In-Cell EPR Distance Measurements on Ubiquitin Labeled with a

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In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag Yin Yang, Feng Yang, Xia-Yan Li, Xun-Cheng Su, and Daniella Goldfarb J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11442 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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The Journal of Physical Chemistry

In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag Yin Yang1, Feng Yang2, Xia-Yan Li2, Xun-Cheng Su2*, and Daniella Goldfarb1* 1 Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 76100, Israel, 2 State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China

Corresponding authors : Daniella Goldfarb , [email protected], Xun-Cheng Su, [email protected]

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Abstract Double electron-electron resonance (DEER) measures distances between spin labels attached at welldefined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS). Gd(III)Gd(III) DEER measurements were carried out on cells frozen after different incubation times following delivery to test the stability of the spin label inside the cell. For both delivery methods it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd(III) and considering the presence of Mn(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd(III)/Mn(II) exchange with endogenous Mn(II). These experiments also showed that the relative Gd(III)/Mn(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.


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Introduction Protein structure and dynamics and their modification by interactions with ligands, other proteins, and nucleic acids are often key factors delineating their functions. Currently, protein structure and dynamics are mostly evaluated in vitro, under conditions that are often dictated by the physical method applied to extract the information. These conditions differ considerably from the cellular milieu, where molecular crowding, sub-organelle localization, post-translational modifications, and specific and nonspecific associations with cellular components may inevitably affect the structure and conformational equilibria of proteins.1-2 Therefore, there is a growing interest in tracking protein conformation and dynamics inside live cells. EPR spectroscopy, in particular double electron-electron resonance (DEER)3is a method that can provide structural information on proteins. It comprises labeling the protein with two spin labels (usually identical) by site-directed spin labeling (SDSL) and measuring their dipolar interaction, which is inversely proportional to the cube of their inter-spin distance. The method is usually applicable to frozen solutions and generates distance distributions with atomic level accuracy and has the potential to become an efficient tool for in-cell structural studies of proteins. It can complement NMR when both are applicable4 or it can be used alone in the many cases where NMR is not applicable. The first proof-of-concept reports of in-cell DEER was by injecting a nitroxide labeled DNA or protein into oocytes.5-7 These pioneering early works used standard spin labels based on a tetramethyl 5-membered pyrroline ring, which is susceptible to reduction in the cellular environment and this leads to reduced sensitivity.6-9 This excludes cases where the nitroxide spin label is not exposed to the incell environment but rather, to its external environment.10-11 To overcome this stability problem new spin probes were designed, replacing the tetramethyl substitution with tetraethyl substitution

12

and

recently, in-cell DEER on a protein labeled with such a redox resistant nitroxide was reported after injection into oocytes.13

In the last few years trityl spin labels for nucleic acids and proteins have

been reported,14-17 and the first in-cell measurement on a trityl-Fe(III) pair has been demonstrated.18 Other attractive spin labels for in-cell DEER applications are based on Gd(III) complexes. They feature both chemical stability and high sensitivity when measured at high spectrometer frequencies (for example, W-band, 95 GHz).19-23 The chemical and spectroscopic properties of the Gd(III) label determine the efficiency of in-cell distance measurements. The ideal spin label should exhibit a 3 ACS Paragon Plus Environment


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chemically stable conjugation to the protein, a high binding constant to prevent leakage of Gd(III) from the label, a narrow central transition and a long phase memory time for sensitivity, and a rigid and short tether to the protein for high distance resolution. In case of the latter, the labeling site should be carefully chosen to ensure that this rigidity does not interfere with the protein structure. In-cell Gd(III)-Gd(III) DEER distance measurements were demonstrated on proteins labeled with maleimideDOTA-Gd(III)24-25 , BrPSPy-DO3MA-Gd(III)26 and BrPSPy-DO3A-Gd(III) 27 delivered into human HeLa cells by electroporation (EP) or hypotonic swelling (HS). A different approach is the in-situ production in bacterial cells of a protein fused with two lanthanide binding peptides and supply Gd(III) through the growing media.28 In addition, Gd(III) labeled protein can be delivered by injection into oocytes as reported for a 4-vinyl-PyMTA conjugated peptide.29 Recently a reactive Gd(III) labeling tag based on the PyMTA chelate, 4PS-PyMTA (see Fig. 1) with a redox stable and rigid tether30 has been introduced for in vitro Gd(III)-Gd(III) distance measurements.31 The advantage of the PyMTA-Gd(III) label stems from its somewhat smaller size compared to BrPSPy-DO3MA-Gd(III) (see Fig 1) or BrPSPy-DO3A-Gd(III), while a potential disadvantage is its lower binding constant.32-33 To further establish in-cell DEER as a method for tracking protein conformational changes in eukaryote cell lines we explored the performance of 4PSPyMTA-Gd(III) for in-cell DEER Gd(III)-Gd(III) distance measurements. We investigated the in-cell behavior of three doubly labeled ubiquitin mutants, G35C/E64C, D39C/E64C and L73C/E64C as a function of the time they spend in the cell after delivery by EP and HS before freezing the cells. We report that in-cell DEER measurements can be carried out up to 12 h after delivery using both delivery methods with an estimated in-cell labeled protein concentration as low as ~5 M, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, the behavior was different for the two delivery methods. In the case of EP the distance distributions for all three mutants were independent on the incubation time following delivery, whereas for HS one of the three mutants, G35C/E64C, reported shortening of the distance with increasing incubation time. Comparison with analogous in-cell distance measurements of G35C/E64C labeled with BrPy-DO3MA-Gd(III) and in vitro exchange measurements with Mn(II) suggest that this change arises from an exchange of some of the coordinated Gd(III) with endogenous Mn(II). Why this happens only with HS remains an open question. We conclude that while 4PS-PyMTA-Gd(III) is a viable label for in-cell measurements, 4 ACS Paragon Plus Environment

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caution should be exercised when a distance change is observed and the effect of exchange with endogenous Mn(II) need to be considered.

Figure 1. a) Chemical structures of the 4PS-PyMTA-Gd(III) and BrPSPy-DO3MA-Gd(III) tags (the leaving groups are indicated in red) and the space filling models of PyMTA-Gd(III) and BrPy-DO3MA-Gd(III). (C (grey), H (white), N (blue), O (red), Gd (black), S (yellow), Br (orange)). b) Ribbon structural representation of human ubiquitin (PDB code: 1UBI34) indicating the mutation sites for anchoring the paramagnetic tags at two sites (one red sphere and one blue sphere). c) The central transition region of the 10 K W-band ED-EPR spectrum of ubiquitin D39C/E64C-PyMTA-Gd(III) in vitro (dashed line) and in cells, frozen after 7 hour incubation following cell delivery by HS (black) and EP (red). The positions of the pump (1) and observer (2) frequencies in the DEER experiment are denoted by arrows.

Methods Protein preparation Three double mutations, G35C/E64C, D39C/E64C, and L73C/E64C from the human ubiquitin gene were prepared in a pET28a vector. E. coli BL21 (DE3) Rosetta was transformed for protein overexpression. Grown in 2 L LB medium at 37 °C until mid log phase, the bacterial cells were induced by addition of 0.5 mM IPTG for protein expression overnight at 25 °C. The bacterial cells were collected by centrifugation and then lysed by sonication. The target protein was purified by DEAE column (DEAE Sepharose FF, GE Healthcare Biosciences), followed by a size exclusion column 5 ACS Paragon Plus Environment


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(HiLoad 16/600 Superdex 75, GE Healthcare Biosciences). Pure ubiquitin fractions were determined by SDS-PAGE. Protein labelling The ubiquitin mutants were ligated with 4PS-PyMTA as previously reported.31 The protein-PyMTAGd(III) samples were prepared by mixing 10 equivalent of 4PS-PyMTA into the labelling mixture and 2.2 equivalents of Gd(NO3)3 in 20 mM MES buffer at pH 6.5. The excess of free tags and Gd(NO3)3 was removed by a desalting column PD10 (GE Healthcare Biosciences). BrPSPy-DO3MA-Gd(III) was labeled following the protocol previously described.26 The completion of the ligation reactions was confirmed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometry. For in vitro EPR measurement, the solution of spin-labeled proteins was lyophilized and then re-dissolved in D2O/glycerole-d8 (7/3 v/v). The final protein concentration was determined spectroscopically using the absorption at 280 nm. For labeling with ATTO488-maleimide (ATTO488M) (Sigma-Aldrich), 1.0 mL of a 1.0 mM solution of ubiquitin mutants was mixed with 3.0 mM ATTO488-M and 1.0 mM TCEP in 20 mM MES buffer at pH 6.5. The mixture was incubated at room temperature for 4 h. Unreacted fluorescence label was removed by size exclusion chromatography (HiLoad 16/60 Superdex 30, GE Healthcare Biosciences). Cell extract preparation HeLa cell extract was prepared as described previously.35 The cells were split into 16 culture dishes 24 h before preparing the extracts. During this time the cells grew exponentially, reaching 60–70% confluency. Then the HeLa cells were detached with a cell scraper, washed 2 × with 10 mL of ice-cold PBS (100 mM, pH 7.2), and sedimented by centrifugation at 300xg for 5 min. The cell pellet was resuspended in 500 µL of cell lysis buffer (PBS in D2O, supplemented with 150 mM NaCl, 1% NP-40, protease inhibitor cocktail III (Calbiochem™), pD 7.2) and then incubated on ice for 15 min. The cell extract was snap-frozen and stored in liquid nitrogen until use. Cells and extracts were kept on ice during all stages of preparation. G35C/E64C-PyMTA-Gd(III) was added such that its bulk concentration in the sample was 100 μM and the sample contained extract from about 50,000 cell/μL. This amounts to about 1 mM protein per cell. At this stage glycerol-d8 was added to yield a 20% v/v solution. The samples were incubated under the same conditions as for the living cell and for different times. 6 ACS Paragon Plus Environment

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Delivery of labeled ubiquitin by HS The protein delivery followed the procedure reported earlier.24 HeLa cells were suspended in 200 μL of PBS hypotonic solution (90 mOsm, pH 7.2) containing 0.1 mM labeled ubiquitin complexes and incubated at 37 °C for 60 min. Next, 200 μL PBS solution (480 mOsm, pH 7.2) was added to restore the isotonic conditions (280 mOsm) and incubated at 37 °C for another 60 min. The osmolarity of the solutions was monitored with a Vitech Scientific 3300 Advanced Micro Osmometer. The cell volume is fully recovered in 10-15 min after the washout of the hypotonic solution. 24, 36-37 The cells were then transferred onto collagen-treated dishes and incubated at 37 °C for different times. Thereafter, the cells were detached from culture dishes with trypsin/EDTA (0.05%/0.02%), centrifuged at 1000xg for 5 min at 25 °C, washed 2 × in PBS buffer to remove the non-internalized protein and dead cells, and then incubated at 37 °C for 15 min in PBS-D2O buffer. Finally, they were washed and incubated for 10 min in PBS buffer in D2O and glycerol-d8 (7/3 v/v). To increase the incell echo decay rate, which is crucial for DEER measurements, the HeLa cells were washed well in 90% deuterated buffer before freezing. This results in an echo decay rate that is not considerably shorter than that of a 100 μM solution of labeled protein in vitro, as can be seen for D39C/E64C-PyMTAGd(III). The cells were counted on a haemocytometer (Ncells), placed in the EPR capillary (quartz, 0.6 mm I.D.x0.8 mm O.D.) centrifuged at 1500xg for 30 min. The final volume of the cell pellet inside the EPR capillary after centrifugation was recorded (Vsample). And then the capillary was cut to a size that fits the EPR probe-head (~ 1.5-2 cm) and then slowly frozen in an isopropanol rack at −80 °CIn the case that Western blots were carried out, the cell sample was halved before the last centrifugation, half of the cells were for Western blots and the other half for the EPR.

Delivery of labeled ubiquitin by EP. The protein delivery followed the procedure reported earlier.4 Briefly, HeLa cells were suspended in 100 μL of PBS electroporation buffer (100 mM sodium phosphate, 5 mM KCl, 15 mM MgCl2, 15 mM HEPES, 2 mM ATP, 2 mM reduced glutathione, pH 7.4) containing 0.25 mM labeled ubiquitin complex, and then transferred into a 2 mm cuvette and electroporated by Nucleofector™ 2b (Lonza) with pulse program B28 for HeLa cells given by Lonza. The cells were then transferred onto collagen7 ACS Paragon Plus Environment


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treated dishes and incubated at 37 °C for different times. EPR samples were prepared following the same procedure for HS. The minimum recovery time of the cells after EP is reported to be 4 h based on visualization of attachment and cell spreading. 4,38 Western blots Western blot (WB) experiments were used to quantify the intracellular ubiquitin levels.31 HeLa cells were lysed in 50 µL lysis buffer (20 mM Tris buffer pH 7.4, 100 mM NaCl, 2 mM EDTA, 1% NP40) supplemented with protease inhibitor cocktail III (Calbiochem™). The number of cells used are listed in Table S2. After incubation on ice for 10 min, a soluble extract was obtained by centrifugation at 10 000xg for 10 min at 4 °C. The lysate was boiled in SDS loading buffer and resolved by SDS/PAGE. The calibration series of 20-1000 ng of recombinant ubiquitin were run on the same gel to determine the average amount of ubiquitin in cells. Immunoblot analysis was performed with rabbit polyclonal anti-ubiquitin (ABR-PA511325, Thermo Fisher Scientific) and goat anti-rabbit IgG (H+L) secondary antibody (DyLight 488, Thermo Fisher Scientific). The immunofluorescence was visualized with Typhoon FLA 9500.

Mn(II) exchange in vitro experiments The 50 M solution of G35C/E64C-PyMTA-Gd(III) in D2O/glycerole-d8 (7/3 v/v) was mixed with 40 M MnCl2 and 100 M MnCl2, respectively. For the D39C/E64C-PyMTA-Gd(III) sample, a solution of 100 M labeled protein in D2O/glycerole-d8 (7/3 v/v) was mixed with 80 M MnCl2 and 200 M MnCl2, respectively. For the BrPy-DO3MA-Gd(III) labeled G35C/E64C and D39C/E64C samples, a solution of 50 M labeled protein in D2O/glycerole-d8 (7/3 v/v) was mixed with 100 M MnCl2, respectively. A stock solution of 10 mM MnCl2 in H2O was used. EPR measurement All EPR measurements were carried out on a home-built W-band spectrometer (94.9 GHz) at 10 K.39-40 W-band echo-detected EPR (ED-EPR) spectra were recorded using π/2 and π pulse durations of 15 and 30 ns, respectively, with an echo delay of 550 ns and a repetition time of 1 ms. Echo decays were measured by a Hahn echo sequence (π/2-τ-π-τ-echo) by setting the magnetic field to the maximum of the ED-EPR spectra. DEER measurements were recorded using the standard four-pulse DEER sequence.41 The pump pulse 8 ACS Paragon Plus Environment

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duration was 15 ns, and the observer pulse durations were 15 and 30 ns, respectively. The frequency difference between the pump (1) and observer pulses (2) was 100 MHz, with the pump pulse set to the maximum of the EPR spectrum, as shown in Fig. 1c. The delay time, τ, was 400 ns, the step of t (pump pulse position) was 20 ns, and the repetition time was 800 μs. An eight-step phase cycle was employed to remove the instrumental artefacts and to compensate for DC offset. The accumulation time ranged from 1 to 6 h for in vitro measurements, and 10 to 20 h for in-cell DEER measurements. Chirp-pulse DEER measurements were recorded using a modified 4-pulse DEER sequence and the details on the general setup have been reported earlier.27 The observer pulse was set to the maximum of the EPR spectrum, whereas the /2 and  pulse durations were 15 and 30 ns, respectively. The pump pulse was set on both sides of the maximum of the central transition with a frequency offset of 100 MHz. The bandwidth of the chirp pump pulse was 300 MHz and the duration was 96 ns. The second delay time was 3 μs, the step of t was 20ns, and the repetition time was 800 μs. The accumulation time ranged from 12-14 h for in-cell DEER measurements. The DEER data were analyzed using the program DeerAnalysis 2015.42 Distance distributions were obtained using Tikhonov regularization with a regularization parameter of 100 for in vitro and 1000 for in-cell measurements, respectively. The validations were performed by varying the background start range from 15% to 80% of the primary time window in 15 trials.

Estimation of in-cell protein concentrations The ED-EPR spectra of HeLa cells without exogenous protein (SHe𝐿𝑎 (x)) and of 50 M G35C/E64CPyMTA-Gd(III) in solution (𝑆𝑣𝑖𝑡𝑟𝑜 (x)) were recorded. The ED-EPR spectra of G35C/E64C-PyMTAGd(III) in-cell samples (𝑆𝑒𝑥𝑝 (x)) were simulated as 𝑆𝑒𝑥𝑝(𝑥) = 𝑎 ∗ 𝑆𝐻𝑒𝐿𝑎(𝑥) +𝑏 ∗ 𝑆𝑣𝑖𝑡𝑟𝑜(𝑥) with a and b as fitting parameters. The bulk concentration of labeled protein in the sample was estimated as Cbulk(EPR) =b*50 M (all samples occupies to full active volume of the cavity). The error in Cbulk(EPR) were estimated from the standard deviation of a calibration curve of G35C/E64C-PyMTA-Gd(III) (10200 M) in vitro assuming 100% labeling efficiency. The intracellular concentrations, Ccell(EPR) was first estimated by using the Mn(II) signal as an internal standard as described earlier26 : Ccell(EPR)=Cbulk(EPR)*VHeLa/(a*Vcell*NHeLa) where Vcell is the volume of a single HeLa cell taken as ~2,000 m3 43, VHeLa is the pellet volume of the HeLa cells 9 ACS Paragon Plus Environment


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sample without exogenous protein and NHeLa is the number of cells in EPR the capillary. Ccell(EPR) was also estimated as follows : Ccell(EPR)=Cbulk(EPR)*Vsample/(Vcell*Ncell) where Vsample is the volume of the cell pellet

and Ncell is the number of cells in the EPR capillary (Table S2). Finally,

Ccell(EPR) was calculated as the average of the results from above two methods and the difference between the two values was used to estimate the error. The Western blot experiments gave the amount of monomeric ubiquitin in the cells (Wsample) using a calibration curve prepared from purified recombinant ubiquitin. The intracellular ubiquitin concentrations (Ccells(WB)) were abstained as : Ccell(WB)= Wsample/(MW*Vcell*Ncell). For comparison with the EPR bulk concentration, we also estimated the bulk protein concentration from the Western blots according to : Cbulk(WB)= Wsample/(MW*Vsample). The volumes of the cell pellet and the cell number in the EPR capillary and the Western blots were the same (see Table S2). The error range of Ccell(WB) and Cbulk(WB) were estimated from the standard deviation of the calibration curve of the Western blot. The error in cell counting, estimated from the standard deviation of several measurements was estimated to be low, only 5% and was not taken into account. Additional error arises from the cell volume used, which was not taken into account. Accordingly, the errors in the incell concentrations may be underestimated. Immunofluorescence Microscopy For immunofluorescence imaging, delivery of ubiquitin-ATTO 488 was carried out by the HS and EP procedures used for spin-labeled ubiquitin. The cells were recovered in the incubator on collagentreated 25 mm cover slips for 1 h, 7 h, and 12 h, respectively. Cells were rinsed 3 × in PBS and then fixed in PBS containing 4% PFA for 15 min. After having been washed 3 × in PBS, coverslips were mounted with a drop of PBS and sealed with nail polish. Confocal images were taken using an automated Olympus microscope X83 with a 60× oil objective (Olympus, plan apo, 1.42 numerical aperture) coupled to a spinning disk confocal scanner (Yokogawa W1). A 488 nm laser (Toptica, 100 mW) was used for fluorescence excitation, and a green LED was used for brightfield imaging. The emission filter-sets used for brightfield and fluorescence images were identical (520/28, Chroma). Images were recorded on a Hamamatsu Flash4 camera.


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Results The EPR Spectra and echo decay rates. The in vitro EPR and DEER characteristics of 4PS-PyMTA-Gd(III)-labeled G35C/E64C, D39C/E64C, and L73C/E64C (see Fig. 1b) have been described recently.31 NMR measurements on the double labeled mutants showed that the mutations and labeling did not disturb the ubiquitin structure.31 In addition,

15N-HSQC

NMR spectra of

15N-labeled

wild type and G35C ubiquitin revealed chemical

shift changes at only a few residues close to G35C whereas the overall 15N-HSQC spectra remained unchanged, indicating that the mutation did not introduce obvious structural perturbation (Fig. S1, supplementary information). Furthermore, comparison of the 15N-HSQC NMR spectra of wild type ubiquitin and G35C conjugated to PyMTA-Y3+ suggests that also with the label the protein retains the overall structural fold, since no obvious chemical shift perturbations were observed. (see Fig. S2). The doubly PyMTA-Gd(III)-labeled ubiquitin mutants were delivered into HeLa cells by HS24 and EP4 and the spread of the protein within the cytosol was confirmed using fluorescence microscopy (see Fig. S3). The cells were incubated for different times after delivery and then they were frozen for various EPR measurements. The W-band echo-detected EPR (ED-EPR) spectra of ubiquitin D39C/E64C-PyMTA-Gd(III) delivered into HeLa cells, compared to the spectrum in vitro, is shown in Fig. 1c. The in-cell spectra are a superposition of the delivered Gd(III)-labeled protein and endogenous Mn(II). The Gd(III) central transition has the same width (120 MHz at half height) as in the in-vitro sample. The in-cell spectra of the other ubiquitin-PyMTA-Gd(III) mutants are similar and are shown in Fig. S4. Generally, the spectra exhibited reduced signal intensities of Gd(III) with increasing incubation time. This will be further discussed later. Echo decay rates are shown in Fig. S5 and phase memory times are listed in Table S1.

DEER measurements. DEER measurements were carried out on all three doubly PyMTA-Gd(III)-labeled ubiquitin mutants delivered into HeLa cells via EP and HS as a function of incubation time after delivery, prior to freezing. The DEER data after background removal and the derived distance distribution, are depicted in Fig. 2. The primary DEER data and distance uncertainties of these traces are presented in Figs. S611 ACS Paragon Plus Environment


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S8. In the case of delivery by EP (Fig. 2a-c), all three mutants show in-cell distance distributions that, within experimental uncertainty, do not reveal any significant change with time and are similar to those obtained in vitro.31

What does change with time is the modulation depth, λ. The in-cell samples

reveal a generally lower λ value than do the in vitro ones31 and this is further reduced as the incubation time increases. The reduction in  with time is attributed to the increasing relative contribution of the Mn(II) to the EPR signal as reported earlier26 and potential loss of Gd(III) from the protein-PyMTAGd(III) complex. Additionally, degradation of the labeled ubiquitin can generate singly labeled products, which will also lead to a decrease in λ.

Figure 2. W-band DEER results of PyMTA-Gd(III)-labeled ubiquitin mutants in HeLa cells frozen at different times after delivery by EP (a-c) and HS (d-f). (a, d) G35C/E64C-PyMTA-Gd(III). (b, e) D39C/E64C-PyMTAGd(III). (c, f) L73C/E64C-PyMTA-Gd(III). Background-corrected DEER traces are shown on the left with the fitted data (red trace) and the corresponding distance distributions obtained using DeerAnalysis42are shown on the right. The corresponding distance distributions of the in vitro samples are indicated in light gray for comparison. The traces in (b) was measured with chirp pump pulses for better SNR. The corresponding rectangular-pulse DEER results are shown in Fig. S7.

For HS delivery the D39C/E64C and L73C/E64C samples behave like their counterparts delivered by EP. No significant change in distance distribution over time was noted and there was a high similarity 12 ACS Paragon Plus Environment

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to the in vitro distance distribution (Fig. 2). Surprisingly, G35C/E64C-PyMTA-Gd(III) revealed a systematic shift in the distance distribution to smaller distances with time and therefore it was measured at more time points. A duplicate experiment performed with the same protein concentration (250 M) in the hypotonic media reported the same shift (Fig. S9). An additional experiment was carried out for the long 12 h time point at a lower protein concentration (100 M) in the media, again revealing the short distance (Fig. S10). In Fig. S11 we compare the normalized DEER form factors for G35C/E64C-PyMTA-Gd(III) ES and HS delivery (Fig. S11a,b, respectively) and the three repeats of the latter after 12 h; the shortening of the distance after 12 h can be recognized also in these traces. The reduction in  with time was observed for all samples delivered using HS. To determine whether the change in the distance distributions with time reported by G35C/E64CPyMTA-Gd(III) is tag related, we repeated the experiment using the BrPSPy-DO3MA-Gd(III) tag. This DOTA-like tag was tested earlier for in-cell distance measurements on the D39C/E64C mutant using these two delivery methods and no differences in the distance distribution with time were reported.26 We now extended these measurements to G35C/E64C-BrPy-DO3MA-Gd(III). The DEER measurements and the derived distance distributions are depicted in Fig. 3 and S12. We observed some small, non-systematic variations in the maxima of the distributions as a function of incubation time, which can be considered within experimental uncertainty, as suggested by the distance uncertainties shown in Fig. S12. All measurements showed some variations in the width of the distance distribution but considering the limited SNR of the in-cell data we considered these within experimental error. A reduction in λ with increasing incubation time is observed also for the BrPy-DO3MA-Gd(III)labeled samples, though to a lesser extent than for the PyMTA-Gd(III)-labeled samples. Note that in vitro, λ is lower than for PyMTA-Gd(III)31 for a similar labelling efficiency because of the broader EPR linewidth (180 MHz at half height for the central transition, compared with 120 MHz for PyMTA).



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Figure 3. W-band DEER results of ubiquitin G35C/E64C-BrPy-DO3MA-Gd(III) inside HeLa cells incubated for different times after delivery by EP (a-b) and HS (c-d). (a, c) Background-corrected DEER traces and the fitted trace. (b, d) The corresponding distance distributions obtained using DeerAnalysis. The DEER distance distribution of G35C/E64C-BrPy-DO3MA-Gd(III) in vitro is shown in light gray.

To further rationalize the different behavior of G35C/E64C-PyMTA-Gd(III) delivered with HS, we performed DEER measurements

in cell extracts (see Figs S13). Unlike the in-cell measurements, the

distance distributions of G35C/E64C-PyMTA-Gd(III) in cell extracts were the same as in vitro and did not change with incubation time. The significant reduction in  detected for in-cell samples was not observed either. In-cell concentration determination. We estimated the Gd(III) bulk concentration, Cbulk(EPR), in the cell samples and the in-cell concentration, Ccell(EPR), of G35C/E64C-PyMTA-Gd(III) from the EPR signal intensity (see details in the Methods section and Fig. S14). These concentrations are given as 0.5*[Gd(III)] considering that there are two Gd(III) per protein molecule. We note that the cells are extensively washed before freezing such that anything that is expelled out of the intact cells, be it the protein, its degradation products or released Gd(III), did not remain in the sample. Figure 4 shows that Cbulk(EPR) and Ccell(EPR) decrease as the incubation time increases for both HS and EP deliveries.

Naturally,

Ccell(EPR) > Cbulk(EPR ) because the cells occupy only part of the sample volume. While these results show that the amount of Gd(III) in the cell is reduced with time, it does not necessarily indicate a reduction in in-cell concentration of the delivered protein because, in principle, the Gd(III) can leak 14 ACS Paragon Plus Environment

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out of the labeling tag. To address this we determined the in-cell concentration of G35C/E64CPyMTA-Gd(III) also by Western blots (see Method section and Fig. S15).

Our Western blot

experiments had a sensitivity too low to detect native monomeric ubiquitin in HeLa cells and therefore all signals observed were due to the delivered protein (Fig. S15). The total amount of ubiquitin in Hela cells was reported to be 3.2  0.45 μg/mg protein44. Taking into account the volume of an Hela cell (2 pL)43 and that a Hela cell contains a total of 150 pg proteins45 we estimated the in-cell concentration of native ubiquitin (all forms) to be around 30 μM. Based on our Western blot calibration curve the concentration of free ubiquitin is lower than 2.6 μM. Figure 4 shows that for both HS and EP the in-cell and the sample bulk concentrations (Ccell(WB) and Cbulk(WB), respectively) of monomeric ubiquitin obtained from Western blots were reduced as the incubation time increased. Comparison of Gd(III) concentrations determined from EPR and the protein concentrations determined from Western blots reveal that Cbulk(WB) > Cbulk(EPR) and Ccell(WB) > Ccell(EPR). This implies that in addition to the reduction of the in-cell concentration of the delivered protein, some additional loss of Gd(III) from the PyMTA tag took place. Similar measurements carried out on G39C/E64C-BrPy-DO3MA-Gd(III) delivered into Hela cells with HS and EP did not reveal a loss of Gd(III) from the tag as no significant difference was reported between Ccell(WB) and Ccell(EPR)26). This is in agreement with the larger reduction in λ observed for PyMTA-Gd(III) as compared to BrPy-DO3MA-Gd(III). The reduction of the in-cell concentration of the delivered protein most probably arises from degradation46 and followed by expulsion from the cell. This in turn can generate protein fragments with only single labels that also contribute to the reduction in λ.

Figure 4. In-cell and bulk concentrations of ubiquitin G35C/E64C-PyMTA-Gd(III) determined by Western blots and EPR spectroscopy as a function of incubation time after delivery. a) EP. b) HS. 15 ACS Paragon Plus Environment


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The effect of Gd(III) exchange in the presence of Mn(II) We have seen in this work, as in previous ones26-27 , that with time the delivered protein concentration in the cell is reduced and the relative in-cell Gd(III)/Mn(II) concentration decreases. Therefore we have considered the potential effect of the presence of the Mn(II) on the DEER results. Obviously, the overlapping Gd(III) and Mn(II) signals will affect the modulation depth26 but Mn(II) could also exchange some of the coordinated Gd(III) if Mn(II) also has a high binding constant to PyMTA. Recently we have evaluated the potential of PyMTA-Mn(II) as a spin-label for distance measurements in proteins and compared the distance distributions obtained with Gd(III) and Mn(II) for the three ubiquitin mutants studied in the present work.31 For both G35C/E64C and D39C/E64C the Mn(II)Mn(II) distance was shorter and broader than their Gd(III)-Gd(III) counterparts. This was attributed to Mn(II)’s smaller size and lower coordination number, which does not restrict Mn(II) to a well-defined position within the chelate. The absence of a change in L73C/E64C was attributed to the relative orientation of the two labels, which affects the sensitivity of the distance to the location of Mn(II) within the chelate.31 In these measurement the labeled protein was loaded either with Gd(III) or Mn(II) but competition experiments were not carried out. To test the possibility of Mn(II)/Gd(III) exchange we carried out in vitro DEER measurements on G35C/E64C-PyMTA-Gd(III) and D39C/E64CPyMTA-Gd(III) in the presence of 0.4 and 1 equivalent of Mn(II) (see Figs. 5 and Fig. S16). For G35C/E64C-PyMTA-Gd(III) we observed a change in the distance distribution; 1 added equivalent Mn(II) caused a shifted of the maximum of the distance distribution to ~ 2.5 nm, similar to the HS incell measurements after 12h (see Fig. 2). Surprisingly, for D39C/E64C-PyMTA-Gd(III) the distance distribution remained unchanged upon the addition of Mn(II), consistent with the in-cell measurements. Since both mutants have labels at E64C we attribute the difference to a higher relative binding affinities of Gd(III)/Mn(II) to PyMTA in D39C compared to D35C due to different stabilizing effects excreted by nearby protein residues. To substantiate this further, we carried out additional atomic absorption measurements on the singly labeled mutants D39C-PyMTA and G35C-PyMTA, which were first treated with 1 eq. Gd(III), 1 eq. Mn(II), and 2 eq. EDTA, and then we washed out the excess of small molecules. We used atomic absorption spectrometry, since we could not quantify the loaded metal concentration with NMR methods. The results, given in Table S3, show that G35C-PyMTA contained a small but significant 16 ACS Paragon Plus Environment

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amount of Mn(II), whereas for D39C-PyMTA the Mn(II) amount was negligible, supporting the different binding affinities of G35C-PyMTA and D39C-PyMTA to Mn(II) and Gd(III). The small amounts of Mn(II) in both samples relative to Gd(III) are due to the binding competitions between Gd(III) and Mn(II) for PyMTA and the removal of free metal ions by addition of EDTA. These results are in agreement with the DEER data showing that the ratio of the Gd(III)/Mn(II) binding constant to PyMTA is larger for D39C-PyMTA than for G35C-PyMTA. We also carried out similar Mn(II) exchange experiments on G35C/E64C-BrPy-DO3MA-Gd(III) and D39C/E64C-BrPy-DO3MA-Gd(III), where no change in the distance was observed upon addition of Mn(II) into the samples (see Fig. S17). This is expected as the affinity of cyclen ligands to Gd(III) is much higher than the Mn(II).32-33

Figure 5. W-band EPR results of PyMTA-Gd(III) labeled ubiquitin mutants with different amount of MnCl2 in solution (no Mn(II) (black), 0.4 equivalent Mn(II) (red), 1 equivalent Mn(II) (blue)). (a-c) G35C/E64C. (d-f) D39C/E64C. (a, d) ED-EPR spectra of the central transition region. The positions of the pump (1) and observer (2) frequencies are denoted by arrows. (b, e) Background corrected DEER trace with the fitted trace (grey). (c, f) The corresponding distance distributions. Discussion In-cell distance measurements by EPR are still in the early stages of development and the search for a spin label exhibiting high distance resolution, chemical stability, and high sensitivity is ongoing. Similarly, the best method for producing or delivering the labeled protein into the cell has not yet been 17 ACS Paragon Plus Environment


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established. The main purpose of this work was to evaluate the performance of the 4PS-PyMTA-Gd(III) tag, with emphasis on the long-term stability in the cell, which is essential for following long-term processes in cells. The PyMTA-Gd(III) tag was reported to be stable in oocytes following the injection of a peptide, polyproline helix II, doubly labeled with 4-vinyl-PyMTA and Q-band DEER measurements carried out on samples frozen 5 min and 1 h after delivery.29 The 4PS-PyMTA tag presented in this work has higher reactivity towards cysteine and it features a more rigid linker to the protein, designed to increase distance resolution, as compared to 4-vinyl-PyMTA.

Here we focused

on HeLa cells and carried out DEER measurements as a function of the time the delivered protein spent in the cells before freezing. Two delivery methods, EP and HS were tested on three different ubiquitin mutants and we were able to obtain in-cell distance distributions up to 12 h after delivery by both methods, at in-cell Gd(III) concentrations as low as ~ 10 M (~ 5 M labeled protein). Our additional experimental observations can be summarized as follows: (i) A general reduction in the incell concentration of the delivered protein with incubation time for both delivery methods. This could be due to the cell proliferation and degradation/expulsion of the delivered ubiquitin from the cell. (ii) Estimation of the in-cell Gd(III) concentration by EPR as compared with the protein concentrations obtained from Western blots, along with a somewhat larger reduction of the modulation depth with time for PyMTA-Gd(III) as compared to BrPSPy-DO3MA-Gd(III) tag suggest that some Gd(III) leaks from the PyMTA tag. (iii) The in-cell samples of all mutants, except G35C/E64C-PyMTA-Gd(III) delivered by HS, showed distance distributions similar to the in vitro ones. (iv) Our most interesting and puzzling observation was the shortening of the in-cell distance distribution of G35C/E64CPyMTA-Gd(III) with time, when HS is applied. This change was neither observed for L73C/E64CPyMTA-Gd(III) and D39C/E64C-PyMTA-Gd(III) nor for G35C/E64C-BrPy-DO3MA-Gd(III). It is tempting to attribute the distance changes to a conformational change in the protein or as a sign of oligomerization, but we excluded this possibility because (i) the

change was not observed for

G35C/E64C-BrPy-DO3MA-Gd(III), which features the same linker to the protein, (ii) it was not reported by D39C/E64C-PyMTA-Gd(III), where residue D39, situated only 4 residues away from G35, is expected to be affected by the conformational change to some extent. Based on the in vitro Mn(II)/Gd(III) exchange experiments it is more likely that the distance distribution changes as a consequence of Mn(II) replacing some of the Gd(III) coordinated to PyMTA. 18 ACS Paragon Plus Environment

We hypothesize that

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as the in-cell concentration of the delivered protein decreases with time while the Mn(II) concentration remains constant, the exchange equilibrium between Mn(II) and Gd(III) favors Mn(II) as the incubation time increases. Consequently, whereas initially we indeed measured in-cell Gd(III)-Gd(III) distances, as time progressed Gd(III)-Mn(II) distances contributed, and after a long time some Mn(II)Mn(II) contributions may also be present. To provide an unambiguous evidence for this hypothesis, the delivered protein should be purified and subjected to EPR measurements showing the presence of Mn(II). Unfortunately this is not a realistic experiment as it would require a very large number of cells delivered with proteins. Moreover, it is not obvious that the chelated Mn(II) will survive the purification procedure. Similarly, the overlapping spectra of the Gd(III) and Mn(II) and the low SNR of in-cell DEER prevent spectral editing of the DEER data, such that Mn(II)-Gd(III) and Mn(II)-Mn(II) distance distribution can be distinguished, as this would requires placing the pump and observed pulses in the Mn(II) spectral region, which has low intensity. We also note that the spectrum of Mn(II)PyMTA is quite broad and therefore difficult to resolve under the sharp features of Mn(II) appearing the in-cell spectra.31 This hypothesis immediately raises the question of why this exchange is not observed for the other mutants. In the case of L73C/E64C-PyMTA there is no difference between the Mn(II)-Mn(II) and Gd(III)-Gd(III) distance distributions and therefore the exchange will not affect the distance distribution.31 In the case of D39C/E64C-PyMTA-Gd(III) a reasonable explanation would be that Gd(III) binding constant is considerably larger than that of Mn(II) and the exchange is negligible as observed in vitro. It is interesting that Gd(III)/Mn(II) binding constants depend on the labeling position and are affected by the protein structure. The question that remains open is why does the Mn(II) exchange take place with HS and not with EP? We do not know and can just swelling, which leads to an increase in the cell volume,

47-49

speculate that hypotonic

and the lower osmotic pressure in the

extracellular space, might affect metal homeostasis in cytoplasm that is tightly controlled under normal conditions. Our measurements were carried out on only one protein, ubiquitin, and only one mutant out of three, labeled with 4PS-PyMTA behaved anomalously when delivered with HS. Nonetheless, these results call for caution when this label is used for in-cell measurements. While its Gd(III) binding constant is sufficiently high for in vitro measurements it may not be for in-cell measurement, particularly because 19 ACS Paragon Plus Environment


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of the presence of endogenous Mn(II). Therefore a good practice would be to test the Mn(II)/Gd(III) exchange in vitro a priory, or refrain from using HS as a delivery method.

Conclusions In-cell Gd(III)-Gd(III) distance distribution of ubiquitin doubly labeled with PyMTA-Gd(III) can be carried out up to 12 h after delivery by EP and HS for in-cell protein concentrations as low as 5 M . The in-cell concentration of the delivered protein was reduced with time and some of the Gd(III) was found to leak out of the tag. The distance distribution of the three ubiquitin mutants studied did not show any significant dependence on time after delivery by EP and so did two mutants delivered by HS. This indicates no obvious conformational differences under in vitro and in-cell conditions. In contrast, for the G35C/E64C mutant we observed a

shortening of the distance over 12 hours after

delivery. Such a change was not observed for analogous experiments with the BrPy-DO3MA-Gd(III) label.

We hypothesized that this change occurs because of the exchange of Gd(III) coordinated to

PyMTA with endogenous Mn(II). This was supported by in vitro exchange experiments that showed the binding constant of the metal ion depends on the labeling position in the protein. The reason why this distance change occurs as a consequence of hypotonic swelling remains unknown We can conclude that delivery by EP is preferred for proteins labeled with PyMTA. Labeling with DO3MAGd(III) is immune to these effects because of the higher Gd(III) binding constant and both delivery methods can be applied. Finally, although the reason for the change observed in the in-cell DEER derived distances distributions is unlikely to reflect a conformational change, this observation is significant as it means that with this method it will be able to track conformational changes in cells.

Supporting information : NMR spectra, fluorescence imaging, ED-EPR spectra and echo –decay curves, primary DEER data, duplicate DEER measurements, primary DEER data of ubiquitin G35C/E64C-BrPy-DO3MA-Gd(III), ED-EPR spectra and DEER data in cell extracts, Simulations of ED-EPR spectra to extract the Gd(III) bulk concentration, Western blots,

Mn(II)/Gd(III) exchange

experiments for ubiquitin labeled with PS-PyMTA and BrPSPy-DO3MA in vitro. Acknowledgements 20 ACS Paragon Plus Environment

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In-Cell EPR Distance Measurements on Ubiquitin Labeled with a

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