Advances in EPR Approaches for Studying Structural Properties of Membrane Proteins

Abstract

Electron paramagnetic resonance (EPR) in connection with site-directed spin labeling is a structural biology tool that can be employed to obtain structural and conformational properties of various biological systems. Recent advances in methodological and technical improvements have made EPR spectroscopy a rapidly growing tool for gleaning important structural and conformational dynamics of membrane proteins. In this review, we discuss advancements in the popular site-directed spin labeling EPR approaches in brief and their applications to study the structure and conformations of biologically important membrane proteins. Recent examples of electron spin echo envelope modulation (ESEEM), double electron–electron resonance (DEER), and In-cell EPR studies for addressing structural and conformational-related questions of membrane proteins will be highlighted.

1 |. EPR Spectroscopy

In early stages, electron paramagnetic resonance (EPR) spectroscopy was used to study molecular species containing unpaired electrons such as transition metal complexes, free radicals, and paramagnetic oxygen due to the requirement of an unpaired electron to be detected by EPR. The application of EPR was restricted to limited protein systems containing metal clusters or free radicals. The limitation was due to difficulties in the incorporation of unpaired electrons into proteins. The advancements in spin labeling methods significantly broadened the application of EPR to most protein systems [1, 2]. While traditional spin labeling methods primarily target thiol moieties, the possibility of labeling other residues and other biomolecules has opened up new perspectives [3].

EPR measures an interaction of the unpaired electron with microwave radiation in the presence of an external magnetic field. When combined with site-directed spin labeling (SDSL), EPR can provide structural features and conformational dynamics of membrane proteins within physiologically relevant lipid bilayer membrane environments [4–8]. Spin label EPR spectroscopy is a very useful technique to characterize the assembly of the protein and lipid membrane [6, 9, 10]. Furthermore, SDSL EPR approaches are widely used to investigate protein–protein interactions as well as the oligomerization states of membrane proteins [11–20].

Biophysical studies of membrane proteins remain challenging due to their low expression yields, hydrophobic behavior, dynamics, and limited stability [21]. Nuclear magnetic resonance (NMR) spectroscopy is a very useful biophysical technique to study structural and dynamic properties of numerous membrane proteins in a noncrystal environment. However, there is a major challenge associated with size limitation in solution NMR spectroscopy (restricted to < 50 kDa) [22–25]. Solid-state NMR spectroscopy is very successful for studying structure, interactions, and dynamics of membrane proteins [26–28]. NMR also requires isotropic samples to minimize line broadening effects that introduce challenges in studying some membrane proteins in a closest membrane mimetic such as proteoliposomes [26].

In this context, EPR spectroscopy has emerged as a powerful and complementary structural biology tool that minimizes many of these challenges and provides important structural and dynamic properties of membrane proteins. EPR uses a similar working principle to NMR where EPR utilizes unpaired electrons whereas NMR utilizes isotopic nuclear spin of the individual atom. Several EPR approaches have been used to study structural and conformational dynamics of membrane proteins such as continuous wave (CW)-EPR spectroscopy (i.e., EPR spectral measurements and EPR power saturation measurements) and pulse EPR spectroscopy (i.e., electron spin echo envelope modulation [ESEEM] spectroscopy and double electron–electron resonance [DEER] spectroscopy). CW-EPR measurements are usually carried out at room temperature while ESEEM and DEER measurements are carried out at cryogenic temperatures.

2 |. EPR Approaches and Recent Advances

EPR is responsive to the motion and environment of the molecular species containing individual electrons. Significant improvements have been made in several aspects of EPR approaches including instrumentation, methods, types of spin labels, and molecular biology techniques [2, 6, 29]. The commonly used microwave frequency bands and corresponding approximate frequencies are L-band (1.5 GHz), S-band (3 GHz), X-band (9.5 GHz), K-band (23 GHz), Q-band (36 GHz), W-band (95 GHz), and J-band (263 GHz) [30]. In connection with the SDSL, EPR can be used to characterize any size of the protein [31]. Proteins solubilized in various solvent conditions including aqueous solutions, membrane suspensions, tissue samples, and a wide range of temperature conditions including frozen samples can be studied using EPR approaches [32]. EPR is sensitive to a wide range of sample volumes and concentrations ranging from ~70 nL to mLs or small animals [33, 34]. Different methods of EPR spectroscopy can be used to glean pertinent structural dynamics and conformation of membrane proteins [35–44]. For example, CW-EPR spectral lineshape analysis of a series of nitroxide spin-labeled sites along the protein sequence can be used to study structural dynamics of nitroxide labeled side chains and to infer secondary structure features of the protein in an asymmetric environment (e.g., a helix that is crowded on one side and surface exposed on the other). EPR power saturation and ESEEM approaches can be used to study the solvent accessibility. Similarly, CW-EPR dipolar line broadening and DEER approaches can be used to perform distance measurements between two spin labels. The CW-EPR spectral lineshape analysis can be used to study the protein–protein interactions [45].

This topic can easily fill one or more books. Here, we present a brief discussion of the most popular EPR techniques and their recent employment to solve membrane protein-related questions. Authors refer the detailed discussion of EPR techniques to these excellent literatures [4, 38, 46].

2.1 |. Technical Approaches of CW-EPR Spectroscopy

A simplest EPR active system is one that contains an unpaired electron spin located in a molecular orbital. The electron can occupy one of the two quantum states +1/2 and −1/2 known as degenerate states having the same energy in the absence of an external magnetic field. The energy associated with the −1/2 state decreases and the energy associated with the +1/2 state increases as a function of the magnetic field strength in the presence of external magnetic field. The energy level diagram of a nitroxide spin label containing unpaired electrons is shown in Figure 1. Figure 1 indicates that the energy level-splitting is dependent on the strength of the applied magnetic field (B). The difference in the energy of these two levels is ΔE = g_e β B with g_e being an electronic g-factor (analogous in role to the chemical shift parameter in NMR), and β is the electron Bohr magneton. When microwave radiation is applied to the system, resonance occurs when the energy difference between these two spin levels (g_e β B) matches with the energy of the applied microwave radiation (hf). This is known as resonance condition with the relation hf = g_e β B [44, 47, 48], where h is Planck’s constant and f is microwave radiation frequency.

FIGURE 1 |.

Energy level diagram of a nitroxide spin label in the presence of an external magnetic field (B) (A), and the corresponding absorption (upper panel) and first derivative (lower panel) EPR spectra in the absence and presence of a ¹⁴N (I = 1) hyperfine interaction (B) [6, 44, 47–49]. EPR spectra were simulated with a routine from nonlinear least square (NLSL) package [50, 51].

In a typical CW-EPR spectroscopic measurement, a magnetic field (B) is varied at a constant microwave frequency. When a resonance condition is reached, a transition occurs and an absorptional signal is recorded (Figure 1B [upper panel]) [47]. The modulation of the magnetic field is employed to improve the signal-to-noise ratio (S/N) and obtain the first derivative of the EPR spectrum (Figure 1B [lower panel]). The EPR signal at the particular magnetic field is dependent on the Zeeman interaction, which is characterized by the g-factor. The slope of the energy levels as a function of the magnetic field is determined by the g-value. For a free electron, the g-value is 2.0023, whereas for an unpaired electron in a molecule, the g-tensor may be anisotropic due to spin-orbit coupling affected by the local electronic environment [47]. In the absence of the interaction of the electron with the nearby nucleus, the CW-EPR spectrum appears as an individual line (Figure 1B [left panel]). Nitroxide spin labels are generally used for most biological systems. The interaction of a nitroxide spin label with the nitrogen nucleus having a nuclear spin of one (I = 1) is known as hyperfine interaction, characterized by hyperfine coupling constant A. The hyperfine interaction is controlled by several EPR spectral parameters including the amount of the spin density of the electron at the nucleus, the distance between the electron spin and the nucleus, and the angle between electron spin-nucleus separations with respect to the magnetic field (B). Due to the presence of the hyperfine interaction, there are transitions allowed between energy levels of the electrons, resulting in the splitting of the EPR lines into multiple lines controlled by the state of the nuclear spin. The three spectral lines are observed due to the hyperfine coupling of an electron spin to an individual nucleus as shown in Figure 1B (right panel). The EPR spectrum is also influenced by the electron–electron coupling between two sets of spins. This can provide the information about distances of biological systems. This method can provide distance measurements below 20 Å using frozen samples [52]. Nitroxide spin labels, such as MTSL, offer high stability and a unique ability to probe local environment [24, 52, 53]. However, the nitroxide spin label rotamers and backbone dynamics of the protein have a significant impact on the range of distance measurements. The rigid spin labels such as 3,4-bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy radical (HO-1944) bifunctional spin label (BSL) and 2,2,6,6-tetra methyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) spin label reduce the spin label rotamers and backbone fluctuations [54–56]. But the incorporation of BSL and TOAC spin labels in membrane proteins is still challenging. The examples of application of the CW-EPR line broadening method will be discussed in Section 3.

2.2 |. SDSL EPR

SDSL is essential for studying the structural and conformational properties of membrane proteins. SDSL provides a tag for a spin label probe that contains an unpaired electron at the target location of the amino acid sequence of the protein. Natural cysteine is replaced with serine or alanine, and site-directed mutagenesis is used to generate the cysteine at the target amino acid location of the protein. The structural integrity and/or functional activity of the mutant should be verified following mutagenesis. This is further reacted with a sulfhydryl-based nitroxide reagent to obtain a stable side chain of the spin label [35, 57, 58]. The most commonly used nitroxide spin label is the methanethiosulfonate spin label (MTSL) [37]. Additional spin labels such as iodoacetamido-PROXYL and maleimido-PROXYL have also been used in SDSL-EPR spectroscopy [59, 60]. We refer to recent literature for details of nitroxide spin labels used for SDSL-EPR spectroscopy [44, 61].

CW-EPR spectral features are highly sensitive to the motion of the spin label side chain that can be used to understand local dynamics and conformational changes within a protein system [38, 62]. The analysis of CW-EPR spectra can provide dynamic parameters such as relative mobility represented by the inverse central linewidth and motional parameter characterized by rotational correlation times [36, 38, 63–65]. Figure 2 shows the CW-EPR spectra simulated for different dynamic motions of nitroxide spin labels using various rotational correlation times. For spin labeling sites with isotropic motion with lower rotational correlation times (0.0017 ns) (Figure 2A), the spectral line shape is very sharp and narrow. As the motion of the spin label decreases, the spectral lineshape gets broader and approaches the rigid limit with frozen samples (Figure 2B–E) [66].

FIGURE 2 |.

CW-EPR spectra at different motions of spin label side chains with various rotational correlation times. Spectra were simulated with a routine from NLSL package [50, 51].

SDSL-based CW-EPR power saturation technique can be used to study solvent accessibility of the protein with respect to the membrane bilayer [11, 37, 67–69]. EPR power saturation accessibility data can also be used to understand conformational changes observed in the membrane proteins [70, 71]. The peak-to-peak amplitude of the first derivative resonance line (m_I = 0) is obtained by analyzing the CW-EPR power saturation data and plotted against the square root of the incident microwave power. The power saturation curves are obtained for samples (1) equilibrated with nitrogen (control); (2) equilibrated with 20% oxygen (air) (lipid-soluble paramagnetic agent); and (3) equilibrated with nitrogen containing NiEDDA (water-soluble paramagnetic agent). The membrane depth parameters (Φ) for the spin labeling sites with respect to the membrane surfaces are calculated from the power (P_1/2) where the first derivative amplitude of the CW-EPR spectrum is reduced to half of its unsaturated value using the equation below.

$$\Phi =\text{ln}\left(\frac{\Delta P_{1/2}\left({\text{O}}_2\right)}{\Delta {\text{P}}_{1/2}(\text{NiEDDA})}\right),$$

where ΔP_1/2(O₂) = difference in P_1/2 values of samples exposed to air and nitrogen and ΔP_1/2(NiEDDA) = difference in P_1/2 values of samples in the presence of NiEDDA and nitrogen.

2.3 |. ESEEM Spectroscopy

ESEEM is an advanced EPR spectroscopic approach that can detect a weak interaction between magnetic nuclear spin and unpaired electrons. The formally forbidden electron–nuclear spin transitions become slightly allowed if the hyperfine coupling has the same order of magnitude as the nuclear Zeeman interaction. This is the foundation of ESEEM spectroscopy. ESEEM detects weak hyperfine interactions between the unpaired electron of a spin label and isotopically labeled nuclei, observed as periodic modulations in the electron spin echo amplitude. The deuterium nucleus can be utilized as an isotopically labeled nucleus to obtain a distance of up to 8 Å between a spin label and an individual ²H nucleus to study the structural properties of macromolecules [72]. It has been employed to study numerous biological systems, for example: KcsA Potassium Channel, ABC Transporter HisQMP(2) [73–90]. A three-pulse ESEEM sequence has been very popular for the investigation of biological systems. As shown in Figure 3, in the three-pulse ESEEM sequence, a stimulated echo amplitude modulation is produced at a time after the third pulse where the time (τ) between the first and second pulse is kept constant and the time (T) between the second and third pulse is increased. In the ESEEM experiment, the echo amplitude is recorded against the time (T + τ). The three-pulse ESEEM experiments provide improved resolution in the ESEEM signal with the increased value of spin–lattice relaxation time (T₁) [89]. The modulation depth observed in the three-pulse ESEEM time domain signal depends inversely on the sixth power of the distance between the weakly interacting nuclei and unpaired electron spin (∝1/r⁶) [91, 92]. A detailed theory of ESEEM spectroscopy and methods for obtaining modulation depth parameters have been discussed in the literature [91–93].

FIGURE 3 |.

Three pulse ESEEM sequence.

The function of membrane proteins is very sensitive to the local secondary structure of the protein, especially the packing assembly and interaction of the proteins are influenced by the nature of the local secondary structure of the protein. Nitroxide-based spin labeling ESEEM spectroscopy can be used to directly obtain the local secondary structures including α-helix, β-sheet, 3₁₀ helices of the membrane proteins and peptides in aqueous solution as well as in membrane mimetic environments [78–87, 94]. This method uses a connection of SDSL and ²H-labeled side chains.

2.4 |. DEER Spectroscopy

DEER spectroscopy is a very popular structural biology tool to measure distance distributions in the nanometer range [95–99]. DEER detects weak dipolar coupling between a pair of electronic spins separated at a distance greater than 20 Å [100]. A distance range of 20–60 Å can be obtained using nitroxide-based SDSL DEER spectroscopy for membrane proteins and the range extends up to 160 Å for soluble proteins when both solvents and the proteins are deuterated [95, 101–103]. During the detection of dipolar coupling between the two spins, one set of spins is observed at a first microwave frequency while another set of spins is excited with a second microwave frequency [91, 95, 104]. The dipolar coupling frequency is dependent on the inverse of the cubic power of the radial distance between two spins (∝1/r³) [91, 105]. During DEER measurements, intramolecular dipolar coupling can occur between spin labels attached to the same protein. Alternatively, intermolecular dipolar coupling can occur between spin labels on different protein molecules, and this interaction modulates the spin echo decay of the detected spins. The former process produces a periodic oscillating echo yielding the average distance while the latter process contributes to decaying background that decreases the oscillation. During the data analysis, the background effect is usually minimized in the echo decay resulting in a distance distribution from which the weighted average distance and standard deviation can also be derived. DEER experiments for biological systems are usually carried out at cryogenic temperatures (50–80 K) to enhance phase memory time [106, 107]. Recently, DEER distance measurements have also been conducted at room temperature on model systems having immobilized biomolecules [108, 109]. Schiemann et al. have provided community standards of details of the guideline for instrumentation, sample conditions, and data analysis for obtaining distance distribution and uncertainty estimates for SDSL based EPR DEER measurements [110]. Recently, Sweger et al. introduced a Bayesian probabilistic inference approach of data analysis to obtain full quantitative information of uncertainty in DEER distance distribution parameters using a multi-Gauss mixture model [111]. The detailed theory behind DEER spectroscopy and the pulse sequences used during DEER distance measurements have been discussed in the recent literature [6, 101, 110, 112].

3 |. Studying Structure and Conformation of Membrane Proteins via SDSL-EPR Methods

SDSL associated with EPR spectroscopy is a widely used biophysical tool to glean structural and conformational dynamic properties of proteins, peptides, macromolecules, and nucleic acids [1, 2, 31, 35, 37, 38, 113–119]. For a more detailed discussion of this topic, we refer to several excellent reviews [1, 6, 24, 31, 38, 113, 118, 120–123].

3.1 |. SDSL-EPR for Studying Conformational Dynamic Properties of Membrane Proteins

Membrane proteins are very crucial for biological functions required for the life of living organisms [124, 125]. They undergo multiple conformations including dynamic changes during their function. For the case of membrane proteins, multiple conformations of the protein while interacting with lipid bilayers are observed as two motional components in the CW-EPR broadening spectrum which is the superposition of the signals arising from multiple motional components [11, 64, 67, 69, 126, 127]. Some examples of membrane proteins studied by CW-EPR approaches to understand the conformational dynamics and topology of the proteins include ABC cassette transporter MsbA [128], integrin β_1a transmembrane domain [67], Escherichia coli ferric citrate transporter FecA [129], the prokaryotic potassium channel KcsA [130], KCNE1 [64], KCNE3 [65, 131, 132], KCNQ1 voltage sensor domain (VMD) [133], functional amyloid Obr2A [134], GM2 activator protein [135], C99 domain of the amyloid precursor protein [136], lactose permease protein [137], bacteriorhodopsin [68, 138], gp28 [139], YiDC [71], GTPase domain of HydF [140], bacteriophage lambda S holin [141], bacterial phospholipase ExoU [142], KvAP voltage-sensing domain [63], and phospholamban (PLB) [143].

A latest illustration of employing CW-EPR spectroscopy is a study of solvent accessibility of YidC protein in a lipid bilayer [71]. The YidC family of proteins is membrane insertases involved in catalyzing the translocation of the periplasmic domain of membrane proteins through a hydrophilic groove residing within the inner leaflet of the membrane. In bacteria, YidC can function on its own or together with the Sec system for protein insertions into the cytoplasmic membrane [144, 145]. YidC is involved in the removal of transmembrane (TM) proteins from the Sec channel and helps in the formation of membrane protein α-helical bundles [146, 147]. E. coli YidC protein contains five TM segment core domains (TM2–TM6) with an unusual hydrophilic cavity within the inner leaflet of the membrane. This cavity is accessible from the cytoplasm and lipid bilayer, but not from the periplasm. Chen et al. performed CW-EPR power saturation experiments on nitroxide spin labeled sites of YidC to determine the water accessibility of residues 366 and 517 under WT conditions (R366C and Y517C) and mutated conditions (Y517N/R366C, Y517I/R366C, Y517C/R366N, and Y517C/R366I) reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposome [71]. The results of EPR power saturation experiments combined with 200-ns molecular dynamics simulations suggested that the positive charge at site 366 in E. coli is required to preserve a hydrophilic microenvironment in the channel when Y517 is replaced with an apolar residue. This is critical for YidC function by adjusting for the polar regions of substrates during the process of insertion.

Another recent example of nitroxide spin labeling CW-EPR spectroscopy is a study of bacteriophage lambda S holin [141]. The bacteriophage lambda S protein holin is a type I holin, containing three TM helices (TMD1, TMD2, and TMD3) and a loosely structured C-terminal domain extending into the cytoplasm [148]. It is produced by the prophage that assembles in the host membrane during late gene expression. It controls the first step in lysing the host cell at the last stage of the viral life cycle by forming large holes which permeabilize the host membrane to allow the escape of virions. Moris et al. performed CW-EPR experiments on the nitroxide spin labeled sites of the span of the S105 protein to examine the topology of the S protein in the assembled hole structure [141]. The CW-EPR spectral lineshape analysis data on the six spin labeled sites of the S105 in POPC/POPG liposomes suggested that the sites in the TM helices (A15C, G20C, T75C, and F78) have broader spectra with minimized backbone motions in these regions [141]. The EPR spectrum of the site A95C showed sharper lineshape indicating no steric hindrance of the label at that site and a highly flexible peptide backbone characteristic of an unstructured region. The site A55C on TMD2 has substantially more constrained motion close to the rigid limit when compared to other TMD sites. The CW-EPR power saturation data collected on several spin label sites indicated that the sites A15C and A95C having negative depth parameters were solvent exposed while sites G20C, A55C, T75C, and F78C having positive depth parameters were buried inside the membrane. Figure 4 shows the helical diagram of the three helices of S105, CW-EPR spectra of MTSL labeled S105 mutants in POPC/POPG liposomes, and EPR power saturation membrane depth parameters profile of MTSL-labeled S105 mutants in POPC/POPG liposomes [141].

FIGURE 4 |.

(A) Helical diagram of the three TMDs of S105 indicating spin labeled positions of residues. The residues associated with the aqueous face are shown with blue. (B) CW-EPR spectra of MTSL labeled S105 mutants in POPC/POPG liposomes. (C) The membrane depth parameter profile obtained from the power saturation EPR measurements of MTSL-labeled S105 mutants in POPC/POPG liposomes. (Adapted from Morris et al. [141] with permission).

The dynamic properties of a potassium channel accessory protein KCNE3 have been recently studied using SDSL EPR spectroscopic techniques [65]. KCNE3 is a single-pass TM β-subunit that modulates the gating and conductance of voltage-gated potassium channels such as KCNQ1. KCNE3 acts as a modulatory subunit that regulates KCNQ1 from a voltage-dependent to a constitutively open channel. This modulation depends on how KCNE3’s TM helix and juxtamembrane regions move and interact with the channel, which are dynamic and lipid-sensitive processes. Reynolds et al. employed SDSL CW-EPR spectroscopic technique to probe the dynamic properties of the KCNE3 protein reconstituted into POPC:POPG (3:1) bilayered vesicles [65]. CW-EPR spectral lineshape analysis was performed on 50 nitroxide spin labeled residue sites of the KCNE3 amino acid sequence including 19 sites on the TM domain and 31 sites on the N- and C-termini of KCNE3 to determine the site-specific motions of KCNE3. The sidechain mobility was quantified by calculating the inverse central linewidth of each EPR spectrum. Figure 5 displays the predicted topology model of KCNE3 based on EPR studies and a plot of inverse central linewidth and motion parameter as a function of residue position. The sidechain mobility data indicated that the sites within the TMD of KCNE3 (57–82) have lower inverse central linewidth values with broader linewidth periodicity pattern profiles when compared to the N- and C-termini. The C-terminal sites display the overall highest inverse central linewidth profile compared to the N-terminus, containing lower values in the helical region. An empirical motion parameter (τ) calculated from the CW-EPR spectrum of each residue site further suggested the slower tumbling motion of spin-labeled sidechains within the KCNE3 TM domain relative to the extracellular termini, with the C-terminal sidechains tumbling more rapidly than those at the N-terminus. Additionally, a structural topology model of KCNE3 was predicted based on EPR results.

FIGURE 5 |.

(A) Predicted topology of the KCNE3 in a lipid bilayer based upon EPR studies with highlighted spin labeling sites in green, (B) inverse of the central EPR resonance linewidth (m_I = 0) plotted against the residue position, obtained from the CW-EPR spectra of KCNE3, and (C) motional parameter (τ) plotted against the residue position derived from the CW-EPR spectra of KCNE3. (Adapted from the Reynolds et al. [65] with permission).

3.2 |. Local Secondary Structure of Proteins and Peptides

In ESEEM approach, a nitroxide spin label is generated at the second, third, and fourth amino acid positions from the ²H-labeled leucine or valine (i) in positive or negative directions ([i ± 2], 3 [i ± 3], or 4 [i ± 4]) on the protein sequence. For an alpha helix of a protein, a feature of 3.6 residues per turn with a pitch of 5.4 Å yields a specific feature in the ESEEM spectra. The frequency domain data (Fourier transform [FT] of time domain data) shows a peak at approximately 2.9 MHz for the i ± 3 or i ± 4 samples, whereas the i ± 2 samples or the control sample containing no ²H-label show no ²H peak [87]. The nitroxide spin labels are further away from the ²H-labeled leucine or valine to be detected for i ± 2 samples. For a β-sheet, a linear pattern of amino acid sequences keeps spin labels within the ESEEM detection limit from the i ± 1 or i ± 2 position of the ²H-labeled leucine (i). This allows deuterium modulation to be observed in the ESEEM spectrum. Similarly, the linear pattern of amino acid sequences of the β-sheet keeps spin labels outside the ESEEM detection limit from the i ± 3 or i ± 4 position of the ²H-labeled leucine (i), causing absence of deuterium modulation in the ESEEM spectrum. The ESEEM approaches can be also used to differentiate the local secondary structures of an α-helix and a 3₁₀-helix [85]. The 3₁₀-helix has a feature of 3.1 residues per turn with a pitch between 5.8 and 6.0 Å. The specific turn features of the α-helix and 3₁₀-helix can produce an opposite deuterium modulation pattern with a larger ²H-ESEEM peak in the FT frequency domain data for the i + 4 samples in comparison with the i + 3 samples for the α-helix, while the opposite trend can be observed for the 3₁₀-helix. This ESEEM approaches can provide local secondary structural information on protein/peptides [84, 85]. More site-specific secondary structural information is revealed with this ESEEM technique when compared with other secondary structure determination biophysical methods including circular dichroism (CD) [82, 84]. CD spectroscopy provides global secondary structural information on proteins, while the ESEEM method can provide local secondary structural information on proteins. Additional NMR active isotopic nuclei such as ¹³C-labeled leucine have been utilized to study local secondary structures of peptides [87]. The Lorigan lab has employed this ESEEM approaches to study several membrane protein/peptide systems including Acetylcholine receptor (AchR) M2δ peptide [79–81, 84, 86, 87], Human KCNE1 [82], LRL₈ peptide [85], Ubiquitin peptide [78], Bacteriophage S²¹ pinholin [94], and gp28 [149].

A recent example of an application of nitroxide based SDSL ESEEM approach is a study of bacteriophage S²¹ pinholin [94]. Holins are small membrane proteins that assemble in the membrane until they are programmed into holin gene [150]. Pinholin S²¹ is a cytosolic membrane protein encoded by the S²¹ holin gene of the lambdoid bacteriophage Φ21 [151]. Pinholins make nanoscale holes that allow the passage of protons to dissipate the protein motive forces leading to the release and activation of the membrane-tethered SAR endolysin for its muralytic action [151, 152]. Pinholin S²¹ contains two holin proteins namely active pinholin and inactive antipinholin, each containing two TMDs. Drew et al. utilized a three-pulse ESEEM approach to determine the local secondary structure of the pinholin S²¹ in its active and inactive conformations [94]. Authors collected three-pulse ESEEM data sets for the two positions (L25 and L50) with deuterated Leu (d₁₀-Leu) on both active and inactive pinholin in 1,2-dimyristoyl-sn-glycero-3-phospho choline (DMPC)/1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles and DMPC liposomes. Figure 6 shows the three-pulse ESEEM frequency domain data of active pinholin S²¹68 (blue) and inactive antipinholin S²¹IRS (red) for TMD1 and TMD2 in DMPC/DHPC bicelles and in DMPC liposomes [94]. These deuterium-labeled sites were chosen to cover for both TM domains (TMD1 and TMD2) of active pinholin S²¹68 and inactive antipinholin S²¹IRS. The ²H-ESEEM modulation peaks were observed for i − 3 and i − 4 positions, but not for the i − 2 positions, for both deuterated leucine sites (L25 and L50). These data suggested that the TMD1 (L25) and TMD2 (L50) adopt α-helical secondary structures in both the active and inactive forms of the pinholin S21. The ESEEM data reported in this study and previous studies provide a useful reference library for applying the ESEEM technique to investigate more complex proteins.

FIGURE 6 |.

(A) Topology model of inactive and active forms of pinholin S²¹, (B) Normalized ²H frequency domain intensity for active pinholin S²¹68 (blue) and inactive antipinholin S²¹IRS (red) for TMD1 (top) and TMD2 (bottom) in DMPC/DHPC bicelles, (C) Normalized ²H frequency domain intensity for active pinholin S²¹68 (blue) and inactive antipinholin S²¹IRS (red) for TMD1 (top) and TMD2 (bottom) in DMPC liposomes. (Adapted from Ahammad et al. [70] and Drew et al. [94] with permission).

Another recent example of using nitroxide-based SDSL ESEEM approaches is the study of a biologically significant phage-encoded lytic protein, gp28 [149]. This protein represents the first member of a new class of phage proteins known as disruptins, which lack spanin genes and provide an iterative mechanism for degrading the outer membrane (OM). gp28 is a cationic antimicrobial peptide (CAMP) that contains three predicted alpha helices and is produced by φKT phage to complement lambda lysis defects. It plays an important role in the lysis process of bacteriophages that lack spanins. Rochit et al. synthesized gp28 constructs using solid-phase peptide synthesis (SPPS) and applied ESEEM spectroscopic technique to study the local secondary structure of the peptide in a POPC/POPG bilayer membrane [149]. ESEEM experiments were performed on a series of three strategically deuterated leucine residues: L18 on helix 1, L30 on helix 2, and L46 on helix 3, with the nitroxide spin label strategically placed at positions i ± 2, i ± 3, and i ± 4 relative to the deuterated site. A ²H-ESEEM modulation signal was observed for the i ± 3 and i ± 4 samples but not for i ± 2. These results indicated that all three predicted gp28 helices adopt α-helical conformation in POPC/POPG bilayer vesicles.

3.3 |. Structural and Conformational Properties of Membrane Proteins Utilizing SDSL-EPR Distance Measurements

EPR spectroscopy in combination with SDSL can be utilized to obtain distances between two spin labels for investigating secondary, tertiary, and quaternary structures of membrane proteins [6, 32]. In addition, these approaches can provide relative orientations between spin labels [34, 55, 56].

3.3.1 |. CW Dipolar Broadening EPR for Distance Measurement

CW dipolar broadening EPR can provide important structural and dynamic information over an intermediate distance range of 8–20 Å [153]. The measurement of magnetic dipolar interactions can yield distances between two spin labels on biomolecules. The dipolar interaction energy inversely varies with the cube of the distance (r³). The electron–electron interaction induces a broadening in the CW-EPR spectrum if the distances between the spin labels are less than 20 Å. Several CW-EPR spectral lineshape analysis tools are available in the field to estimate the dipolar interaction strength from the spectral line broadening function to determine corresponding distances between spin labels [32, 34, 53, 100, 154–157]. Some examples of the available spectral lineshape analysis programs are short distances, Fourier deconvolution, and global data analysis [32, 34, 53, 100, 154–156]. SDSL-CW dipolar broadening EPR has been applied to study several important membrane protein systems such as bacteriorhodopsin [158, 159], erythroid β spectrin [155, 160], bacterial K⁺-translocating protein KtrB [161], WALP peptide [162], sensory rhodopsin II (NpSRII)/transducer NpHtrII from natronobacterium pharaonis [163], magainin 2 peptide [55], AchR M2δ peptide [56], E. coli integral membrane sulfurtransferase (YgaP) [164], and human KCNE1 [54].

One of the important examples of the application of the CW-EPR dipolar broadening approach is a study of AchR M2δ peptide [56]. AchR M2δ is a membrane-spanning peptide that forms a pore-lining region of the ligand-gated ion channel nicotinic acetylcholine receptor (AChR). It has 23 amino acids and forms an α-helical structure in phospholipid bilayers having a helical tilt angle of 12° relative to the membrane bilayer normal [165, 166]. Ghimire et al. performed an X-band CW-EPR dipolar broadening experiment on a doubly TOAC labeled AChR M2δ peptide mechanically aligned using DMPC lipids on planar quartz support [56]. The authors employed global data analysis and rigorous spectral simulations to analyze CW-EPR spectra obtained for randomly dispersed samples and mechanically aligned samples of the doubly TOAC labeled AChR M2δ peptide to determine the distance between the TOAC spin labels and angles determining the relative orientations of the two nitroxide spin labels [56]. The results revealed a distance of 14.6 Å between the TOAC spin labels situated at Positions 7 and 13 that also matched closely the 14.5 Å distance obtained from the molecular modeling of the double TOAC labeled peptide. Figure 7 shows an example of the CW-EPR data on the double TOAC-labeled AChR M2δ peptide in DMPC bilayers and the comparison of the best-fit results to the predicted model of the doubly labeled peptide [56].

FIGURE 7 |.

CW-EPR dipolar line broadening spectra for doubly TOAC-labeled AChR M2δ peptide in DMPC bilayers at 318 K for (A) randomly dispersed sample, (B) parallel aligned (n||B₀) sample, (C) perpendicular aligned (n_┴B₀) sample, and (D) the side view of the comparison of the best-fit data to the predicted model of the doubly labeled peptide. The predicted helical model is indicated in black color. The oxygen, nitrogen, and nearby carbon atoms of the TOAC spin labels are shown by stick models. The TOAC spin labels at residue Positions 7 and 13 are represented in gray and black colors, respectively. The calculated x-, y-, and z-axes of the TOAC spin labels are overspread on the TOAC for the model are represented in red, green, and blue colors, respectively. The set of additional axes represents the position and orientation of TOAC 7 relative to TOAC 13 as back-calculated for the best-fit values of the distance and angles. (Adapted from Ghimire et al. [56] with permission).

3.3.2 |. SDSL-Based DEER Measurements

Biological systems undergo structural and conformational changes during their functional activities. The distance and distance distribution measurements can be widely used to study the structural and conformational dynamics of numerous biological systems including membrane proteins [12, 49, 95, 96, 101, 102, 106, 108, 112, 164, 167–171]. There are technical challenges in DEER spectroscopic measurements that introduce difficulties in obtaining distances with accurate and precise values on membrane protein systems. These challenges include (1) EPR active membrane protein sample preparations in a native bilayer membrane environment at optimum pH condition; (2) high effective concentration of membrane proteins in two-dimensional environments of liposome samples introduces a strong contribution of the background yielding a decrease in the sensitivity, phase memory time, range of distance, and experimental throughput [101, 171]; (3) the nitroxide spin label rotamers and backbone dynamics of the protein have a significant impact on the width of DEER distance distribution measurements; (4) the spin diffusion of the proton due to the presence of hydrogen in the lipid acyl chain solvents and proteins may decrease the values of phase memory times [172]. Great advances have been made in recent years to minimize these difficulties. Membrane proteins have been incorporated into several membrane mimetic systems including bicelles, nanodiscs, lipodisq nanoparticles (SMALPs), mixing with wild-type proteins in the liposomes, low protein to lipid molar ratios during the membrane reconstitutions, rigid/restricted spin label probes, deuterated proteins and solvents, and Q-band pulse EPR approaches [10, 106, 170, 172–179]. An arbitrary waveform generator (AWG) has also been used to enhance pulse sequences in DEER experiments [180]. The modulation depth of the DEER data can be enhanced by increased width of the excitation band of the linear chirp pump pulses that increases the sensitivity of the DEER experiments [172, 180, 181]. Further, extended DEER pulse sequences including 5-pulse and 7-pulse sequences can be used to increase the dipolar evolution window [172, 180, 182]. The refinement of membrane protein structures has been carried out using computational methods of molecular dynamics simulations, alphafold2 (AF2), and DEER distance restraints [13, 63, 170, 183–187]. These advancements have made DEER spectroscopy a very powerful and widely used biophysical tool to study the structure and conformation of numerous membrane proteins. Nitroxide based DEER approach has been employed to study several biologically significant membrane proteins including rhodopsin [188], pentameric ligand-gated channel [189], E. coli integral membrane sulfurtransferase (YgaP) [164], KCNE1 [170, 190], homodimer protein [191], KCNQ1 voltage sensing domain (KCNQ1-VSD) [192], KCNE3 [132, 193], C99 amyloid precursor protein [194], KvAP voltage-sensing domain [63], human dihydroorotate dehydrogenase enzyme (HsDHODH) [195], influenza A M2 protein [14, 196], cardiac Na⁺/Ca²⁺ exchange (NCX1.1) protein [197], Na⁺/Proline transporter PutP E. coli [198], oligomeric anabaena sensory rhodopsin (ASR) [13], tetrameric potassium ion channel KcsA [174], α-synuclein [175, 183], pinholin S²¹ [70, 184], HCN channel [199], voltage-gated sodium channel [200], aspartate transporter Glt_Ph [201], mechanosensitive channel of small conductance (MscS) [202], helical-bundle membrane protein GlpG [203], ABC transporter P-glycoprotein [204], and ABC transporter MsbA [173]. SDSL-DEER spectroscopy has been employed to investigate oligomerization states of numerous membrane proteins including KcsA [15], NhaA Na⁺/H⁺ antiporter of E. coli [12], M2 TM domain [11, 14], β₁-adrenergic receptor (β1AR) [205], LptA [206], anabaena sensory rhodopsin (ASR) [13] and proteorhodopsin [207].

A recent example of utilizing SDSL-DEER spectroscopy is an investigation of the conformational changes induced by the mutations of Pinholin S²¹ [70]. Pinholin S²¹ belongs to the holin family known as a class-II holin. It contains two types of proteins: pinholin S²¹68 (lysis effector) and antipinholin S²¹71 (lysis inhibitor). Pinholin proteins have two TM domains (TMD1 and TMD2). TMD2 of pinholin S21 [68] resides in the lipid bilayer, and TMD1 partially externalizes from the bilayer with some residues resided on the surface. In the case of antipinholin S²¹71, both TMDs reside inside the lipid bilayers [184]. Ahammad et al. utilized DEER and CW-EPR power saturation spectroscopic techniques to directly investigate the effects of mutations on the structure and conformational changes of antipinholin S²¹71 [70]. Authors collected DEER distance data on seven double spin labeled mutants of antipinholin S²¹71 with or without additional substitution of wild-type amino acids. Two spin labels were attached at S8 and L53 in S²¹71 denoted as IRS_S8R1/L53R. Three more constructs were designed with single, double, and triple mutations denoted as IRS_S8R1/L53R1_A17Q, IRS_S8R1/L53R1_A17Q/G21Q, and IRS_S8R1/L53R1_G14Q/A17Q/G21Q with the spin labels at the positions S8 and L53. DEER distance data of all mutant constructs indicated a shorter distance peak centered around 26 Å matching with that of the IRS_S8R1/L53R1, and additional distance population at around 49 Å. The longer-distance population increased with increasing mutation variants. These results suggested the increased hydrophobicity at these residue positions increased the externalization of TMD1 and that the subpopulation was moving away from TMD2 and others remained closely in the inactive conformational form. The tendency for externalization increased with increased hydrophilicity and the effect was increased with the addition of mutations. Figure 8 shows the inactive and active conformation of S²¹71 and DEER distance distributions of IRS_S8R1/L53R1 with or without additional mutations inducing the conformational changes.

FIGURE 8 |.

(A) Inactive and active conformation of S²¹71. Spin label positions are shown in green and mutational points are shown as a red font in the orange circles. (B) Distance probability distributions are color-coded having the same color of the construct name. (Adapted from Ahammad et al. [70] with permission).

Another important example of using DEER spectroscopy is an investigation of the conformational properties of a voltage-gated sodium channel [200]. Voltage-gated sodium channels (Na_vs) are TM proteins responsible for generating the Na⁺ currents underlying the initiation and propagation of action potentials in nerves and muscle fibers [208]. Na_vs are a well-known molecular target in the treatment of neuropathic pain, cardiac arrhythmia, and epilepsy [209–211]. Chatterjee et al. applied SDSL CW-EPR and DEER spectroscopic measurements on membrane-incorporated prokaryotic Na_v homologues to investigate the conformational dynamics of the selectivity filter region in the conductive and slow-inactivated states to regulate the molecular events associated with Na_v gating [200]. The authors performed Q-band DEER experiments on the NaVSp1 (prokaryotic Na_v homologue from Silicibacter pomeroyi) and NaVSp1-P SF regions to measure average distances and distance distributions to determine the magnitude of the conformational differences between these two subunits. The distance distribution data revealed at least two main components: One corresponds to a short adjacent distance, and the other to the long nonadjacent distance. The distance distribution results suggested a rearrangement of the P1 and P2 helices with respect to each other, with the P1 helix moving closer to the pore axis, whereas the P2 helix is moving away from it [200].

Another recent example of utilizing SDSL-DEER spectroscopy is an investigation of the conformational ensemble of the mechanosensitive channel of small conductance (MscS) [202]. The MscS protein is found in the bacterial cell membranes and acts as a safety valve that helps protect the cell from bursting due to changes in osmotic pressure. The MscS channel serves as an essential model system for understanding the function of mechanosensitive (MS) channels in both bacteria and eukaryotes [212]. Lane et al. employed nitroxide-based DEER distance measurements to investigate the structural ensemble of MscS under molecular tension when the channel reconstituted into nanodiscs (ND) in the presence of β-cyclodextrin (CD) [202]. The authors attached MTSL spin labels at sites D76C along the TM helix (TM2) and M47C along the TM helix (TM1) to discriminate between open and closed states and performed DEER distance measurements to determine changes in the equilibrium of MscS conformational states. The DEER distance measurements showed a change in opposite directions for D67R1 and M47R1, with the average D67R1 distance reduced and the average M47R1 distance increased upon opening. The authors utilized DEER distance data to confirm that the MscS conformational ensemble shifts upon lipid depletion and further quantified the associated lipid movement in real time using NMR spectroscopic measurements.

A recent oligomerization study using DEER measurements is the study of β₁-adrenergic receptor (β₁AR) [205]. β₁AR is a TM protein containing seven TM helices. β₁AR belongs to the member of the adrenoreceptor family of class A G protein-coupled receptors (GPCR) and is involved in the stimulation of the sympathetic nervous system [205]. It is mostly expressed in cardiac tissue and regulates the heart rate and strength of cardiac contraction. Kubatova et al. utilized DEER distance measurements between a pair of R1 nitroxide spin labels to investigate the effect of two different sterol analogs of cholesterol (cholesteryl hemisuccinate [CHS] and sodium cholate) on the structural and oligomeric properties of β₁AR in n-dodecyl-β-D-maltoside (DDM) micelles [205]. The results of this study showed that the soluble cholesterol analog CHS binds to spin-labeled β₁AR (C163-R1/C344-R1) solubilized in DDM micelles, while sodium cholate promotes dimerization of spin-labeled β₁AR (C163-R1/C344-R1) in a cooperative manner with an equilibrium dissociation constant of ~35 mM and a Hill coefficient of ~2.5. Additionally, the authors suggested that dimerization of β₁AR in the presence of cholate and its inhibition by specific binding of CHS are unique findings that will help to understand GPCR oligomerization and the mechanism of GPCR signal transduction [205].

4 |. In-Cell EPR Spectroscopy

Understanding the structural dynamics and conformation of biomolecules in a physiological intracellular environment is one of the most challenging aspects for structural biologists. Despite the great advancement of EPR spectroscopy in vitro, its application to membrane proteins in vivo conditions is still very challenging. Recent advances in structural biology approaches including spin labeling methods and different types of spin labels have shown great promise toward the expansion of EPR techniques to study the structure and conformational dynamics of membrane proteins directly inside cells [44, 213–221]. EPR DEER measurements have been very successful using the combination of orthogonal spin labels such as triarylmethyl (TAM) label and nitroxide spin label for detecting protein and ligand interactions in native lipid bilayers [222]. Micro-injection of spin labeled bio-macromolecules into Xenopus laevis oocytes has been used to obtain CW-EPR and DEER distance measurements on protein/peptides inside eukaryotic cells [223, 224]. Recently, Pierro et al. investigated the structural dynamics of the bacterial chaperon NarJ using nitroxide-based CW-EPR spectroscopy in its E. coli natural host [225]. Authors performed CW-EPR spectral lineshape analysis of data obtained on several individual nitroxide spin labeled NarJ derivatives in E. coli cells [225]. The results suggested a decrease in spin label sidechain mobility in E. coli cells when compared with that in vitro. In addition, authors performed DEER distance distribution measurements on doubly nitroxide spin labeled NarJ variants in E. coli cells. Recently, Braun et al. utilized rapid-scan (RS) EPR spectroscopy to investigate the interaction of human α-synuclein (αS) with negatively charged vesicles in vitro and that incorporated into oocytes of X. laevis [226]. Dual nitroxide spin labels, such as MTSL, have been successfully used on OM proteins to perform DEER measurements in intact cells [227]. These spin labels are incorporated into the intact cells at the lower cell density (OD₆₀₀ less than ~10) and at the optimum spin label concentration for achieving maximum spin labeling efficiency [227].

One excellent example of EPR study of membrane proteins in intact cells is an investigation of E. coli vitamin B₁₂ transporter, BtuB in intact cells [228]. E. coli vitamin B₁₂ transporter, BtuB, is a TonB dependent OM protein involved in absorbing vitamin B₁₂ (or cobalamin) [228]. BtuB forms a β-barrel with inner luminal domain and extracellular loops that allow passing cobalamin [229]. Nilaweera et al. performed CW-EPR and DEER distance measurements on the E. coli vitamin B₁₂ transporter, BtuB in intact cells to reveal structural changes that occurred in the core domain during the substrate binding and transport [228]. The authors utilized nitroxide spin labels to produce double spin label sites on BtuB for DEER distance measurements. The DEER distance measurements revealed a substrate-induced structural change in BtuB in the substrate binding loop 3 (SB3) on extracellular apex of the core. Further, this study revealed an alternative and more dramatic structural change in SB3 upon substrate binding with approximately 2-nm displacement of SB3 toward the periplasmic surface of BtuB when the ionic interaction between R14 in the core and D316 in the barrel is broken. The authors remarked that these structural changes are no longer observed when BtuB is reconstituted into a phospholipid bilayer. Figure 9 shows the DEER distance measurements on the V90R1-T188R1 spin pair in cells. Both the apo (blue) and vitamin B12 bound (red) distributions yield two main intramolecular peaks at 2.4 and 3.2 nm, with a substrate-dependent shift observed toward the shorter component [228]. This study highlighted the significance of studying bacterial membrane proteins in their native cell environment.

FIGURE 9 |.

(A) A side view of BtuB showing the spin labeling site V90 in SB3 of the core domain (yellow) and the site T188 near the apex of the 3/4 extracellular loop (light blue). (B) Background-corrected double electron–electron resonance (DEER) data for the apo (blue) and substrate bound states (red) of the V90R1-T188R1 spin pair, where the red traces represent the fits to the data. The resulting distance distributions are shown in (C) with the histograms represent predicted distance ranges. (D) This structural change titrates between two states (labeled 1 and 2) with the addition of vitamin B₁₂. (Adapted from Nilaweera et al. [228]).

5 |. Advantages and Limitations of EPR Spectroscopy for Studying Membrane Proteins

Advantages

1. EPR spectroscopy is a sensitive biophysical approach for studying structural and conformational dynamic properties of membrane proteins. It uses an electron as a spin probe and is more sensitive compared to NMR spectroscopy, which uses a proton nuclear spin.

2. It can be used for any size of the protein as the nitroxide spin labels can be attached to the protein using SDSL.

3. EPR spectroscopy can be performed in any membrane environments including lipid bilayers, nanodiscs, and intact cells. However, NMR spectroscopy can be used mostly in detergent micelles.

4. It requires a lower amount of protein samples and data acquisition times when compared with other structural detection approaches such as NMR.

5. It can provide a wide range of distance measurements on membrane proteins such as < 8 Å using ESEEM approaches, 8–20 Å using CW-EPR dipolar line broadening method, and 20–60 Å using DEER spectroscopy. A longer distance can also be obtained using DEER spectroscopy for the deuterated protein samples in deuterated buffer.

Disadvantages

1. The site-specific spin labeling of membrane protein using nitroxide spin labels may cause structural and functional perturbations. So care should be taken while choosing the particular site for spin labeling on membrane proteins.

2. The incorporation of nitroxide spin labels may be challenging for certain sites of the protein. So additional optimization may be required for achieving optimum spin labeling efficiency

3. The sample optimization may be required for incorporating protein into native-like vesicles for obtaining superior quality of EPR data for structural studies.

6 |. Conclusions

In this review, we highlighted some important advances in EPR spectroscopic approaches and their latest utilization to reveal the structure and conformation-related properties of important membrane protein systems. Nitroxide-based SDSL-ESEEM approach is very useful for understanding local secondary structure of membrane proteins/peptides. DEER distances between nitroxide spin labels and corresponding distance distributions can provide pertinent information about the structure and conformation of complicated membrane protein systems. In-cell EPR spectroscopy can be used to obtain structural and conformational properties of important biological systems in native membrane environment, which is very challenging using in vitro experimental conditions. Although there have been great advances in the technical and methodological development of EPR approaches for studying membrane proteins, researchers still need to work on the universal sample conditions for membrane proteins suitable for EPR measurements to obtain structural and conformational properties of the majority of membrane proteins.

Abbreviations:

CD

circular dichroism

CW

continuous wave

DEER

double electron–electron resonance

DHPC

dihexanoylphosphatidylcholine

DMPC

1,2-dimyristoyl-sn-glycero-3-phosphocholine

DOPC

1,2-dioleoyl-sn-glycero-3-phosphocholine

EPR

electron paramagnetic resonance

ESEEM

electron spin echo envelope modulation

FT

Fourier transform

MTSL

S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate

NLSL

nonlinear least square

NMR

nuclear magnetic resonance

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPG

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)

SDSL

site-directed spin labeling

TM

transmembrane

TMD

transmembrane domain

TOAC

2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.