Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for

Letter pubs.acs.org/JPCL

Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins Christoph Gmeiner,† Daniel Klose,† Elisabetta Mileo,*,‡ Valérie Belle,‡ Sylvain R. A. Marque,§,∥ Georg Dorn,⊥ Frédéric H. T. Allain,⊥ Bruno Guigliarelli,‡ Gunnar Jeschke,† and Maxim Yulikov*,† †

Laboratory of Physical Chemistry, ETH Zurich, Zurich 8093, Switzerland Aix Marseille Univ, CNRS, BIP, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille 13402, France § Aix Marseille Univ, CNRS, ICR, Institut de Chimie Radicalaire, Marseille 13397, France ∥ N. N. Vorozhtsov Novosibirsk Insititute of Organic Chemistry, 630090 Novosibirsk, Russia ⊥ Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich 8093, Switzerland ‡

S Supporting Information *

ABSTRACT: Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron−electron resonance (DEER) EPR measurements were performed for both the nitroxide−nitroxide and Gd(III)−nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

D

for instance, for homooligomeric proteins, where distance distributions between sites in the same protomer may be required for unambiguous modeling of the structure. We already encountered such a case for the N-terminal domain of the trimeric major light-harvesting complex LHCII of green plants and solved it with only a single label type using an elaborate double-tag purification strategy.37 This strategy becomes even more elaborate for oligomers with more protomers and then also leads to a low fraction of spin-labeled complexes. Spectroscopically, orthogonal labeling simplifies the distance distributions analysis because it allows us to obtain interprotomer and intraprotomer distance information on the same sample. However, for such strategy, the spin labeling chemistry at different protein sites also needs to be “orthogonal”. Practically, this means that one label needs to be attached to a residue different from cysteine and with a sufficiently different chemical reaction to ensure site selectivity. Existing solutions for SDSL of proteins that are inaccessible to cysteine labeling and for controlled spectroscopically orthogonal labeling within the same protein molecule exploit unnatural amino acids.21,38−42 This approach requires more effort in protein mutant design and expression as compared

ouble electron−electron resonance (DEER) spectroscopy1,2 in combination with site-directed spin labeling (SDSL)3−7 provides access to the distance distributions in biomolecules in the nanometer range.8−12 Such data are of significant importance in structural biology as they help to determine structures or structural models,13−15 conformational heterogeneity,16,17 and, in some cases, conformational dynamics18 of biomolecules. Among other alternatives to nitroxide spin labels, the labels based on Gd(III) chelate complexes were proposed.19,20 Sitespecific attachment of spin labels to proteins is typically performed via reaction of cysteine residues with a thiol-reactive linker of the spin label.3−7 This strategy, however, requires alternatives whenever the protein of interest contains functional and structural cysteine residues that cannot be mutated out. Furthermore, spectroscopically orthogonal DEER experiments require the site-selective attachment of different types of spin labels.21−23 Such experiments allow for determination of multiple distances in the same sample and are particularly well-suited for studies of protein interactions with other biomolecules. Initiated by the demonstration of a very good spectroscopic selection in Gd(III)−nitroxide pairs,24−26 a large number of new spectroscopically orthogonal spin label pairs and distance measurements were reported, especially in the last 3 years.27−36 Here we focus on the situation where two spectroscopically orthogonal spin labels need to be attached to the same protein molecule.21,22 Such a strategy can be useful, © XXXX American Chemical Society

Received: August 22, 2017 Accepted: September 21, 2017 Published: September 21, 2017 4852

DOI: 10.1021/acs.jpclett.7b02220 J. Phys. Chem. Lett. 2017, 8, 4852−4857

Letter

The Journal of Physical Chemistry Letters with the conventional recombinant protein expression. It was previously demonstrated that with use of a three-component Mannich-type reaction combined with an isoindoline-based nitroxide, tyrosine spin labeling of proteins can be performed, which thus offers an alternative SDSL protocol without the need for unnatural amino acids.43,44 Here we present a proofof-principle example of orthogonal spin labeling of proteins using the specific reactivity of cysteine and tyrosine, along with the corresponding DEER measurements and their analysis. To this end, we performed SDSL on the isolated RNA Binding Domains (RBDs) 1 (∼13.36 kDa) and 34 (∼23.34 kDa) of the alternative splicing regulator Polypyrimidine-Tract Binding Protein 1 (PTBP1).45,46 RBD1 contains three (Y113, Y114, and Y127) and RBD34 two (Y361, Y430) native tyrosine residues. The structure of the full complex of PTBP1 and the Internal Ribosomal Entry Site (IRES) of the Encephalomycarditis Virus (EMCV) is not yet known, and thus studying the intramolecular and intermolecular interactions in this system and in its individual domains is of practical interest. Here we use single RBDs with a priori known structures as model system to test the approach. For measuring nitroxide−nitroxide and nitroxide−Gd(III) DEER we introduced a single cysteine residue by site-directed mutagenesis in both constructs (RBD1−T109C, RBD34−Q388C) and labeled them with different types of spin labels following the conventional cysteine-based SDSL approach.3,4,7 Tyrosine SDSL was carried out via a three-component Mannich-type reaction by attaching an in-house synthesized nitroxide radical (5-amino-1,1,3,3-tetramethyl-isoindoline-2yloxyl, abbreviated in the following as “Nox”) to the native tyrosine residues in RBD1 and RBD34.43−46 The details of the SDSL, sample purification, and analysis for all types of spin labels can be found in the Supporting Information. In aqueous solution tyrosines labeled by Nox are present in two forms, a closed form (referred as cyclic Nox, “Nc”) with a formed heterocyclic ring arising from the reaction with two molecules of formaldehyde and a linear form, where this ring is open (referred to as linear Nox, “Nx”, Scheme S1) and corresponding to the reaction with only one molecule of formaldehyde. The two forms of Nox differ by a mass of 12 Da (Nx shows an increase in mass of 217 and Nc of 229 Da), which can be distinguished by mass spectrometry (see Figure S7). To account for the conformational flexibility of these two forms we computed libraries of Nx and Nc conformations using the known rotamer library approach (see the Supporting Information for details).47 The computed rotamer libraries were included in the multiscale modeling of macromolecular systems (MMM) package for Matlab,48 and the distance distributions were modeled based on the available liquid-state NMR structures for RBD1 (PDB code: 2AD9) and RBD34 (PDB code: 2ADC).45,46 On the basis of the structures of RBD1 and RBD34, we assumed that the residues Y113 and Y114 in RBD1 and Y361 in RBD34 are unlikely to be labeled as they are protected by other residues and embedded in α-helices, whereas Y127 (RBD1) and Y430 (RBD34) are more solvent-exposed and should have higher accessibility for the Nox radical (see Figures 1 and 2 for RBD34 and Supporting Information Figure S1 for RBD1 labeling sites). Indeed, simulations of the Nx and Nc conformations with MMM indicated that the labeling of Y113, Y114, and Y361 might fail due to the tightness of these sites (see Table S4). To prove our considerations, we produced the single mutants RBD34 Y361F and Y430F and labeled them

Figure 1. Labeling of tyrosine residues with different accessibility in RBD34. (a) X-band (9.5 GHz) room-temperature CW spectra of Noxlabeled RBD34 single mutants Y430F (Y361 labeled) and Y361F (Y430 labeled) and cysteine-less RBD34 (Y361/Y430 labeled) are compared with free Nox radical (0.5 mM, red dashed line). (b) QBand (35 GHz) DEER results of Nox-labeled cysteine-less RBD34 (Y361/Y430 labeled) measured at 50 K in 50% d8-glycerol. Order from top to bottom: Experimental DEER data (top, black) including background correction (top, red). Background-corrected DEER data (center, black) including form factor fitting (center, red dashed line). Distance distribution (bottom, black) obtained by Tikhonov regularization in DeerAnalysis49 and comparison with the MMMsimulated distance distribution (bottom, green solid line) between residues Y361 and Y430 using the generated rotamer library for the linear Nox (Nx). Note that the rotamer approach predicts that Y361 cannot be labeled with the cyclic Nox (Table S4). (c) 3D solution NMR structure of RBD34 (PDB code: 2ADC) showing the two native tyrosine residues Y361 and Y430. (d) Structure of linear and cyclic Nox radical attached to tyrosine residue.

with the Nox radical. Surprisingly, CW-EPR spectroscopy showed that both single mutants could be labeled (Figure 1). However, in line with expectations, the mutant Y430F (Y361 4853


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Figure 2. Nitroxide−nitroxide labeling at a tyrosine and a cysteine site. (a) 3D solution NMR structure of RBD34 (PDB code 2ADC) showing the two native tyrosine residues (black) and the position Q388 (red) for cysteine mutation. (b) Background-corrected DEER data (top, black) including form factor fit (top, red dashed line) and distance distribution of Q388C IAP/Y361 Nox/Y430 Nox (bottom, black). The experimental distance distribution was compared with its simulated counterparts Q388C−Y361 (Nx: bottom, violet solid line) and Q388C−Y430 (Nc: bottom, green dashed line; Nx: green solid line). (c) 3D solution NMR structure of RBD1 (PDB code 2AD9) showing all three native tyrosine residues (black) and the position T109 (red) for cysteine mutation. (d) Background-corrected DEER data (top, black) including form factor fit (top, red dashed line) and distance distribution of T109C IAP/Y127 Nox (bottom, black). The experimental distribution was compared with simulated T109C−Y127 distance distributions based on either of the two Nox libraries (Nc: bottom, green dashed line; Nx: bottom, green solid line). All DEER measurements were performed in Q-band (35 GHz) at 50 K with the addition of 50% d8-glycerol to the protein solutions.

In contrast, cysteine-less Nox-labeled RBD1 (Y113/Y114/ Y127 Nox), even after very long DEER signal accumulation of ∼60 h, did not reveal any dipolar modulation (Figure S17), although the rotamer approach predicted small number of Nox rotamers for the Y113 and Y114. From this DEER results and the performed rotamer analysis, we conclude that Y127 is very likely the only accessible tyrosine in this RBD, and Y113/Y114 could not be labeled. No single tyrosine mutants were designed for the RBD1. Note also that positions Y113 and Y114 are too close to the designed cysteine position T109C to produce detectable dipolar oscillations in the DEER experiment. To combine tyrosine and cysteine SDSL, we produced the single cysteine mutants T109C (RBD1) and Q388C (RBD34) and labeled them using three different spin labels: MTSSL (referred to as R1 when attached to a cysteine), iodoacetamido-proxyl (IAP), and Gd(III)-maleimido-DOTA (Gd(III)-DOTA). The two different nitroxide spin-label types were chosen as the most popular options to form reversible and irreversible bond to the cysteine. Good spin labeling efficiency was proven by CW-EPR spectroscopy and mass spectrometry (MS) for nitroxide- and by MS for Gd(III)-labeled samples (Figure S5−S7). In addition, we also tested a potential influence of the spin label to the overall folding of the protein by circular dichroism (CD) spectroscopy (Figure S8). SDSL did not show a significant change in the CD spectra, leading to the conclusion that the labeling did not alter the folding of the RBDs. The rotamer library modeling for the tyrosine Y127 of RBD1 in the case of Nc revealed some bimodality in the distance distribution to the site T109C for all three types of cysteinespecific labels. Such a bimodality was not present for Nx form. In the case of RBD34 (tyrosine Y430), the “cysteine−tyrosine”

labeled, Y430 substituted by phenylalanine) delivered a significantly lower labeling efficiency (∼3%) as compared with the Y361F (between 14 and 17%). In both cases, the CW-EPR spectrum of Nox attached to a tyrosine residue has a two-component pattern with one component corresponding to a fast tumbling and the other component corresponding to a much slower rotational tumbling of the spin-labeled side chain. The fast component has a characteristic triplet of lines due to the 14N hyperfine interaction, and the peak-to-peak amplitudes for the three components differ more than in the case of free Nox (Figure 1a, Table S2), indicating slower and, likely, more anisotropic reorientations of the spin label attached to the RBD as compared with the free spin label. This is similar to the original work, where two-component CW-EPR spectra were recorded for the tyrosine spin-labeled CP12 protein.43 It is natural to assume that this might be a manifestation of the coexistence of the cyclic Nc and linear Nx form of Nox at each labeling site, yet this cannot be concluded with certainty because conformational dynamics of each form of Nox might be complex on its own. We performed DEER measurements on the cysteine-less Nox-labeled RBD34 because CW-EPR results suggested that a small fraction of the doubly labeled protein should be present (Figure 1a). Indeed, a weak oscillation of the DEER echo with a modulation depth λ of ∼1% could be obtained, leading to a distance distribution in the range of 3 to 4 nm, which is in agreement with the rotamer library prediction (Figure 1b). Strong oscillations in the beginning of the DEER trace represent a distribution in a shorter distance range that might result from spectrometer instabilities or from the poor signalto-noise ratio. 4854


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The Journal of Physical Chemistry Letters distance distributions modeled for the Nc and Nx forms of Nox were very similar (see Supporting Information Figure S9). Rotamer analysis data for all labels are summarized in the SI (Table S4). Because cysteine-less tyrosine-labeled RBD1 did not show any DEER modulation depth, we expected a single distance distribution in the RBD1 T109C measurements, labeled with IAP/Nox and Gd(III)-DOTA/Nox. In the first case the main peak of the experimentally obtained distance distribution between 2 and 4 nm (Figure 2) matches well with the simulated distance distribution T109C−Y127 using both rotamer libraries (Nc and Nx), which is thus in line with a potential presence of both Nox forms. This is also in line with the assumption above that Y127 is labeled predominantly. The orthogonal DEER measurement for Gd(III)-DOTA-labeled RBD1 T109C delivered a distance distribution in agreement with the prediction by the Nx and Nc rotamer library (Figures S9 and S17 and Table S6). Note that the prediction with the Nc library covers the range of the experimental distances and shows a significantly broader distribution than Nx with a not fully resolved but clearly visible bimodality. Because the DEER data of cysteine-less Nox-labeled RBD34 showed a measurable fraction of spin-labeled site pairs Y361 and Y430 (Figure 1), we expected two or three peaks in the DEER distance distribution from the sample where cysteine and tyrosine SDSL were combined. In this case, for cysteine labeling we used MTSSL, IAP, or Gd(III)-DOTA. MMM simulations predicted a short distance peak in the range of 2 nm between Q388C and Y361 because these sites are in close vicinity, whereas the Q388C−Y430 distance was predicted to be in a range of 3 to 4 nm. Indeed, a contribution of the Q388C−Y361 distance to the overall distance distribution could be detected in the measurements Q388C IAP/Nox and Q388C R1/Nox (Figures 2b and 3b). Note that a contribution from the Y361−Y430 distances would also be present in the nitroxide−nitroxide DEER data. This additional peak overlays in both cases with the cysteine−tyrosine distance distributions. In Figure 3, the combination of R1-Nox and Gd(III)-DOTANox DEER measurements is shown. In the first case, a broad distance distribution was obtained, which encompasses both simulated distances, Q388C−Y361 around 2 nm and Q388C− Y430 around 3 nm. From the Gd(III)-nitroxide DEER data we obtained a distance distribution between 2.5 and 3.8 nm. The main experimental distance distribution peak is in reasonable agreement with the MMM-predicted distribution for Q388C− Y430 and Q388C−Y361 (see also Table S6). The width of this peak, is smaller than in the R1-Nox case, and the two MMMpredicted peaks are placed at its shoulders (Figure 3). The modulation depth between 4 and 2% was determined for the IAP/Nox- and R1/Nox-labeled samples, and modulation depth of ∼0.7% was obtained for the Gd(III)/Nox-labeled sample. Table S2 lists all nitroxide labeling efficiencies determined by CW-EPR spectroscopy. Furthermore, successful attachment of the spin labels can be seen in the ESI−MS spectra (see Figures S6 and S7). In all tested cases nitroxide radicals are pumped, so we can assume the same inversion efficiency pNO. With the labeling efficiencies f C and f Y for cysteine and tyrosine labeling, respectively, the observer-spin weighted modulation depth λNO−NO for the nitroxide−nitroxide case is

Figure 3. Nitroxide−nitroxide versus Gd(III)−nitroxide labeling of the same RBD34 construct. (a) Background-corrected DEER data (Q388C−I: top, 50 K, black; Q388C−II: top, blue, 10 K) including form factor fits (top, red dashed lines) measured on a high power QBand (35 GHz) spectrometer.51 Q388C−I = Q388C R1/Y361 Nox/ Y430 Nox and Q388C−II = Q388C Gd(III)-DOTA/Y361 Nox/Y430 Nox. (b) Experimentally obtained distance distribution of Q388C R1/ Nox (center, black) and distributions predicted for Q388C−Y361 (Nx: center, violet solid line) and Q388C−Y430 (Nc: center, green dashed line; Nx: center, green solid line). (c) Experimental distance distribution of Q388C Gd(III)−DOTA/Nox (bottom, blue solid line) and predictions for Q388C−Y361 (Nx: bottom, violet solid line) and Q388C−Y430 (Nc: bottom, green dashed line; Nx: bottom, green solid line). (d) 3D solution NMR structure of RBD34 (PDB code: 2ADC) showing spheres for the conformational distributions of the labels, the cysteine-labeled residues (magenta and brown), and residue Y430 labeled with linear (green) and cyclic (black) Nox (overlaid).

λNO − NO =

fC fY pNO + fY fC pNO fC + fY

=

2fC fC + fY

·fY pNO

(1)

whereas the modulation depth λGd−NO in the Gd(III)−nitroxide case is λGd − NO = fY pNO

(2)

because the echo signal stems only from molecules where cysteine labeling succeeded. Accordingly, a larger modulation depth for the nitroxide−nitroxide case implies f C > f Y. Note that the experimental modulation depth λGd−NO ≈ 0.007 is, for instance, in the same range of values as in Gd(III)−Gd(III) DEER.19,20,50 Furthermore, observation on Gd(III) has the advantage of a larger observer spin polarization at 10 K and of faster permissible repetition of the experiment due to a shorter longitudinal relaxation time. The lower modulation depth of the orthogonal DEER experiment thus does not necessarily imply lower sensitivity. In summary, despite the relatively low dipolar modulation depths, we demonstrated the combination of tyrosine and cysteine orthogonal SDSL to obtain distance distributions in the DEER experiment. Because cysteine labeling might fail if native cysteine residues are important for the protein function or folding, SDSL of native or engineered tyrosine residues can be used as an alternative to cysteine labeling. The tyrosine spin labeling therefore further expands the use of DEER spectros4855


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Systems and the Sensitized Photolysis of Decalin. Chem. Phys. Lett. 1984, 110, 67−72. (2) Pannier, M.; Veit, S.; Godt, A.; Jeschke, G.; Spiess, H. W. DeadTime Free Measurement of Dipole-Dipole Interactions Between Electron Spins. 2000. J. Magn. Reson. 2011, 213, 316−325. (3) Altenbach, C.; Marti, T.; Khorana, H. G.; Hubbell, W. L. Transmembrane Protein Structure: Spin Labeling Of Bacteriorhodopsin Mutants. Science 1990, 248, 1088−1092. (4) Hubbell, W. L.; Lopez, C. J.; Altenbach, C.; Yang, Z. Y. Technological advances in site-directed spin labeling of proteins. Curr. Opin. Struct. Biol. 2013, 23, 725−733. (5) Qin, P. Z.; Haworth, I. S.; Cai, Q.; Kusnetzow, A. K.; Grant, G. P. G.; Price, E. A.; Sowa, G. Z.; Popova, A.; Herreros, B.; He, H. Measuring Nanometer Distances in Nucleic Acids Using a SequenceIndependent Nitroxide Probe. Nat. Protoc. 2007, 2, 2354−2365. (6) Shelke, S. A.; Sigurdsson, S. Th. Site-Directed Spin Labelling of Nucleic Acids. Eur. J. Org. Chem. 2012, 2012, 2291−2301. (7) Feintuch, A.; Otting, G.; Goldfarb, D. Gd3+ Spin Labeling for Measuring Distances in Biomacromolecules: Why and How? Methods Enzymol. 2015, 563, 415−457. (8) Schiemann, O.; Prisner, T. F. Long-Range Distance Determinations in Biomacromolecules by EPR Spectroscopy. Q. Rev. Biophys. 2007, 40, 1−53. (9) Jeschke, G.; Polyhach, Y. Distance Measurements on SpinLabelled Biomacromolecules by Pulsed Electron Paramagnetic Resonance. Phys. Chem. Chem. Phys. 2007, 9, 1895−1910. (10) Jeschke, G. DEER Distance Measurements on Proteins. Annu. Rev. Phys. Chem. 2012, 63, 419−446. (11) Bordignon, E. Site-Directed Spin Labeling of Membrane Proteins. Top. Curr. Chem. 2011, 321, 121−157. (12) Goldfarb, D. Gd3+ Spin Labeling for Distance Measurements by Pulse EPR Spectroscopy. Phys. Chem. Chem. Phys. 2014, 16, 9685− 9699. (13) Alonso-Garcia, N.; Garcia-Rubio, I.; Manso, J. A.; Buey, R. M.; Urien, H.; Sonnenberg, A.; Jeschke, G.; de Pereda, J. M. Combination of X-ray Crystallography, SAXS and DEER to Obtain the Structure of the FnIII-3,4 Domains of Integrin α6β4. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71, 969−985. (14) Duss, O.; Michel, E.; Yulikov, M.; Schubert, M.; Jeschke, G.; Allain, F. H. Structural Basis of the Non-coding RNA RsmZ Acting as a Protein Sponge. Nature 2014, 509, 588−592. (15) Bleicken, S.; Jeschke, G.; Stegmueller, C.; Salvador-Gallego, R.; García-Sáez, A. J.; Bordignon, E. Structural Model of Active Bax at the Membrane. Mol. Cell 2014, 56, 496−505. (16) Hänelt, I.; Wunnicke, D.; Bordignon, E.; Steinhoff, H.-J.; Slotboom, D. J. Conformational Heterogeneity of the Aspartate Transporter GltPh. Nat. Struct. Mol. Biol. 2013, 20, 210−214. (17) Georgieva, E. R.; Borbat, P. P.; Ginter, C.; Freed, J. H.; Boudker, O. Conformational Ensemble of the Sodium-Coupled Aspartate Transporter. Nat. Struct. Mol. Biol. 2013, 20, 215−221. (18) Endeward, B.; Marko, A.; Denysenkov, V. P.; Sigurdsson, S.Th.; Prisner, T. F. Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids. Methods Enzymol. 2015, 564, 403−425. (19) Raitsimring, A. M.; Gunanathan, C.; Potapov, A.; Efremenko, I.; Martin, J. M.; Milstein, D.; Goldfarb, D. Gd3+ Complexes as Potential Spin Labels for High Field Pulsed EPR Distance Measurements. J. Am. Chem. Soc. 2007, 129, 14138−14139. (20) Abdelkader, E. H.; Lee, M. D.; Feintuch, A.; Cohen, M. R.; Swarbrick, J. D.; Otting, G.; Graham, B.; Goldfarb, D. A New Gd3+ Spin Label for Gd3+-Gd3+ Distance Measurements in Proteins Produces Narrow Distance Distributions. J. Phys. Chem. Lett. 2015, 6, 5016−5021. (21) Garbuio, L.; Bordignon, E.; Brooks, E. K.; Hubbell, W. L.; Jeschke, G.; Yulikov, M. Orthogonal Spin Labeling and Gd(III)Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme. J. Phys. Chem. B 2013, 117, 3145−3153. (22) Lueders, P.; Jager, H.; Hemminga, M. A.; Jeschke, G.; Yulikov, M. Distance Measurements on Orthogonally Spin-Labeled Membrane

copy in studies of biomolecules. While tyrosine spin labeling required a rather high (80-fold) excess of both Nox and formaldehyde, the measured distances are in line with the available structures of single RBDs. It is thus likely that protein fold was not affected by spin labeling. The generated rotamer libraries for the in-house synthesized Nox radical reveal a good correspondence of the label-to-label distance simulations and the experimentally obtained distance distributions. Besides nitroxide−nitroxide DEER, we also showed the performance of the tyrosine labeling approach in orthogonal DEER with a Gd(III)-DOTA label at the cysteine residue. As reported in previous studies, the maximal obtained labeling efficiency for Nox so far is ∼40%.43 In this study, we obtained a maximum value of about 14 to 17%, implying that either the native tyrosine residues of RBD1 and RBD34 of PTBP1 are not fully accessible for the spin labels or the SDSL conditions were not perfectly optimized (see the SI). Future studies should aim to improve the labeling efficiency, which would also increase the sensitivity of the corresponding DEER measurements. However, already at the reported labeling efficiencies, the approach appears to work well enough for a number of applications. We propose that tyrosine SDSL alone or in combination with cysteine SDSL offers a useful opportunity for systems where only few native tyrosine residues are present and either native cysteine residues cannot be labeled or orthogonal spin labeling experiments are required.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b02220. Materials and methods, mass spectroscopy data, CWEPR data, circular dichroism data, further description of the tyrosine SDSL and the rotamer library for the Nox radical, and supplementary figures. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*M.Y.: E-mail: [email protected]. *E.M.: E-mail: [email protected]. ORCID

Elisabetta Mileo: 0000-0002-0883-6043 Sylvain R. A. Marque: 0000-0002-3050-8468 Maxim Yulikov: 0000-0003-3275-0714 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.G., M.Y., and G.J. thank ETH Zurich for financial support (Grant No. 0-2066-14). M.Y., G.J. and F.A. also thank the Sinergia grant CRSII5-170976 for financial support. We acknowledge mass spectrometry analysis done by Dr. Serge Chesnov at the Functional Genomics Center Zurich (FGCZ). Prof. Glockshuber (ETH Zurich) is acknowledged for the help with CD spectroscopy measurements. We are grateful to the EPR facilities available at the national TGE RPE facilities (IR 3443) in Marseille.

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REFERENCES

(1) Milov, A. D.; Ponomarev, A. B.; Tsvetkov, Yu. D. Electron Electron Double Resonance in Electron-Spin Echo - Model Biradical 4856


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The Journal of Physical Chemistry Letters Spanning WALP23 Polypeptides. J. Phys. Chem. B 2013, 117, 2061− 2068. (23) Yulikov, M. Spectroscopically Orthogonal Spin Labels and Distance Measurements in Biomolecules. Electron Paramag. Res. 2014, 24, 1−31. (24) Lueders, P.; Jeschke, G.; Yulikov, M. Double Electron-Electron Resonance Measured Between Gd3+ Ions and Nitroxide Radicals. J. Phys. Chem. Lett. 2011, 2, 604−609. (25) Yulikov, M.; Lueders, P.; Warsi, M. F.; Chechik, V.; Jeschke, G. Distance Measurements in Au Nanoparticles Functionalized With Nitroxide Radicals and Gd3+−DTPA Chelate Complexes. Phys. Chem. Chem. Phys. 2012, 14, 10732−10746. (26) Kaminker, I.; Yagi, H.; Huber, T.; Feintuch, A.; Otting, G.; Goldfarb, D. Spectroscopic Selection of Distance Measurements in a Protein Dimer with Mixed Nitroxide and Gd3+ Spin Labels. Phys. Chem. Chem. Phys. 2012, 14, 4355−4358. (27) Abdullin, D.; Duthie, F.; Meyer, A.; Müller, E. S.; Hagelueken, G.; Schiemann, O. Comparison of PELDOR and RIDME for Distance Measurements between Nitroxides and Low-Spin Fe(III) Ions. J. Phys. Chem. B 2015, 119, 13534−13542. (28) Akhmetzyanov, D.; Plackmeyer, J.; Endeward, B.; Denysenkov, V.; Prisner, T. F. Pulsed Electron−Electron Double Resonance Spectroscopy Between a High-Spin Mn2+ Ion and a Nitroxide Spin Label. Phys. Chem. Chem. Phys. 2015, 17, 6760−6766. (29) Kaminker, I.; Bye, M.; Mendelman, N.; Gislason, K.; Sigurdsson, S. Th.; Goldfarb, D. Distance Measurements Between Manganese(II) and Nitroxide Spin-Labels by DEER Determine a Binding Site of Mn2+ in the HP92 Loop of Ribosomal RNA. Phys. Chem. Chem. Phys. 2015, 17, 15098−15102. (30) Meyer, A.; Schiemann, O. PELDOR and RIDME Measurements on a High-Spin Manganese(II) Bisnitroxide Model Complex. J. Phys. Chem. A 2016, 120, 3463−3472. (31) Meyer, A.; Abdullin, D.; Schnakenburg, G.; Schiemann, O. Single and Double Nitroxide Labeled Bis(Terpyridine)-Copper(II): Influence of Orientation Selectivity and Multispin Effects on PELDOR and RIDME. Phys. Chem. Chem. Phys. 2016, 18, 9262−9271. (32) Joseph, B.; Tormyshev, V. M.; Rogozhnikova, O. Yu.; Akhmetzyanov, D.; Bagryanskaya, E. G.; Prisner, T. F. Selective High-Resolution Detection of Membrane Protein−Ligand Interaction in Native Membranes Using Trityl−Nitroxide PELDOR. Angew. Chem., Int. Ed. 2016, 55, 11538−11542. (33) Di Valentin, M.; Albertini, M.; Dal Farra, M. G.; Zurlo, E.; Orian, L.; Polimeno, A.; Gobbo, M.; Carbonera, D. Light-Induced Porphyrin-Based Spectroscopic Ruler for Nanometer Distance Measurements. Chem. - Eur. J. 2016, 22, 17204−17214. (34) Hintze, C.; Bücker, D.; Domingo Köhler, S.; Jeschke, G.; Drescher, M. Laser-Induced Magnetic Dipole Spectroscopy. J. Phys. Chem. Lett. 2016, 7, 2204−2209. (35) Motion, C. L.; Lovett, J. E.; Bell, S.; Cassidy, S. L.; Cruickshank, P. A. S.; Bolton, D. R.; Hunter, R. I.; El Mkami, H.; Van Doorslaer, S.; Smith, G. M. DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR. J. Phys. Chem. Lett. 2016, 7, 1411−1415. (36) Giannoulis, A.; Oranges, M.; Bode, B. E. Monitoring Complex Formation by Relaxation-Induced Pulse Electron Paramagnetic Resonance Distance Measurements. ChemPhysChem 2017, 18, 2318−2321. (37) Fehr, N.; Dietz, C.; Polyhach, Y.; von Hagens, T.; Jeschke, G.; Paulsen, H. Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR. J. Biol. Chem. 2015, 290, 26007−26020. (38) Fleissner, M. R.; Brustad, E. M.; Kalai, T.; Altenbach, C.; Cascio, D.; Peters, F. B.; Hideg, K.; Peuker, S.; Schultz, P. G.; Hubbell, W. L. Site-Directed Spin Labeling of a Genetically Encoded Unnatural Amino Acid. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21637−21642. (39) Kucher, S.; Korneev, S.; Tyagi, S.; Apfelbaum, R.; Grohmann, D.; Lemke, E. A.; Klare, J. P.; Steinhoff, H. J.; Klose, D. Orthogonal Spin Labeling Using Click Chemistry for In Vitro and In Vivo Applications. J. Magn. Reson. 2017, 275, 38−45.

(40) Schmidt, M. J.; Borbas, J.; Drescher, M.; Summerer, D. A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements. J. Am. Chem. Soc. 2014, 136, 1238−1241. (41) Schmidt, M. J.; Fedoseev, A.; Bücker, D.; Borbas, J.; Peter, C.; Drescher, M.; Summerer, D. EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels. ACS Chem. Biol. 2015, 10, 2764−2771. (42) Abdelkader, E. H.; Feintuch, A.; Yao, X.; Adams, L. A.; Aurelio, L.; Graham, B.; Goldfarb, D.; Otting, G. Protein Conformation by EPR Spectroscopy Using Gadolinium Tags Clicked to Genetically Encoded p-Azido-L-Phenylalanine. Chem. Commun. 2015, 51, 15898− 15901. (43) Mileo, E.; Etienne, E.; Martinho, M.; Lebrun, R.; Roubaud, V.; Tordo, P.; Gontero, B.; Guigliarelli, B.; Marque, S. R. A.; Belle, V. Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective SpinLabeling of Tyrosine Using an Isoindoline-Based Nitroxide. Bioconjugate Chem. 2013, 24, 1110−1117. (44) Lorenzi, M.; Puppo, C.; Lebrun, R.; Lignon, S.; Roubaud, V.; Martinho, M.; Mileo, E.; Tordo, P.; Marque, S. R.; Gontero, B.; Guigliarelli, B.; Belle, V. Tyrosine-Targeted Spin Labeling and EPR Spectroscopy: an Alternative Strategy for Studying Structural Transitions in Proteins. Angew. Chem., Int. Ed. 2011, 50, 9108−9111. (45) Oberstrass, F. C.; Auweter, S. D.; Erat, M.; Hargous, Y.; Henning, A.; Wenter, P.; Reymond, L.; Amir-Ahmady, B.; Pitsch, S.; Black, D. L.; Allain, F. H. Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation. Science 2005, 309, 2054−2057. (46) Vitali, F.; Henning, A.; Oberstrass, F. C.; Hargous, Y.; Auweter, S. D.; Erat, M.; Allain, F. H. Structure of the Two Most C-Terminal RNA Recognition Motifs of PTB Using Segmental Isotope Labeling. EMBO J. 2006, 25, 150−162. (47) Polyhach, Y.; Bordignon, E.; Jeschke, G. Rotamer Libraries of Spin Labelled Cysteines for Protein Studies. Phys. Chem. Chem. Phys. 2011, 13, 2356−2366. (48) Jeschke, G. MMM: A toolbox for Integrative Structure Modeling. Protein Sci. 2017, DOI: 10.1002/pro.3269. (49) Jeschke, G.; Chechik, V.; Ionita, P.; Godt, A.; Zimmermann, H.; Banham, J.; Timmel, C. R.; Hilger, D.; Jung, H. DeerAnalysis2006 - a Comprehensive Software Package for Analyzing Pulsed ELDOR Data. Appl. Magn. Reson. 2006, 30, 473−498. (50) Yagi, H.; Banerjee, D.; Graham, B.; Huber, T.; Goldfarb, D.; Otting, G. Gadolinium Tagging for High-Precision Measurements of 6 nm Distances in Protein Assemblies by EPR. J. Am. Chem. Soc. 2011, 133, 10418−10421. (51) Polyhach, Y.; Bordignon, E.; Tschaggelar, R.; Gandra, S.; Godt, A.; Jeschke, G. High Sensitivity and Versatility of the DEER Experiment on Nitroxide Radical Pairs at Q-band Frequencies. Phys. Chem. Chem. Phys. 2012, 14, 10762−10773.

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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for

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