Temperature-dependent DEER effects are observed as a function of methyl rotation by either leucine- or nitroxide-specific protonated methyl groups. Both species induce a site specific enhancement in the apparent Tm-relaxation of the paramagnetic nitroxide label. The presence of a single protonated methyl group in close proximity (4–10Å) to only one of the two nitroxide rotamer ensembles in protein A results in a selective and substantial decrease in Tm, manifested by differential decay of the peak intensities in the bimodal P(r) distance distribution as a function of the total dipolar evolution time, temperature or both. Temperature assisted Tm filtering will capture the DEER structural analysis of biomolecular systems heterogenic conformations, including complexes involving multimeric proteins.
Research Article
Temperature dependent rotation of protonated methyl groups in otherwise deuterated proteins modulates DEER distance distributions
https://doi.org/10.21203/rs.3.rs-4818320/v1
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Pulsed double electron–electron resonance (DEER) EPR spectroscopy provides means to accurately measure long-range distances between pairs of paramagnetic labels up to 170 Å under deuterated conditions, and therefore is an invaluable tool for conformational analysis of proteins.[1–3] The 4-pulse DEER experiment (Fig S1) produces a Hahn-echo at the observer frequency with an electron-electron distance modulation introduced by an ELDOR pulse at the pump frequency; however, the overall echo intensity largely depends on the electron phase-memory relaxation time, Tm.[4, 5] The Tm is anticorrelated to the number of near protonation sites, thus protonated moieties (e.g. methyl groups) in close vicinity to the spin label decreases the DEER echo.[6, 7] Here, protonated methyl group(s) attached to either the protein sidechain (less than 10 Å from the electron) or the paramagnetic label itself in a deuterated background permits the acquisition of a pseudo-3D DEER spectrum.[8] The increased dimensionality, comprising temperature (T), Tm-relaxation (τ2 period) and dipolar evolution time (tmax), permits resolving multimodal distance distributions as in proteins displaying several labelling sites, oligomeric complexes or conformational heterogeneity.[9, 10] This approach relies on the chemical environment dependent methyl-rotation and their unique activation energy barriers (Ea) that subsequently modulates the Tm of paramagnetic spins specific to their molecular origin (Fig. 1A).[11–15]
The model protein, AviTag-Protein A (Fig. 1A), has the MTSL spin label (R1) attached to engineered cysteine residues (Q39C/K88C) within the ordered Protein A domain.[16] Previous measurements showed two resolved distances in the P(r) distribution at 33 and 38 Å, arising from the Q39C-R1 label occupying two distinct regions of the conformational space (respectively labeled a and b in Fig. 1A), as judged by the predicted P(r) distribution generated from the atomic coordinates (PDB: 1bbd[16]) using the spin-label rotamer program MMM, ChiLife or Xplor-NIH (Fig S2).[17–19] Protonation of Leu64 or MTSL resulted in dramatic changes in the apparent distance population as observed by Tm-edited DEER. Here we extended the expression to include the modulation factor kapp capturing methyl group rotation as a function of temperature.[4] The chemical origin of the relaxation components was disentangled through site-specific protonation of either MTSL and Leu64 methyl groups (Fig. 1B). The three-site jump model predicts the longitudinal relaxation rate for methyl groups based on the jump constant k3 that distinguishes the methyl conformers by their unique values of activation energy barrier (Fig. 1C).
The ratio of the b to a components in the P(r) distributions, P(rbC,τ2,T)/P(raC,τ2,T), for [Leu-CH3/2H]-AviTag-Protein A (Q39C-R1/K88C-R1) shown in Fig. 1, is expected to decay as:
where pb is the Q39C-R1 nitroxide population in the b state, and Rbm (= 1/Tbm) and Ram (= 1/Tam) are the phase-memory relaxation rates for Q39C-R1 in the b and a states (Fig S3). Interestingly, the rotamer specific phase-memory relaxation rates are modulated by kaapp and kbapp, respectively. While both labeling sites are influenced by methyl rotation the DEER data only present the difference thereof, hence it is practical to present the relaxation difference from the frame of the slowest relaxing electron by setting kaapp to 1 and k0 to the same for all conformers. Further, the electron Tm decreases with increasing temperature, the presented analysis permits to maintain the same Tm for all temperatures (42 µs[4]) based on a fully deuterated sample. Figure S3A highlights the P(r) relationship between the 2τ2 time interval and the temperature induced methyl rotation as outlined by Eq. 1; the induced relaxation pathway shifts the apparent P(r) conformation from state A (blue) to D (red) by cumulating Tm modulations at 80K with a 2τ2 of 40 µs (Fig S3B). The individual changes in P(r) ratio along the Tm-relaxation pathway due to either temperature or 2τ2 are outlined in Fig S3C and S3D, respectively. Overall, Fig S3 highlights the selection of values of temperature and 2τ2 to achieve the desired P(r) ratio based on localized protonated methyl groups and their rotation.
Data collection was performed in a pseudo three dimensional fashion whereby temperature, 2τ2 interval and dipolar evolution time serve as independent parameters. The conjoint fitting of those variables describes the relaxation contributions by protonated methyl groups attached to either label, leucine, or the combination thereof. Here, the following isotope labeling schemes were tested: 2H-MTSL/2H-Leu64 (Fig. 2A), 1H-MTSL/2H-Leu64 (Fig. 2B), 2H-MTSL/1H-Leu64 (Fig. 2C), and 1H-MTSL/1H-Leu64 (Fig. 2D). In Fig. 2A, the Tm-edited DEER experiment showed only minor changes in the distance population ratios for all set values of evolution time and temperature for fully deuterated proteinA covalently linked to deuterated MTSL.[7, 20] This is not surprising as in the absence of electron-proton coupling neither spin diffusion nor methyl rotation can affect electron Tm. In comparison, the addition of protons to the MTSL label affected the distance distribution at temperatures above 60 K (Fig. 2B); the population ratios decreased with increasing temperature and evolution time originating from induced methyl rotation. DEER population ratios at 2τ2 set to 10 µs did not present significant variations for all temperatures, however upon increasing the 2τ2 to longer evolution times the ratio decreased with increasing temperature to a minimum ratio of 0.6 at 85 K and a 2τ2 of 40 µs. Methyl rotation, experienced by Q39C-R1 nitroxide rotamer a and b, were fit to the dipolar time traces by Eq. 1, while varying the activation energy, <Ea>, and its distribution, σ. The apparent < Ea > of 11.9 kJmol-1 for the b-rotamer is induced by chemical environment dependent effects on methyl rotation of the MTSL rotamer population, such as steric hindrance and hydrophobicity at various labeling sites.
Localized Tm relaxation can be modulated by introducing amino acid specific protonation, subsequently adjusting it via spin diffusion and methyl rotation of the proton-harboring amino acid.[4] Here, protonated leucine methyl groups reduced the relative DEER distance population inversely related to 2τ2 and temperature in the Tm-edited DEER time traces. Figure 2C depicts deuterated MTSL in the vicinity of protonated leucine methyls; the temperature driven rotation of the methyl groups decreased the distance ratios with increasing evolution time. In contrast to the previous case (Fig. 2A, 2B), spin diffusion presents a temperature independent relaxation component of the b-rotamers (Tbm = 29 µs), hence 2τ2 associated population ratios will not converge at low temperatures (20 K). In the presented fits, activation energy for the leucine methyl groups is 12.5 kJ mol-1 with a distribution of 3.5 kJmol-1, the increase in σ<Ea> compared to previous values originates from the Leu64 methyl groups in vicinity to the MTSL. Protonation of both MTSL and leucine were conjoined in Fig. 2D; the spin diffusion rate is enhanced due to the increase in coupled protons in close vicinity (< Ea > = 13.4 kJmole-1) with no change in σ<Ea>. The similarities to 2H-MTSL/1H-Leu64 presents the dominance the leucine methyl groups exhibit over the nitroxide relaxation behavior. Overall, we present evidence that amino acid methyl causes both spin diffusion and methyl rotation.
|
ProteinA |
MTSL |
<Ea> (kJmol-1) |
σ<Ea> (kJmol-1) |
Tbm (µs) |
Pbc/Pac |
X2 |
|---|---|---|---|---|---|---|
|
2H |
2H |
- |
- |
- |
1.1#/ 1.2* |
|
|
2H |
1H |
11.9 ± 0.1 |
1.1 ± 0.2 |
- |
0.54 ± 1 |
1.0#/1.3*/1.3✝ |
|
1H-Leu-CH3/2H |
2H |
12.5 ± 0.5 |
3.5 ± 0.5 |
29.3 ± 0.5 |
0.54 ± 1 |
1.4#/1.4*/1.4✝ |
|
1H-Leu-CH3/2H |
1H |
13.4 ± 0.2 |
3.6 ± 0.4 |
20.9 ± 0.4 |
0.55 ± 1 |
1.1#/1.1*/1.2✝ |
# Averages for model-free fits to the individual DEER echo curves using DeerLab with validated Tikhonov regularization (n = 1000).
* Gaussian global fits based on the program DD/GLADDvu, incorporated into an inhouse Python program to the complete set of Q-band DEER echo curves recorded over a series of temperate and τ2 values. For the presented two-Gaussian global fit, only the mean distances and corresponding widths are constrained to be invariant.
✝ In the two-Gaussian global fit, the dependence of the fractional populations of the distance peaks on temperature and τ2, as specified by the three-site jump rate and the resulting weighted exponentials as in eq S4 (Fig. S2), were included as an optimized parameter.
DEER on large protein complexes is fostered by orthogonal labeling, in which distances between two chemically distinct paramagnetic species are measured (e.g. MTSL-Cu or MTSL-Gd). While R1p is similar to MTSL with the addition of the 4-pyridyl which increases its size, hence presents a narrower rotamer distribution.[21] Here, the R1p nitroxide label iterated the effect on Tm relaxation as a function of temperature and 2τ2 on the DEER distance distribution. In Fig. 3A, protonated R1p label is in a fully perdeuterated environment upon increasing the temperature a dispersion in population height marks a temperature effect, however due to the large population difference the ratio change is rather small. Leu64 was protonated in addition to the R1p label in Fig. 4B; the protonated methyl groups increased susceptibility to spin diffusion and temperature which was mirrored by the difference between 10 and 40 µs DEER traces with increasing temperature. We did not attempt to fit the resulting DEER traces, as it is unknown how the pyril group contributes to observed spin-diffusion and temperature effects. While the leucine methyl group exhibited similar temperature/2τ2 dependencies as observed for the MTSL samples, the extent of the modulation converges between 70 to 80K that differs from MTSL. Therefore Tm/Temperature-modulated DEER provides means to differentiate chemical similar paramagnetic labels during orthogonal labeling (e.g. MTSL-R1p) for distance measurements of homodimeric complexes.
In summary, temperature titration of nitroxide labeled proteins distinguishes multimodal distance distributions by DEER spectroscopy. We characterized the relationship between temperature induced methyl rotation and differential relaxation of the paramagnetic labels that ultimately modulate a bimodal P(r) distribution in AviTag-Protein A as proof of principle. Conjoined titration of temperature and evolution time resulted in apparent populations of two DEER distance distributions modulated by rotational diffusion of protonated methyl group(s) to either protein sidechain (less than 10 Å from electron) or even the paramagnetic label itself in an otherwise fully deuterated background. Here, the localized chemical environment of individual protonated nitroxide labels allows temperature driven spin-differentiation without sidechain protonation which is enhanced upon the introduction of protonated leucine moieties (L64). Vast applications towards protein complexes and their equilibrium kinetics are foreseen; for example, structural information on an equilibrium of homodimeric proteins will present multiple spin labels, therefore the assignment of DEER distance distribution to their paramagnetic centers is essential. While Tm-edited DEER in conjunction with selective protonation is used to separate individual DEER distances, the introduction of localized methyl labels in a deuterated background is cumbersome, therefore the use of protonated nitroxide labels in conjunction with temperature titration forms an alternative to assign distance distribution in oligomeric systems. Lastly, DEER distance distribution produced by orthogonal labeling with different nitroxide labels can be distinguished based on temperature titrated Tm-edited DEER data due to their chemical specific methyl rotation. Overall, temperature titration and Tm-edited DEER in conjunction with side specific protonation expands the repertoire to assign DEER distances in oligomeric biomolecular systems.
Expression, purification and labeling of Protein A. Fully deuterated AviTag-protein A, with two surface exposed, engineered cysteine residues (Q39C and K88C) was expressed in E. coli and purified as described previously.[1] The AviTag extends from residues 1–29, and protein A from residues 30–90; residues 1–38 are disordered in solution. Incorporation of protonated methyl groups of Leu (Cδ1H3 and Cδ2H3) in a fully deuterated background was carried out as described previously by growing the bacteria in minimal D2O medium with ammonium chloride as the sole nitrogen source, U-[2H]D-glucose as the main carbon source, and the appropriate α-keto acid precursor for 13CH3 labeling: α-ketoisovaleric acid (13C5, 98%, 3-D1, 98%) for Leu and Val (Cambridge Isotope Laboratories CDLM-4418-PK). Note there are no valines in AviTag-Protein A.[2] Nitroxide (R1 or R1p) spin-labeling was carried out with S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL; Toronto Research Chemicals) or 2,5-Dihydro-2,2,5,5-tetraMethyl-3- [[(Methylsulfonyl)thio]Methyl]-4-(3 -pyridinyl)-1H-pyrrol-1-yloxy (R1p; Toronto Research Chemicals) as described previously. [1] Nitroxide-labeling was verified by electrospray ionization mass spectrometry. The sample for NMR comprised 50 µM AviTag-Protein A, 0.85 mM KH2PO4, 25 mM Na2HPO4, pH 7.4, 75 mM NaCl, and d8-glycerol(30% v/v)/D2O (70% v/v).
Pulsed Q-band EPR spectroscopy. Pulsed EPR data were collected at Q-band (33.8 GHz) and 50 K on a Bruker E-580 spectrometer equipped with a 300 W traveling-wave tube amplifier, a model ER5107D2 resonator, and a cryofree cooling unit, as described previously. DEER experiments were acquired using a conventional four-pulse sequence.[3] The observer and ELDOR pump pulses were separated by ca. 90 MHz with the observer π/2 and π pulses set to 12 and 24 ns, respectively, and the ELDOR π pulse to 10 ns. The pump frequency was centered at the Q-band nitroxide spectrum located at + 40 MHz from the center of the resonator frequency. The τ1 value of 400 ns for the first echo-period time was incremented eight times in 16 ns steps to average 2H modulation; the position of the ELDOR pump pulse was incremented in steps of Δt = 8 ns. The bandwidth of the overcoupled resonator was 120 MHz. All DEER echo curves were acquired for τmax = 4 µs to avoid the persistent “2 + 1” echo perturbation of the DEER echo curves at a time of about τ1 from the final observed π pulse. DEER data were recorded with values of the dipolar evolution time 2τ2 ranging from 10 to 40 µs for [Leu-CδH3/2H] AviTag-Protein A.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Drs. James Baber and John Louis for technical support, and Martin Gelenter, Kent Thurber and Veronica Szalai for scientific discussion. The work was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health.
- M. Pannier, S. Veit, A. Godt, G. Jeschke, and H. W. Spiess, J. Magn. Reson. 142, 331 (2000).
- T. Schmidt, M. A. Wälti, J. L. Baber, E. J. Hustedt, and G. M. Clore, Angew. Chem. Int. Ed Engl. 55, 15905 (2016).
- H. El Mkami and D. G. Norman, Methods Enzymol. 564, 125 (2015).
- T. Schmidt and G. Marius Clore, Chemical Communications 56, 10890 (2020).
- T. Schmidt, J. Jeon, W.-M. Yau, C. D. Schwieters, R. Tycko, and G. M. Clore, Proc. Natl. Acad. Sci. U. S. A. 119, (2022).
- M. Lindgren, G. R. Eaton, S. S. Eaton, B.-H. Jonsson, P. Hammarström, M. Svensson, and U. Carlsson, Journal of the Chemical Society, Perkin Transactions 2 2549 (1997).
- H. El Mkami, R. Ward, A. Bowman, T. Owen-Hughes, and D. G. Norman, J. Magn. Reson. 248, 36 (2014).
- S. M. Jahn, E. R. Canarie, and S. Stoll, J. Phys. Chem. Lett. 13, 5474 (2022).
- T. Schmidt, R. Ghirlando, J. Baber, and G. M. Clore, Chemphyschem 17, 2987 (2016).
- N. Kubatova, T. Schmidt, C. D. Schwieters, and G. M. Clore, Proc. Natl. Acad. Sci. U. S. A. 120, e2221036120 (2023).
- L. Vugmeyster, D. Ostrovsky, A. Khadjinova, J. Ellden, G. L. Hoatson, and R. L. Vold, Biochemistry 50, 10637 (2011).
- L. Vugmeyster, P. J. Nichols, D. Ostrovsky, C. J. McKnight, and B. Vögeli, Magnetochemistry 9, (2023).
- A. Eggeling, J. Soetbeer, L. Fábregas-Ibáñez, D. Klose, and G. Jeschke, Phys. Chem. Chem. Phys. 25, 11145 (2023).
- G. Usevičius, A. Eggeling, I. Pocius, V. Kalendra, D. Klose, M. Mączka, A. Pöppl, J. Banys, G. Jeschke, and M. Šimėnas, Molecules 28, (2023).
- G. Jeschke, Appl. Magn. Reson. 53, 635 (2022).
- H. Gouda, H. Torigoe, A. Saito, M. Sato, Y. Arata, and I. Shimada, Biochemistry 31, 9665 (1992).
- G. Jeschke, Protein Sci. 30, 125 (2021).
- T. Schmidt, D. Wang, J. Jeon, C. D. Schwieters, and G. M. Clore, J. Am. Chem. Soc. 144, 12043 (2022).
- M. H. Tessmer and S. Stoll, PLoS Comput. Biol. 19, e1010834 (2023).
- R. Ward, A. Bowman, E. Sozudogru, H. El-Mkami, T. Owen-Hughes, and D. G. Norman, J. Magn. Reson. 207, 164 (2010).
- N. L. Fawzi, M. R. Fleissner, N. J. Anthis, T. Kálai, K. Hideg, W. L. Hubbell, and G. M. Clore, J. Biomol. NMR 51, 105 (2011).
No competing interests reported.