High Sensitivity In-Cell EPR Distance Measurements on Proteins

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

High Sensitivity In-Cell EPR Distance Measurements on Proteins Using an Optimized Gd(III) Spin Label Yin Yang, Feng Yang, Yan-Jun Gong, Thorsten Bahrenberg, Akiva Feintuch, Xun-Cheng Su, and Daniella Goldfarb J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b02663 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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The Journal of Physical Chemistry Letters

High Sensitivity In-Cell EPR Distance Measurements on Proteins using an Optimized Gd(III) Spin label

Yin Yan1#, Feng Yang]#, Yan-Jun Gong2, Thorsten Bahrenberg1, Akiva Feintuch1, 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

ACS Paragon Plus Environment

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Abstract

Distance measurements by electron-electron double resonance (DEER) carried out on spinlabeled proteins delivered into cells provide new insights into the conformational states of proteins in their native environment. Such measurements depend on spin labels that exhibit high redox stability and high DEER sensitivity. Here we present a new Gd(III)-based spin label, BrPSPy-DO3A-Gd(III), which was derived from an earlier label, BrPSPy-DO3MA-Gd(III), by removing the methyl group from the methyl acetate pending arms. The small chemical modification led to a reduction in the zero-field splitting and to a significant increase in the phase memory time, which, together, culminated in a remarkable improvement of in-cell DEER sensitivity, while maintaining the high-distance resolution. The excellent performance of BrPSPy-DO3A-Gd(III) in in-cell DEER measurement was demonstrated on doubly labeled ubiquitin and GB1 delivered into HeLa cells by electroporation.

TOC GRAPHICS

KEYWORDS Gd(III) tag• DEER • in-cell structure• spin labels • ubiquitin• GB1


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Distance measurements between two well-defined locations within a protein can provide information on its conformation and the changes it undergoes upon ligand binding or its interactions with other proteins and nucleic acids. Such measurements, carried out by pulsedipolar (PD) EPR spectroscopy, employing mainly nitroxide spin labels, are currently routine. These measurements are usually carried out in frozen solutions under well-controlled conditions (e.g., isolated protein, pH and concentration). These conditions, however, differ significantly from those the protein experiences in its native environment, the cell. Therefore, in the last decade there have been considerable efforts to develop this methodology further to allow for incell structural measurements. In-cell measurements are considerably more challenging than their in vitro counterparts, both in terms of the labeling chemistry and the applied spectroscopic methodology. It requires the use of redox stable spin labels, protein conjugation chemistry that is resistant to the reducing environment of the cell, efficient methods of delivering the protein into the cell, and high sensitivity, which allows measurements at protein concentrations approaching physiological concentrations. The first in-cell distance measurements were carried out by DEER (double electron-electron resonance, also called PELDOR) and employing the abundantly used nitroxide spin label, which is reduced in the cell and becomes diamagnetic.1-3 To overcome this difficulty, a new stable redox nitroxide was designed and demonstrated in in-cell DEER measurements after injection into Xenopous oocytes.4 A trityl-based radical has been proposed as well and demonstrated in Fe(III)-trityl distance measurements in Xenopous oocytes.5 The use of Gd(III)-based spin labels is an attractive option, considering its high chemical stability and its high EPR sensitivity at W-band frequencies (~95 GHz).6-7 Moreover, the scope of the chemical modification of relevant Gd(III) tags is high8-14 and should lead to the rational design of Gd(III)


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tags optimized for the most efficient in-cell distance measurements in terms of both in-cell concentration sensitivity and distance resolution.

Figure 1. The chemical structures of the Gd(III) tags studied.

The first in-cell Gd(III)-Gd(III) DEER measurements on a protein delivered into HeLa cells used Gd(III)-maleimide DOTA (DOTA-M) as a spin label.15-16 This tag is characterized by a narrow central transition because of its small zero-field splitting (ZFS, D~650 MHz17) and consequently, high sensitivity, but its linker to the protein is rather flexible and this compromises the distance resolution. A Gd(III)-PyMTA tag18, with a larger ZFS (D~ 1150-1213 MHz17, 19), was used to label a model peptide injected into oocytes.20 A different approach utilized the incell production of a helical bundle peptide fused at both the N- and C-termini with a Gd(III) binding peptide and Gd(III) is supplemented through the growing media.21 While this is an attractive approach, the low binding constant of Gd(III) and the large excess of free Gd(III) limits the SNR. Moreover, the excess of free Gd(III) can potentially affect the cellular metal ion homeostasis.


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DOTA type Gd(III) tags are particularly attractive for in-cell DEER applications because of their very large Gd(III) binding constant.22 Recently we have introduced the BrPSPy-DO3MAGd(III) tag, which features a DOTA-like moiety and a thiol reactive group, 3-bromophenylsulfonylpyridine, which resulted in a very stable and rigid Gd(III) spin label for in-cell DEER measurements (Fig. 1)9. Using this tag, we reported Gd(III)-Gd(III) DEER distance measurements up to 12 h after delivery by either electroporation or hypotonic swelling into HeLa cells. Although these results are exciting, the ZFS of this Gd(III) tag is large (D~1450 MHz) and therefore limits its sensitivity. It is known that DOTA-like-Gd(III) complexes present several isomers mainly in the square antiprism (SAP) and twisted square antiprism (TSAP) configurations.23-24 The rotation about the linker to the protein in a DOTA-like Gd(III) complex in solution can be hindered by additional substitution groups in the acetate arm.24-25 The restricted rotation in the methyl acetate arm results in one dominant isomer in solution, as demonstrated for the protein-BrPy-DO3MALn(III) conjugates by NMR spectroscopy.26 The extent of the paramagnetic effects of lanthanide tags on the NMR spectrum correlates well with the tag-dependent contribution to the DEERdetermined distance distribution, since both depend on the tag’s flexibility. We showed that a rigid paramagnetic tag optimized by NMR offers narrower DEER distance distributions.27 We contend that DEER is less sensitive to the conformational heterogeneity of the paramagnetic tag than is paramagnetic NMR, especially for the protein-DOTA-like tags.28 Therefore, one would expect that replacing the methyl group with a hydrogen, generating the BrPSPy-DO3A tag (Fig. 1), would increase the arm’s rotational flexibility, on the one hand, but would decrease the proton density close to paramagnetic Gd(III), on the other. This should, in turn, increase the phase memory of Gd(III), which will increase DEER’s sensitivity.


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Figure 2. In vitro measurements (at 10 K) of ubiquitin D39C/E64C (50 µM) labeled with the different Gd(III) tags listed in the Figure 1. (a) The central transition region of the W-band EDEPR spectra. (b) Two-pulse echo decay of all tags listed in the figure. BrPy-DO3MA-Gd(III) and BrPy-DO3A-Gd(III) are labeled on the figure as DO3MA-Gd and DO3A-Gd respectively. In the present work, we demonstrated the excellent performance of the new BrPSPy-DO3A tag in in-cell DEER measurements using both doubly labeled ubiquitin D39C/E64C and immunoglobulin G-binding protein 1 (GB1) T11C/V21C and compared the results to those of BrPSPy-DO3MA-Gd(III). We report that by just removing the methyl group from the acetate arms, it is possible to increase considerably the sensitivity of in vitro and in-cell W-band DEER distance measurements. This resulted in an expected increase in the phase memory time and in a less expected reduction in ZFS, manifested by an increase of the EPR signal intensity and the modulation depth. These two effects led to a total increase in the DEER SNR by a factor of about 4, compared with BrPSPy-DO3MA-Gd(III) without compromising the distance resolution or the chemical reactivity needed for high labeling efficiency.29-30 A further increase in SNR by a factor of ~2, compared with standard rectangular pulses, was obtained by applying chirped pump pulses.29-30. Figure 2a compares the echo-detected EPR spectra of ubiquitin D39C/E64C labeled with DOTA-M-Gd(III), BrPy-DO3MA-Gd(III), and BrPy-DO3A-Gd(III) (for experimental details regarding tag synthesis and sample preparation, see the supplementary information). The reduced


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linewidth of BrPy-DO3A-Gd(III) is remarkable, considering that only a methyl group has been removed from the acetate arm. This reduction is associated with a decreased D value from 1450 MHz to 1150 MHz (see simulations in Fig. S2). The narrowest linewidth is reported for DOTAM-Gd(III). Figure 2b compares the echo decays measured at the maxima of the EPR spectra, showing that the removal of the methyl groups has lengthened the phase memory time (from 2.9 µs to 4.2 µs), which is manifested by an increased echo intensity by a factor of about two for τ values relevant for DEER measurements. The effect of methyl groups’ rotation and tunneling on phase relaxation has been documented in the past for other systems.31 This behavior was observed for GB1 T11C/V21C mutant as well (see Fig. S2).

Figure 3. In vitro W-band chirp-pulse DEER data (10 K) after background correction (left) and the distance distributions derived from these traces (right) of (a) ubiquitin D39C/E64C (50 µM) and (b) GB1 T11C/V21C (50 µM) labeled with the tags noted in the figure. The corresponding primary DEER data, along with the background correction, Fourier transforms and distance distribution validations are shown in Fig. S3. BrPy-DO3MA-Gd(III) and BrPy-DO3A-Gd(III) are labeled on the figure as DO3MA-Gd(III) and DO3A-Gd(III) respectively.


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The results of DEER measurements with a shaped pump pulse on ubiquitin D39C/E64C labeled with the three different tags are shown in Fig. 3a. The modulation depth with BrPyDO3MA-Gd(III) is 1.5%, whereas for BrPy-DO3A-Gd(III) it increases to 4% and for DOTA-MGd(III) it is 5% (all measured under the same conditions). The distance distributions reported by BrPy-DO3A-Gd(III) and BrPy-DO3MA-Gd(III) are similar, indicating that contributions from the pseudo-secular terms of the dipolar interactions19, 32 are small for BrPy-DO3A-Gd(III). The distance distribution obtained with the DOTA-M-Gd(III) tag is significantly broader, as expected. Here there could also be contributions to the width owing to neglecting the pseudosecular terms in the data analysis.19, 32 The experimental details for the chirp DEER can be found in the experimental section, and a comparison with standard four-pulse DEER is given in the SI, Fig. S4. The DEER SNR improved by a factor of 4.5 for BrPy-DO3A-Gd(III) compared with BrPy-DO3MA-Gd(III) (see SI, Table S1). Figure 3b reveals the same behavior for GB1 T11C/V21C labeled with BrPy-DO3A-Gd(III) and BrPy-DO3MA-Gd(III). We also carried out RIDME measurements on these samples to verify the extent of broadening due to the contribution of the pseudo-secular dipolar terms,33 and only a slight broadening was detected (see Figs. S5-S7). We found that the DEER data have a better quality than the RIDME data, particularly because of the very strong background decay and the appearance of the multiple quantum harmonics in the distance distribution.34 Although the harmonics can be removed by introducing more free parameters to the data analysis, we preferred not to do so.35,36


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Figure 4. In-cell W-band echo-detected EPR spectra (a,d), chirp-pulse DEER data after background correction (b,e) and the distance distributions derived from these traces (c,f) of ubiquitin D39C/E64C labeled with BrPy-DO3A-Gd(III) (top row) and GB1 T11C/V21C labeled with BrPy-DO3A-Gd(III) (bottom) for cells frozen 5 and 20 h after delivery. The blue traces in b) and c) correspond to ubiquitin D39C/E64C labeled with BrPy-DO3MA-Gd(III) (labeled as .DO3MA on the figure). All measurements were carried out at 10K. The corresponding primary DEER data, along with the background correction, Fourier transforms and distance distribution validations are shown in Fig. S8 and S9. Ubiquitin D39C/E64C-BrPy-DO3A-Gd(III) and GB1 T11C/V21C-BrPy-DO3A-Gd(III) were delivered into HeLa cells using electroporation, following earlier published protocols.15 After delivery of the protein the cells were incubated 5 hours or 20 hours at 37 °C and subsequently frozen. The resulting ED-EPR spectra, presented in Figs. 4a and 4d, show the significant contribution of endogenous Mn(II), whose relative contribution increases with time. This indicates some loss of the delivered protein, as we reported earlier.9 Considering the Mn(II) signal as an internal standard, one can see that the in-cell concentration of delivered ubiquitin is higher than that of GB1 because of the lower GB1 concentration in the media (0.25 mM vs 0.15 mM respectively). High-quality DEER data were obtained for both proteins, revealing a decrease in the modulation depth with time, which is also lower than that of in vitro measurements. This is attributed to the overlap of the Gd(III) signal with the endogenous Mn(II) signal. We observed


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no significant differences in the distance distribution of the in vitro samples and the in-cell samples 5 h and 20 h after cell delivery for both proteins. These results indicate that the overall structural folds of the proteins do not change significantly in the cell, consistent with earlier measurements.9,

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Similar to the in vitro results, the improvement in the in-cell distance

measurements achieved by just removing the methyl groups is remarkable, as depicted in Figs. 4b and 4e. Based on the ED-EPR signal intensity, we estimated the in-cell protein concentration of the delivered BrPy-DO3A-Gd(III)-labeled GB1 to be around 5 µM and 3 µM after incubation at 37oC for 5 and 20 h, respectively, following cell delivery (see Fig. S10 and Table S1). To conclude, in this work we showed that by a small chemical modification, i.e., removal of a methyl group from the acetate pending arms of the cyclene ring, we were able to achieve a ~4 fold SNR improvement while maintaining the narrow distance distribution. This improvement is due to an increase in modulation depth, combined with the increased EPR intensity, because of the reduced ZFS and the longer phase memory. Additional sensitivity improvement, by a factor of ~2, was obtained by the use of chirped DEER, allowing measurements on 3 µM in-cell protein concentrations, which approaches many native protein concentrations under physiological conditions. The coordination of the pyridine nitrogen to the lanthanide ion maintains a rigid protein-tag conjugate.37 This singles out BrPSPy-DO3A-Gd(III) as currently the best spin label for in-cell tracking of a protein’s structural dynamics with high-distance resolution and sensitivity. The methyl groups in BrPSPy-DO3MA-Ln play a major role in the high performance of this tag in paramagnetic NMR measurements because they restrict the arm rotation and lead to one dominant paramagnetic species in solution at ambient temperatures that generates large pseudocontact shifts (PCSs).9 In contrast, the existence of multiple tag isomers is transparent for DEER measurements carried out at low temperatures, as long as the Gd(III) location with respect


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to the protein is highly localized, and indeed, the removal of methyl groups turned out to be crucial for improving DEER sensitivity without compromising the distance resolution. We do not understand, however, why the ZFS is reduced; it could be a steric and/or electronic effect. This highlights the importance of rational tag design aiming at achieving new tags with a longer phase memory time for further improvement of DEER sensitivity for in-cell applications. ASSOCIATED CONTENT Supporting Information. Synthesis of tag, experimental details, EPR measurement and comparison of in vitro and in-cell DEER results are provided. AUTHOR INFORMATION Corresponding author: E-mail: [email protected] and [email protected] Author contributions #

Y. Yang and F. Yang contributed equally.

ACKNOWLEDGMENT This work was supported by an Israel Science Foundation (ISF) - National Natural Science Foundation of China (NSFC) grant (grant number 118768, 21761142004) to X.C.S. and D. G, the Natural Science Foundation of China (21673122 and 21473095) to X. C. S. This research was made possible in part by the historic generosity of the Harold Perlman Family (D. G.). D. G. holds the Erich Klieger Professorial Chair in Chemical Physics. TB acknowledges financial support from the Minerva Foundation.


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