Understanding how proteins adapt in their cellular environments is of immense interest in structural biology. The crowded environment inside cells can affect protein folding and stability. [1][2][3][4][5][6][7] For example, phosphoglycerate kinase (PGK) is more stable in zebrafish tissues 8 and human osteosarcoma cells 5 than in vitro. The increase in stability due to molecular crowding has also been observed with other proteins such as frataxin, 9 ubiquitin, 10 hen egg white lysozyme, 11 and calcineurin. 12 In contrast, the dimerization of the baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis is destabilized in vivo. 13 The destabilization effects are also seen in other protein dimers that are not spherical in shape. 14,15 These experiments are indicators that the in-cell environment modulates protein structure and function, which vary case-by-case. Overall, in-cell experiments are required to understand the behavior of proteins in the context of cellular function.
In this context, ESR has emerged as a widely applicable technique to measure dynamics and distance constraints in vitro and in-cell. For such ESR measurements, the normally diamagnetic proteins can be functionalized with a spin label using site-directed spin-labeling methodologies. [16][17][18][19] The combination of ESR and spin labeling enables the measurement of the dynamics at the labeled site 20,21 and the measurement of distances between the labeled sites of a protein. [22][23][24][25][26][27][28] Distance measurements have been particularly useful for shedding light on the changes in protein conformations, [29][30][31][32][33][34][35][36][37] the assembly of large complexes, [38][39][40][41] and the binding of substrates and metal ions. [42][43][44][45] Additionally, these distance measurements have been performed in-cell for proteins [46][47][48] and DNA. 49,50 The primary challenge for distance measurements in-cell is the reduction of spin labels within the highly reducing cytosolic environment. 51 An intriguing new strategy for in-cell measurements is the use of genetically encoded noncanonical amino acid technology as an in situ labeling strategy. [52][53][54][55] In particular, a photocaged radical amino acid can be incorporated into a protein during translation. 56 Only after the induction of light will the photocage be released to expose the nitroxide radical for ESR measurements. In addition to noncanonical amino acids, reduction-resistant spin labels such as sterically shielded nitroxides, 57,58 Gd(III)-based spin labels, [59][60][61] and triarylmethyls (TAMs, trityls) [62][63][64] have been developed.
Trityls have a lot of potential as a class of spin labels for several reasons. First, trityls are highly resistant to reduction in-cell due to the steric shielding of its radical. [65][66][67] Second, trityls have appreciable relaxation times even at physiological temperatures. 68 Third, trityls have a narrow spectral shape that leads to efficient excitation of the electrons and intense ESR signal. 69 Overall, trityl spin labels have proved suitable for distance measurements at physiological temperatures or in-cell. The most explored trityl spin labels are based on the Finland trityl radical (FT) shown in Figure 1A, which have successfully provided distance measurements at room temperature 70,71 and in-cell. 72,7372,73 However, FT-based spin label usage is still challenging due to the complications in the labeling process.
The spin-labeling process typically entails a reaction between the spin label and a cysteine residue to label the protein at a specific site. However, FT can bind nonspecifically to membranes 74 or proteins. 75 Additionally, FT tends to selfaggregate. 76,77 As a result, efficient labeling of FT requires extensive washing of proteins that are immobilized on a solid support 63 or maintaining FT concentration to be less than 30 μM throughout the process to minimize aggregation. 78 Even after the labeling process, the phase-memory relaxation time (T m ) of FT is significantly reduced upon protein binding, 63,78 which leads to a weaker signal. Additionally, the longitudinal relaxation time (T 1 ) is significantly long at temperatures that are typical for ESR distance measurements (≤50 K), 78 which leads to longer experimental time. Overall, the short T m and long T 1 of FT-based spin labels diminish the sensitivity gain from the efficient excitation of FT. As a result, FT's sensitivity for distance measurements is comparable to distance measurements using commercially available nitroxide spin label. 78 As an alternative, a hydrophilic trityl spin label, based on the OX063 radical shown in Figure 1A, has been recently developed that allows for a straightforward labeling procedure without nonspecific binding or aggregation. 79 Interestingly, deuterated OX063 (OX063-d 24 ) was reported to have the longest transversal relaxation time at 50 K to date (T m = 6.3 μs). 79 Additionally, OX063-d 24 has been shown to also have a sufficiently long phase-memory relaxation time even at 200 K (T m = 3 μs). 46 Because T 1 is generally shorter at higher temperatures, OX063-d 24 has the potential for highly sensitive distance measurements at temperatures higher than 50 K. Despite OX063-d 24 's improvement over FT, OX063-d 24 only utilized a methanethiosulfonate linker so far, which is a limiting factor for in-cell experiments. This linker labels a protein by forming disulfide bonds with cysteines, which can be cleaved inside cells. 48 On the other hand, a maleimide linker reacts with a cysteine to form a thioether bond, which is uncleavable under normal physiological conditions. 47 In response to the need of a hydrophilic trityl with an uncleavable linker, a hybrid of OX063 and short-linker maleimide (SLIM) 73 known as Ox-SLIM was recently developed (Figure 1A). 80 Unlike OX063, the trityl core of Ox-SLIM has one of its bisthioketalaryl moieties remain unhydroxylated to bear the short maleimide linker. The hydrophilicity of Ox-SLIM permitted the labeling efficiency of ∼85%. These results motivate the development of hydrophilic trityl labels with high labeling efficiency for in-cell distance measurements.
To increase the viability of OX063-d 24 -based spin labels, we have developed a new OX063-d 24 spin label with a maleimide linker (mOX063-d 24 ), as shown in Figure 1A. The maleimide linker allows for mOX063-d 24 to maintain its linkage with the protein in-cell. 81 We explored two aspects of mOX063-d 24 for distance measurements in proteins. First, we show how mOX063-d 24 provides highly sensitive distance measurements at temperatures higher than the typical ≤50 K in vitro. Second, we showcase the usage of mOX063-d 24 for experiments in-cell, specifically in Xenopus laevis oocytes. When exploring these two aspects, spin labeling and distance measurements were done on the immunoglobulin binding domain of protein G (GB1), 82 a 56-residue globular protein (Figure 1B).
Synthesis of mOX063-d 24 . OX063-d 24 trisodium salt (112 mg, 0.077 mmol, 1 equiv), synthesized using our previously reported protocols, 83 was dissolved in anhydrous dimethylformamide (DMF) (100 mL) under an argon atmosphere at room temperature. Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (28 mg, 0.054 mmol, 0.7 equiv) in DMF (1 mL) was added; the green solution turned into a red-brown colored solution. Then, N-(2aminoethyl)maleimide trifluoroacetate salt (23 mg, 0.09 mmol, 1.2 equiv) in DMF (1 mL) and N,N-diisopropylethylamine (DIEA) (26.8 μL, 1.4 mmol, 2 equiv) were added. The solution turned back to green. The reaction mixture was The Journal of Physical Chemistry B diluted 20× with deionized water and acidified to approximately pH∼2 with trifluoroacetic acid (TFA). The crude product was loaded into a C18 cartridge and purified by reverse-phase chromatography using a C18 column with a gradient of water/acetonitrile (both containing 0.1% TFA) 95/ 5 to 85/15. The purified product was freeze-dried, dissolved in water, titrated to pH = 7 with NaOH, and freeze-dried again to provide 48 mg (40%) of mOX063-d 24 as a disodium salt. The purity assessed by high-performance liquid chromatography (HPLC) reached >95%, as shown in Figure S1. HRMS characterization is shown in Figure S2.
GB1 Labeling Protocol. E15C/K28C GB1 expression and purification were performed, as previously described. 84 The GB1 mutant was reacted with tris(2-carboxyethyl)phosphine (TCEP) overnight at 4 °C to reduce any disulfide formation. To label the protein, GB1 was run through four 5 mL GE Healthcare Hitrap desalting columns, to remove any TCEP, directly into a solution of mOX063-d 24 . The final solution of 10:1 of mOX063-d 24 /GB1 was allowed to react overnight at 4 °C. The spin-labeled protein was concentrated using Sartorius VivaSpin Turbo 4 centrifugal filter units with a molecular weight cutoff of 5 kDa to remove the unreacted label. The final solution was prepared in PBS, pH 7.4. Concentration and labeling efficiencies were calculated from UV-vis measurement using a Nanodrop2000 Spectrophotometer from Thermo Scientific. The extinction coefficient of GB1 was obtained from the ProtParam tool (https://web. expasy.org/protparam/). Masses were measured by liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS, Bruker Micro TOF, Billerica, MA).
Cellular Extracts and Oocyte Microinjection. Oocytes were obtained from Carolina Biological Supplies. The cytosol was extracted following the previously published protocol. 85 The cytosol sample was prepared at 50 μL containing doubly labeled mOX063- All pulsed experiments were performed on a Bruker ElexSys E680 CW/FT X-band spectrometer equipped with a Bruker ER5106-QT2 resonator for Q-band and a 300 W amplifier. The temperature was controlled using an Oxford ITC503 temperature controller and an Oxford CF935 dynamic continuous-flow cryostat connected to an Oxford LLT 650 low-loss transfer tube. Echo decay experiments used a twopulse sequence, π/2-t-π, where t was increased by a step size of 8 ns for 1024 points. T m values were obtained by fitting the Echo decay results with a stretched exponential decay. The time point where the signal is 1/e of the original intensity is the reported T m value. Inversion recovery experiments followed a three-pulse sequence, π-T 1 -π/2-T 2 -π, where T 2 was 400 ns and T 1 was increased by a step size of 1 or 10 μs for 1024 points. The fitting of inversion recovery data is detailed in the Supporting Information. Double quantum coherence (DQC) 22,86 was performed at the field with the maximum signal intensity of mOX063-d 24 . The DQC experiments followed a six-pulse sequence, π/2-t p -π-t p -π/2-t 1 -π-t 1 -π/2-t 2 -π, where t p and t 2 were increased and decreased, respectively, by 10 ns for 136 points. The initial parameters were set as t p = 1.3 μs, t 1 = 50 ns, and t 2 = 1.5 μs. To remove the unwanted echo signal, the 64-step phase cycle was implemented. 22,23 The DQC time traces were then analyzed using DeerAnalysis 87 by Tikhonov regularization.
SNR was calculated from the raw DQC time traces using the previously published method. 88 In summary, the raw DQC time trace was fitted to a 5th-order polynomial. The fit was subtracted from the time trace to isolate the noise of the time trace. The noise was used by the software SnrCalculator to calculate the final SNR.
Our recent report of the synthesis of the OX063 triarylmethyl radical and its deuterated analogues OX063-d 24 83 enables the synthesis of OX063 derivatives such as spin labels. A short maleimide linker was conjugated to OX063-d 24 using the PyBOP peptide coupling reagent, as depicted in Scheme 1.
The Journal of Physical Chemistry B The mOX063-d 24 was isolated in 40% yield after purification on C18 alongside with 10% of the dimaleimide derivative.
We overexpressed and labeled E15C/K28C GB1 with mOX063-d 24 through a reaction between cysteine residues and the maleimide linker. The labeling reaction occurred by incubating E15C/K28C GB1 and mOX063-d 24 in PBS, pH 7.4, overnight. The solution was filtered through a centrifugal filter with a molecular weight cutoff of 5 kDa to remove free mOX063-d 24 . We first performed ESI-MS to confirm the covalent attachment of mOX063-d 24 to E15C/K28C GB1. The data is shown in Figure S3. The MS showed peaks corresponding to doubly labeled, singly labeled, and nonlabeled E15C/K28C GB1. This result is expected because of the detachment of the spin label during sample preparation in acidic conditions (trifluroacetic acid) for ESI-MS and has been reported before using a maleimide-linked FT. 78 The final product was characterized using UV-vis spectroscopy to assess spin-labeling efficiency. This data is shown in Figure 2. The spectrum features two distinctive peaks at 280 and 469 nm. Only mOX063-d 24 contributes toward the 469 nm peak, 89 while both mOX063-d 24 and GB1 contribute toward the 280 nm peak. The UV-vis spectrum was analyzed using the deconvolution method, as depicted in Figure 2, 78 which fits the mOX063-d 24 -GB1 spectrum using GB1's UVvis spectrum (dash-dotted line) and mOX063-d 24 's UV-vis spectrum (dotted line). The deconvolution allowed us to obtain the absorbance of GB1 at 280 nm (ε 280 = 9970 M -1 cm -1 ) and mOX063-d 24 at 469 nm (ε 469 = 16 000 M -1 cm -1 ), 89 which were used to calculate their concentrations. The final concentrations of GB1 and mOX063-d 24 in the sample are 82.0 and 161.8 μM, respectively. Therefore, the ratio of GB1:mOX063-d 24 purified is about 1:1.97. Overall, our UV-vis results indicate efficient mOX063-d 24 labeling of cysteines on GB1.
To further validate the labeling efficiency, the mOX063-d 24 -GB1 sample was characterized using CW-ESR at room temperature. Figure 3A,B shows the CW-ESR spectrum of mOX063-d 24 bound to GB1 and free mOX063-d 24 . The free mOX063-d 24 contained a superhyperfine interaction with the amide nitrogen (a N ∼ 220 mG) on the linker, depicted as a partially resolved triplet splitting of the ESR lineshape. This nitrogen hyperfine is consistent with the previously published trityls with 14 N-containing linkers. 79,90,91 After mOX063-d 24 reacted with GB1, the superhyperfine nitrogen was broadened and unresolved due to the slower tumbling rate upon protein binding, as seen in Figure 3B. 79 However, the tumbling rate after protein binding is still rapid enough to resolve the satellite 13 C peaks in the CW ESR spectrum of GB1-bound mOX063d 24 , as seen in Figure 3A. This behavior has been described in a previous report of OX063-d 24 spin label. 79 Spin counting of the mOX063-d 24 -GB1 CW ESR spectrum yields a spin concentration of 203 μM. Given the protein concentration of 106 μM, these results indicate a labeling efficiency of 95%, which agrees with the UV-vis data.
More importantly, the mOX063-d 24 -GB1 CW ESR spectrum can be fitted with a narrow single-component simulation without a broad component, commonly seen when using FT. 74,75,78,92 FT's broad component has been attributed to aggregated species of FT 76,77 and nonspecific binding in proteins 75,76,79,93 and membranes. 74 As a result, when using the simple spin-labeling workflow, FT had labeling efficiencies of 24-80% depending on the linker and protein. 67,78,79,92 On the other hand, mOX063-d 24 is highly soluble and does not bind nonspecifically. 79 Therefore, the hydrophilicity of mOX063-d 24 The Journal of Physical Chemistry B D allows ∼100% labeling efficiency using simple protein-labeling protocols. Additionally, the labeling efficiency of mOX063-d 24 is slightly improved from the previously developed hydrophilic trityl spin label, Ox-SLIM, which is reported to have 85% labeling efficiency. 80 Such differences in labeling efficiency could be due to the difference in maleimide linker length between mOX063-d 24 and Ox-SLIM and potentially to the differences in solvent accessibilities between the two sites in the two proteins.
Next, pulsed-ESR was used to measure the relaxation times of GB1-bound mOX063-d 24 since these are critical parameters that dictate the efficacy of the label in pulsed dipolar spectroscopy. These data were acquired at a spin concentration of 5 μM and the sample was prepared in 20 mM PBS buffer at pH 7.4 and contained 20% glycerol. The phase-memory relaxation time (T m ) was measured by echo decay experiments. The measured values of T m are listed in Table 1 and the data is shown in Figure S4A. The T m of GB1-bound mOX063-d 24 is 5.1, 4.3, or 3.6 μs at 80, 150, or 180 K, respectively. These relaxation measurements provided additional data points to the existing measurements from previous studies of OX063-based spin labels (cf. Table S1). For comparison, the T m value of 5.1 μs for GB1-bound mOX063-d 24 at 80 K is longer than the T m value of 1.6 μs for protein-bound FT at 80 K. 78 The longest reported T m of protein-bound FT is 2.9 μs at 50 K. 79 Increasing T m can increase the echo intensity for distance measurements and increase the range of feasible temperature of the experiment.
At higher temperatures, mOX063-d 24 also benefits from the shortening of T 1 . GB1-bound mOX063-d 24 has T 1 values of 1.98, 0.175, and 0.112 ms at 80, 150, and 180 K, respectively (Table 1 and Figure S4B,C). The mechanism for T 1 relaxation of trityl radicals as a function of temperature has been previously studied. 94 As T 1 gets shorter with increasing temperature, the amount of time required for GB1-bound mOX063-d 24 to completely relax becomes shorter, leading to a faster rate of repeating the measurement. For comparison, the T 1 value of 1.98 ms for GB1-bound mOX063-d 24 at 80 K listed in Table 1 is slightly longer than the T 1 value of 1.7 ms for protein-bound FT at 80 K. 78 However, distance measurements using FT are typically done at 50 K or lower, which has T 1 values of 6.3 ms or longer. 78 Therefore, distance measurement using mOX063-d 24 at higher temperature leads to more scans per unit of time than the distance measurement using FT at the typical temperature of 50 K. Consequently, we expect that distance measurements using mOX063-d 24 at higher temperatures benefit from a shorter T 1 .
To showcase the sensitivity of mOX063-d 24 , DQC experiments at 80 K or 150 K were performed on E15C/K28C GB1 doubly labeled by mOX063-d 24 , as shown in Figure 4A. The method to measure SNR is described in the methods section. The DQC time trace achieved sufficiently high SNR at 150 K within approximately 1.5 h of runtime (SNR = 20 min -1/2 ). On the other hand, at 80 K, even after 2 h of runtime, the DQC time trace (SNR = 7 min -1/2 ) is noisier than the 150 K DQC time trace. The higher SNR of 150 K DQC than the SNR of 80 K DQC can be rationalized by the following analysis of SNR for pulsed-ESR experiments 95
where T is the temperature and t tot is the amount of time the electron coherence evolves until the detection of the echo signal. The 1/T term in eq 1 is due to the Boltzmann factor. 96 Based on eq 1, the shorter T 1 = 0.175 ms at 150 K than the T 1 = 1.98 ms at 80 K ( The Journal of Physical Chemistry B higher than the SNR at 80 K. We can see the SNR improvement from the DQC echo comparison between 80 and 150 K, as shown in Figure S5. Overall, the gain in sensitivity due to T 1 was able to overcompensate the loss of echo intensity from the shortening of T m and the reduction of spin polarization. However, increasing the temperature further to 180 K causes the reduction in sensitivity due to reduced spin polarization and T m . For example, the sensitivity at 180 K is ∼79% of the sensitivity at 150 K based on eq 1. These comparisons signify the importance of experimentally evaluating the relaxation times at various temperatures, since the values can be different for different systems. While 80 K is not the most optimal temperature for mOX063-d 24 DQC, its SNR = 7 min -1/2 is comparable to the reported FT's SNR 78 ranging from 7 to 8.9 min -1/2 at 50 K. These comparisons of SNR exemplify the sensitivity gained from performing mOX063-d 24 DQC experiments at the optimal temperature. We analyzed the time traces using the DeerAnalysis 2018 87 package and the Tikhonov regularization method to extract the distance distributions shown in Figure 4B. Expectedly, at both temperatures, the distance distributions were close to identical, with the most probable distance of 3.6 nm. To predict the distance distribution, we built an in silico model using MTSSLWizard. 97 Since the mOX063-d 24 spin label does not exist in the MTSSLWizard package, we first implemented the mOX063-d 24 model into the MTSSLWizard software. Details are provided in Figure S6. The model predicted that the most probable distance is 3.8 nm, as shown in Figure 4B, which is in reasonable agreement with the DQC results. Furthermore, the experimental results have a standard deviation of ∼0.6 nm, which is on par with the standard deviation of ∼0.8 nm obtained using nitroxide on the same GB1 mutant. 85 After the in vitro experiments, the viability of mOX063-d 24 for in-cell experiments was explored. Specifically, the mOX063d 24 -GB1 (200 μM of spins) was subjected to either 10 times excess of ascorbic acid or the cytosol extract of X. laevis (African Bullfrog) oocytes. The cytosol was extracted from oocytes using the previously published protocol. 85 The signal intensity of mOX063-d 24 was monitored over time using CW-ESR, and the maximum intensity of each spectrum was plotted against time in Figure 5A. The signal intensity decays to about 97 and 95% of its original intensity in ascorbate and the cytosol after 5 h, respectively. The stability of mOX063-d 24 is on par with the stability of other trityls. 73,80 The signal persistence of mOX063-d 24 showcases the reduction resistance of mOX063d 24 against the cytosolic antioxidants that play a role in reducing radicals in-cell. 98 After measuring the mOX063-d 24 stability, mOX063-d 24 -GB1 was injected into oocytes and incubated for 30 min after injection before flash-freezing the sample. The 30 min incubation allows for mOX063-d 24 -GB1 to completely diffuse in oocytes. 48 The echo decay experiment measured the T m of mOX063-d 24 at 80 K in oocytes to be 4.3 μs, as shown in Figure S7, which is shorter than the T m of mOX063-d 24 in vitro, as shown in Table 1 and Figure S4A. The lower GB1bound mOX063-d 24 T m in-cell compared to in vitro was expected because of the crowded environment in-cell. The crowded environment can lead to an increase in the local concentration of protons near the radical, which enhances the contribution of electron-nuclei interactions to relaxation. In addition, the presence of paramagnetic metal ions, primarily Mn(II), 99 in the cell can enhance relaxation. However, the T m of mOX063-d 24 in oocytes is surprising since previous reports of other organic spin labels used in-cell (nitroxides 50,57,100 and FT 67,72,73 ) have T m values in the range of 0.6-2 μs. Therefore, mOX063-d 24 also improves the sensitivity of distance measurements in-cell due to the T m that is at least 2 times longer than previously published T m of nitroxide or FT in-cell.
Distance measurements of mOX063-d 24 -GB1 in oocytes were done using DQC at 80 K, as shown in Figure 5B. We observed an artifact that overlaps the desired DQC signal at zero time. Such an artifact has been seen previously and attributed to trityl dimers and to partial labeling of noncysteine residues such as lysine. 101 We repeated the labeling procedure on WT GB1 that has no cysteine residues. After concentrating the sample, UV-vis measurement indicates no presence of mOX063-d 24 , as shown in Figure S8. We expected this result since our previous work using GB1 and maleimide-linked nitroxide (5-MSL) did not show overlabeling of the protein. 85 In addition, we did not see an ESR signal from the WT GB1 sample, as shown in Figure S9, which also excludes the presence of dimers.
We attribute this artifact to the formation of a small echo generated by the first and the fourth pulses in the DQC 6-pulse sequence. This interference can be readily seen in the twodimensional (2D) contour plot of the DQC signal, as shown in . As a result, the DQC time trace contained an artifact shown as a sharp feature at the t = 0, as shown in Figure S10B, which led to improper fitting of the time trace. Additionally, the artifact contributed to a short distance around 2 nm, as shown in Figure S11A. The artifact seemed to be a result of inefficient phase cycling in our DQC experiment and is evident in the in-cell data due to the lower SNR.
To support this hypothesis, we performed the DQC experiment using the same parameters on 300 μM TEMPOL, as shown in Figure S12. The same artifact was seen crossing the desired DQC echo at a slanted angle shown in the 2D contour plot in Figure S12A. As a result, a sharp feature at t = 0 manifested, as shown in Figure S12B. This artifact has not been seen in previous works. The artifact was more prominent in the in-cell experiment than in the in vitro experiment for two reasons. First, the measured echo in the in-cell DQC was half as intense as the measured echo in the in vitro DQC. The lower in-cell echo intensity is due to the shorter T m in-cell than the T m in vitro. Additionally, reduction of mOX063-d 24 can still occur due to the contribution of membrane-associated proteins 85 such as thioredoxin 102 and glutathione reductase, 103 which are not accounted for in our cytosol stability measurement. These two contributions led to a less intense measured echo, causing the artifact to be prominent in the in-cell DQC.
To remove the artifact in the DQC time trace in oocytes, DQC was performed on a sample of free mOX063-d 24 , which contained only the artifact shown in Figure S10B. The free mOX063-d 24 DQC time trace was used to subtract the artifact from the time trace of mOX063-d 24 -GB1 in oocytes, as shown in Figure 5B. The artifact-subtracted time trace was used to extract the distance distribution shown in Figure 5C, which agrees quite well with the in vitro distance measurements in Figure 4B. Furthermore, we were able to repeat the in-cell DQC experiment at 150 K, as shown in Figure S13, and obtain a similar distribution as the 80 K in-cell distribution. Additionally, we repeated our in-cell experiments at 80 K using a different batch of oocytes and newly overexpressed and labeled GB1 to ensure that the in-cell results are reproducible. This data is shown in Figure S14. Overall, we obtained a highly sensitive distance measurement in oocytes using mOX063-d 24 .
In conclusion, this work showed that mOX063-d 24 has a high protein-labeling efficiency of ∼97%. Furthermore, we showed that in vitro distance measurements of mOX063-d 24 are more sensitive at higher temperatures. Finally, we obtained distance measurements using mOX063-d 24 in-cell, which agree with in silico modeling. This work adds to the library of spin labels that can be used for in-cell work. In particular, mOX063-d 24 is similar to Ox-SLIM 80 since both are hydrophilic spin labels with a maleimide linker, as shown in Figure 1A. However, these two spin labels differ in their trityl cores and linker lengths. These differences provide variation in the labeling efficiency, T m , and breadth of distance distribution. In one case, Ox-SLIM's short linker length can provide narrow distance distributions that can readily resolve different protein conformations. 73,80 On the other hand, mOX063-d 24 provides longer T m and higher labeling efficiency leading to the sensitivity improvement in the distance measurements. Overall, Ox-SLIM and mOX063-d 24 are complementary to each other due to their differences.
https://doi.org/10.1021/acs.jpcb.1c02371 J. Phys. Chem. B XXXX, XXX, XXX-XXX