., Nietlispach, D. (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy, Nat Struct Mol Biol 17, 768–774. 7. Sprangers, R., Kay, L. E. (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR, Nature 445, 618– 622. 8. Agarwal, V., Xue, Y., Reif, B., Skrynnikov, N. R. (2008) Protein side-chain dynamics as observed by solution- and solid-state NMR spectroscopy: A similarity revealed, J Am Chem Soc 130, 16611–16621. 9. Hamel, D. J., Dahlquist, F. W. (2005) The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy, J Am Chem Soc 127, 9676–9677. 10. Hajduk, P. J., Augeri, D. J., Mack, J., Mendoza, R., Yang, J., Betz, S. F., Fesik, S. W. (2000) NMR-based screening of proteins containing 13C-labeled methyl groups, J Am Chem Soc 122, 7898– 7904. 11. Ollerenshaw, J. E., Tugarinov, V., Skrynnikov, N. R., Kay, L. E. (2005) Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins, J Biomol NMR 33, 25–41. 12. Kerfah, R., Plevin, M. J., Sounier, R., Gans, P., Boisbouvier, J. (2015) Methyl-specific isotopic labelling: A molecular tool box for solution NMR studies of large proteins, Curr Opin Struct Biol 32, 113-122. 13. Kurauskas, V., Schanda, P., Sounier, R. (2017) Methylspecific isotope labeling strategies for NMR studies of membrane proteins, Methods Mol Biol 1635, 109–123. 14. Lichtenecker, R., Coudevylle, N., Konrat, R., Schmid, W. (2013) Selective isotope labelling of leucine residues by using αketoacid precursor compounds, ChemBioChem 14, 818-821. 15. Mas, G., Crublet, E., Hamelin, O., Gans, P., Boisbouvier, J. (2013) Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins, J Biomol NMR 57, 251-262. 16. Lichtenecker, R., Weinhäupl, K., Reuther, L., Schörghuber, J., Schmid, W., Konrat, R. (2013) Independent valine and leucine isotope labelling in Escherichia coli protein overexpression systems, J Biomol NMR 57, 205-209. 17. Linser, R., Gelev, V., Hagn, F., Arthanari, H., Hyberts, S. G., Wagner, G. (2014) Selective methyl labeling of eukaryotic membrane proteins using cell-free expression, J Am Chem Soc 136, 11308–11310. 18. Henrich, E., Hein, C., Dötsch, V., Bernhard, F. (2015) Membrane protein production in Escherichia coli cell-free lysates, FEBS Lett 589, 1713–1722. 19. Takeda, M., Ikeya, T., Güntert, P., Kainosho, M. (2007) Automated structure determination of proteins with the SAIL-FLYA NMR method, Nat Protoc 2, 2896-2902.

20. Miyanoiri, Y., Takeda, M., Okuma, K., Ono, A. M., Terauchi, T., Kainosho, M. (2013) Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labelled amino acids, J Biomol NMR 57, 237-249. 21. Foshag, D., Henrich, E., Hiller, E., Schäfer, M., Kerger, C., Burger-Kentischer, A., Diaz-Moreno, I., Garcia-Maurino, S. M., Dötsch, V., Rupp, S., Bernhard, F. (2018) The E. coli S30 lysate proteome: A prototype for cell-free protein production, New Biotechnol 40, 245–260. 22. Hurst, G. B., Asano, K. G., Doktycz, C. J., Consoli, E. J., Doktycz, W. L., Foster, C. M., Morrell-Falvey, J. L., Standaert, R. F., Doktycz, M. J. (2017) Proteomics-based tools for evaluation of cell-free protein synthesis, Anal Chem 89, 11443–11451. 23. Hein, C., Löhr, F., Schwarz, D., Dötsch, V. (2017) Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study, Biopolymers 107, e23013. 24. Schedlbauer, A., Hoffmann, B., Kontaxis, G., Rüdisser, S., Hommel, U., Konrat, R. (2007) Automated backbone and sidechain assignment of mitochondrial matrix cyclophilin D, J Biomol NMR 38, 267. 25. Reckel, S., Gottstein, D., Stehle, J., Löhr, F., Verhoefen, M.K., Takeda, M., Silvers, R., Kainosho, M., Glaubitz, C., Wachtveitl, J., Bernhard, F., Schwalbe, H., Güntert, P., Dötsch, V. (2011) Solution NMR structure of proteorhodopsin, Angew Chem 50, 11942–11946. 26. Laguerre, A., Löhr, F., Henrich, E., Hoffmann, B., AbdulManan, N., Connolly, P. J., Perozo, E., Moore, J.M., Bernhard, F., Dötsch, V. (2016) From nanodiscs to isotropic bicelles: A procedure for solution nuclear magnetic resonance studies of detergent-sensitive integral membrane proteins, Structure 24, 1830– 1841. 27. Tugarinov, V., Sprangers, R., Kay, L. E. (2004) Line narrowing in methyl-TROSY using zero-quantum 1H-13C NMR spectroscopy, J Am Chem Soc 126, 4921–4925. 28. Gross, J. D., Gelev, V. M., Wagner, G. (2003) A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes, J Biomol NMR 25, 235– 242. 29. Hiller, S., Malia, T. J., Garces, R. G., Orekhov, V. Y., Wagner, G. (2010) Backbone and ILV side chain methyl group assignments of the integral human membrane protein VDAC-1, Biomol NMR Assignments 4, 29–32. 30. OuYang, B., Xie, S., Berardi, M. J., Zhao, X., Dev, J., Yu, W., Sun, B., Chou, J. J. (2013) Unusual architecture of the p7 channel from hepatitis C virus, Nature 498, 521–525. 31. Gelis, I., Bonvin, A. M. J. J., Keramisanou, D., Koukaki, M., Gouridis, G., Karamanou, S., Economou, A., Kalodimos, C. G. (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR, Cell 131, 756–769. 32. Saio, T., Guan, X., Rossi, P., Economou, A., Kalodimos, C. G. (2014) Structural basis for protein antiaggregation activity of the trigger factor chaperone, Science 344, 1250494. 33. Etezady-Esfarjani, T., Hiller, S., Villalba, C., Wüthrich, K. (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies, J Biomol NMR 39, 229-238. 34. Schwarz, D., Junge, F., Durst, F., Frölich, N., Schneider, B., Reckel, S., Sobhanifar, S., Dötsch, V., Bernhard, F. (2007) Preparative scale expression of membrane proteins in Escherichia colibased continuous exchange cell-free systems, Nat Protoc 2, 2945–2957. 35. Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. (2012) Cell-free protein synthesis: Applications come of age, Biotechnol Adv 30, 1185–1194.

10



36. Amero, C., Schanda, P., Asuncion Dura, M., Ayala, I., Marion, D., Franzetti, B., Brutscher, B., Boisbouvier, J. (2009) Fast

Page 12 of 12

two-dimensional NMR spectroscopy of high molecular weight protein assemblies, J Am Chem Soc 131, 3448–3449.

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Precursor-based selective methyl labelling of cell-free synthesized

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