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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2018 Apr 22;1860(7):1447–1451. doi: 10.1016/j.bbamem.2018.04.001

Utilization of 13C-Labeled Amino Acids to Probe the α-helical Local Secondary Structure of a Membrane Peptide Using Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Lauren Bottorf 1, Indra D Sahu 1, Robert M McCarrick 1, Gary A Lorigan 1,*
PMCID: PMC5957090  NIHMSID: NIHMS963387  PMID: 29694834

Abstract

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 310-helices by strategically using 2H-labeled amino acids and SDSL. In this study, we have explored the use of 13C-labeled residues as the NMR active nuclei for this approach for the first time. 13C-labeled d5-valine (Val) or 13C-labeled d6-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The 13C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no 13C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.

Keywords: EPR spectroscopy, ESEEM, site-directed spin labeling, isotopic labels, NMR active nuclei, local secondary structure, α-helix

Graphical abstract

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1. Introduction

Membrane proteins are responsible for performing vital biological functions that ensure the survival of living organisms [1]. Despite their physiological importance, minimal structural information is currently available as a result of limited biophysical techniques for studying these challenging protein systems [2,3]. More than 70% of membrane proteins with solved 3-D structures contain α-helical secondary structural motifs, which play important roles in assembly, packing, and membrane protein interactions [4]. Several established spectroscopic methods for studying secondary structural motifs like these include circular dichroism (CD), solid-state nuclear magnetic resonance (ss-NMR) spectroscopy, FT-IR, and FT-Raman [5–10]. However, these techniques are not without limitations. CD is only able to provide global secondary structural information and thus, makes it a challenge to identify local secondary structural motifs within a protein or peptide. Additionally, ss-NMR requires large sample concentrations, which can often be unobtainable for membrane proteins [3]. Aside from CD, the results obtained from these biophysical methods are often ambiguous and require extensive data analysis [8–10].

The Lorigan lab has established a powerful electron spin echo envelope modulation (ESEEM) approach coupled with site-directed spin labeling (SDSL) to probe the local secondary structure of membrane proteins that is advantageous compared to other structural biology approaches because of the high sensitivity and short data acquisition times required [1,4]. ESEEM spectroscopy can detect weakly-coupled NMR active nuclei to nearby unpaired electron spins. Traditionally, 2H-labeled amino acids act as the NMR active nuclei; however, 13C-labeled amino acids have been used for the first time in this study. An unpaired electron is introduced via the most common spin label (SL), 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl methanethiosulfonate (MTSL). If the 2H and SL are within the appropriate detection limit (8 Å for 2H), the weak dipolar coupling will produce 2H modulation in the time domain data after which a Fourier transformation will yield a peak at the corresponding 2H Larmor frequency. Alternatively, for 13C, the theoretical estimated distance will be a maximum of up to 7 Å based on the nuclear spin and gyromagnetic ratio of 13C compared to the detectable 2H ESEEM modulation. Thus, the use of 13C-labeled amino acids should show similar patterns to 2H labeled amino acids. Distinct patterns are observed from the ESEEM data because of the unique turn periodicities of various secondary structural components, which provide insights into the type of secondary structures present in membrane proteins. Previously, we utilized this pulsed EPR method to identify site-specific secondary structural motifs such as α-helices [1,4,11], β-sheets [12], and more recently, 310-helices [13]. Site-specific secondary structural information provides a better understanding of the functions, dynamics, and protein-lipid interactions of membrane proteins [4,14]. Not only has this ESEEM approach been successful for model peptides, it has also been utilized in over-expression systems to probe α-helical secondary structural motifs in both a water-soluble protein and a membrane protein, demonstrating the versatility of this technique [15,16].

Despite this, over-expression systems are extremely challenging to express and purify because site-specific isotopic labeling is tedious and can suffer from scrambling [17]. In addition, incorporation rates of deuterium into selected sites in a recombinant protein system can be as low as 56% [18]. Alternatively, higher incorporation rates of up to 95% can be achieved when using other common isotopes such as 13C [18]. The use of 13C has been better characterized, specifically for use in a variety of NMR spectroscopic techniques, which is beneficial in avoiding long periods of optimization that would be required when using 2H [19–23].

This work explores the use 13C as the NMR-active nucleus for this ESEEM spectroscopic technique for the first time. The α-helical M2δ subunit of the acetylcholine receptor (AChR M2δ) was the model membrane peptide used in this study. AChR M2δ was selectively labeled with 13C5-labeled valine (Val) or 13C6-labeled leucine (Leu) (i) and the SL was placed either 2 or 3 amino acid residues away (i-2 and i-3). Here, we have successfully demonstrated that other NMR active nuclei such as 13C can be utilized for membrane protein local secondary structural determination. This ESEEM approach is comparable to rotational echo double resonance (REDOR) solid-state NMR spectroscopy, which can probe an α-helical secondary structure by measuring dipolar coupling between 13C or 15N nuclei, however, does not suffer from the limitations of large sample concentrations and long data acquisition times [11]. ESEEM spectroscopy can be conducted using small sample concentrations (μM) and uses short data acquisition times (~30 minutes). This method provides an alternative approach to this biophysical technique which extends the application of ESEEM spectroscopy to study more challenging recombinant membrane proteins in the future.

2. Materials and Methods

2.1 Solid Phase Peptide Synthesis

The M2δ subunit of the acetylcholine receptor (AChR) was the α-helical transmembrane model peptide utilized for this study. All peptides were chemically synthesized using Fmoc-solid phase peptide chemistry on a CEM Liberty Blue microwave solid-phase synthesizer [24]. This method is suitable for proteins of up to 70 amino acid residues or less [24]. Seven unique peptides were designed including the wild type (WT) peptide with the following amino acid sequence, EKMSTAISVLLAQAVFLLLTSQR. 13C5- labeled valine at position 15 or 13C6- labeled leucine at position 11 were introduced (i) and a single cysteine residue (X) was substituted 2 and 3 successive positions away (i-3 and i-2) for subsequent MTSL attachment. Additionally, a sample containing no 13C-labeled valine or leucine for the i-3 positions were synthesized for each set of peptides as a control. One full set of peptides are as follows: i–3 (EKMSTAISVLLXQAiFLLLTSQR), i-2 (EKMSTAISVLLAXAiFLLLTSQR), and i-3 control (EKMSTAISVLLXQAVFLLLTSQR).

The peptides were cleaved from their solid support using trifluoroacetic acid (TFA), anisole, triisopropylsilane (TIPS), and H2O (85/5/5/5). Following evaporation under N2 gas, peptides were precipitated using methyl-tertbutyl ether. Reverse-phase HPLC was used for purification using a C4 preparation column with a gradient of 5% to 95% solvent B (90% acetonitrile) [4]. The purified peptides were labeled with 5-fold excess of MTSL (Toronto Research Chemicals) in dimethylsulfoxide (DMSO) for ~16 hours. Excess MTSL was removed using RP-HPLC on a C4 semi-preparative column. MALDI-TOF was used to confirm purity and accurate molecular weight of the target peptides. All peptides were lyophilized and stored as a powder at −20°C. The spin labeled AChR M2δ peptides were then incorporated into DMPC/DHPC (3.5/1) bicelles at a 1:1000 molar ratio following the previously described method to observe the peptides in a native mimetic environment [11]. Lipid bicelles were chosen because they provide a bilayer environment that is less heterogeneous than that of liposomes. Liposome samples have much shorter phase memory times, Tm, due to an uneven distribution of the spin labeled peptides within the membrane resulting in local inhomogeneous pockets of high spin concentration [13]. Thus, the use of bicelles avoids the effects of poorer signal-to-noise ratios for ESEEM signals that are observed when using liposomes.

2.2 Circular Dichroism Spectroscopy

The wild type AChR and labeled AChR constructs were solubilized in trifluoroethanol (TFE) at a concentration of 100 μM. CD spectroscopy was performed on an Aviv Circular Dichroism Spectrometer Model 435 in a rectangular 0.1 cm quartz cuvette. Data was collected from 185 nm to 260 nm with an average of 5 scans per sample and 1 nm bandwidth at 25 °C.

2.3 Three-Pulse ESEEM Spectroscopy

X-band CW-EPR (~9 GHz) spectroscopy was used to measure spin concentrations of the MTSL-labeled M2δ peptides (150 μM). All samples showed 80-90% labeling efficiency and the three-line EPR spectrum showed significant lineshape broadening which indicated successful incorporation of peptides into the lipid bilayer. Three-pulse ESEEM measurements were performed on a Bruker ELEXSYS E580 with an ER4188X MS3 resonator. Initially, tau (τ) optimization experiments were conducted to determine the optimal τ value to suppress 1H modulation and enhance 13C modulation. Three different τ values were run at 198 ns, 268 ns, and 408 ns. Following this, all subsequent measurements used a 408 ns τ value to suppress 1H modulation while enhancing 13C modulation. Three-Pulse ESEEM data were collected at ~9.279 GHz at 80 K. A starting T of 386 ns and 512 points in 12 ns increments were used to collect the ESEEM spectra. Approximately 40 uL of bicelle-peptide sample were used to collect the data.

The original ESEEM time domain data were normalized by division through a polynomial fit and subsequent subtraction of unity as described previously [15, 25–27]. The LPSVP algorithm was used for back-prediction of the missing data points [28]. The data were then processed further using Hamming apodization and zero filling [29]. A cross-term averaged Fourier Transformation (FT) was then used on the resulting spectrum to generate the corresponding frequency domain with minimized dead time artifacts as has been previously established [14]. The 13C peaks were observed at 3.5 MHz representing the 13C Larmor frequency.

3. Results and Discussion

3.1 Global Secondary Structure

Circular dichroism (CD) spectroscopy was utilized to examine the global secondary structure of the model peptide, AChR M2δ. Figure 1 shows the CD spectra for the WT AChR M2δ as well as the CD spectra for the peptide constructs labeled with either 13C5- labeled Val or 13C6-labeled Leu and the attached MTSL label. Double minima were observed at 220 nm and 208 nm, which is indicative of α-helical global secondary structure for both the WT AChR and the labeled peptides [5,6]. Additionally, there was not a significant difference in the overall spectra for the peptide constructs that had been labeled. This ensures that the addition of 13C in a single Val or Leu amino acid side chain and the addition of the cysteine residue with the attached spin label did not significantly perturb the secondary structural integrity of these peptides. Due to light scattering, it was difficult to collect the CD spectra for the bicelles, and thus TFE, an α-helical inducing solvent, was used to examine the global secondary structure.

Figure 1.

Figure 1

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Circular Dichroism of AChR 13C5-labeled Vali15 i-3 and AChR 13C6-labeled Leu11 i-3 compared to WT AChR in Trifluoroethanol (TFE)

3.2 Tau Optimization

Figure 2 represents the various experimental τ values used for these ESEEM experiments. In three-pulse ESEEM experiments, the peak amplitudes and modulation depth are dependent upon the tau (τ) value [30]. The τ value is the constant delay time between the first and the second microwave pulses in the three-pulse ESEEM experiment (π/2- τ -π/2-T-π/2-τ+T-echo) [1]. Optimal τ values vary depending on the field and frequency at which the data were collected [1]. Typical three-pulse ESEEM experiments for 2H nuclei are run around a τ value of 200 ns; however because this technique is using 13C nuclei for the first time, the τ value of the experiment had to first be optimized.

Figure 2.

Figure 2

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Frequency domain data collected for AChR 13C labeled Valine15 at the i-3 position in DMPC/DHPC bicelles at several τ values. 408 ns is the optimal τ value where the13C peak at 3.5 MHz (dotted line) is at a maximum. At 268 ns, the suppression of both 13C and 1H is observed. 198 ns did not maximize the 13C peak. The minor peak observed at 5.7 MHz is from the 13P nuclei in the lipid bicelles.

In Figure 2, three different τ values were examined. The τ value at 198 ns, which is typical of 2H three-pulse ESEEM experiments, had both a peak at the 1H Larmor frequency at 14 MHz as well as the peak for the 13C nuclei at 3.5 MHz. In an effort to suppress both the 13C peak and the 1H peak, a τ of 268 ns was chosen. Here, the 13C Larmor frequency peak was completely suppressed, and the 1H peak was slightly enhanced. Ultimately, a τ value of 408 ns was used. This τ value was able to successfully enhance the 13C modulation. For these three-pulse ESEEM experiments, choosing a τ value that will enhance the Larmor frequency of the nuclei being observed is the ideal experimental set-up. In addition, at these various τ values and another minor peak was also observed at 5.7 MHz. This peak is from the 31P nuclei that are present in the DMPC/DHPC bicelles that is weakly coupled to the electron spin of the SL attached to the peptides [29]. The 13C modulation could not be easily observed in the time domain data, however, the FT frequency domain was able to show the 13C signal as seen in Figure 2. The three pulse ESEEM time domain data for AChR 13C labeled Valine15 at the i-3 position in DMPC/DHPC bicelles are shown in supporting information (Figure S1).

3.3 Three-pulse ESEEM for Local α-helical Secondary Structure Characterization

A three-pulse ESEEM experiment was performed using 13C as the NMR-active nuclei in exploring the local secondary structure of the α-helical model membrane peptide, AChR M2δ in DMPC/DHPC bicelles. Figure 3 shows the ESEEM frequency domain data for 13C5 -labeled valine at position 15 (i) and the MTSL attached to a cysteine residue (X) either 2 or 3 amino acids away (denoted i-2 and i-3). Here, a peak was observed at the 13C Larmor frequency at 3.5 MHz for the i-3 sample. For the i-2 sample, no peak was observed because it is outside the detection limit. In addition, in a control sample with no 13C labeled Val, no peak was observed since there are no 13C nuclei. This type of pattern is similar to what has been found for 2H labeled amino acids and are strongly indicative of the α-helical local secondary structure of this model peptide [1,11,13,15].

Figure 3.

Figure 3

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Three-Pulse ESEEM Frequency domain data of AChR 13C labeled Val15 in DMPC/DHPC bicelles. The i-3 sample shows a peak at the 13C Larmor frequency at 3.5 MHz. No peaks are observed for the control sample without 13C Valine or the i-2 sample.

In previous studies, 2H-labeled Leu has been shown to provide better signal to noise when compared to 2H labeled valine [11]. We explored the use of 13C further by also collecting three-pulse ESEEM spectra for 13C6-labeled Leu at position 11 on this model peptide as shown in Figure 4. For the 13C labeled Leu, a peak was observed at 3.5 MHz for the i-3 sample representing the 13C Larmor frequency. No peak was observed for the i-2 sample or the control sample with no 13C. This pattern again matched with the expectation of the known α-helical secondary structure for this peptide. The peak for the i-3 for 13C labeled Leu sample was similar to what was observed for the 13C labeled Val. It is likely that 13C-labeled Leu does not show an improvement in signal intensity like 2H labeled leucine amino acids because the 13C nuclei are in the backbone of the amino acids when compared to 2H, which are on the perimeter and are more mobile and likely to come closer in distance. Since the modulation depth for ESEEM is proportional to 1/r6, this is less variable for 13C-labeled amino acids compared to 2H labeled amino acids11. In addition, Leu only has one more carbon atom than Val whereas Leu has two more 2H atoms than Val. The more nuclei present, the better the signal intensity will be. As a result, for 13C-labeled amino acids, the signal observed is similar for both 13C-labeled Val and 13C-labeled Leu.

Figure 4.

Figure 4

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Three-Pulse ESEEM Frequency domain data of AChR 13C labeled Leu11 in DMPC/DHPC bicelles. The i-3 sample shows a peak at the 13C Larmor frequency at 3.5 MHz. No peaks are observed for the control sample without 13C Leu or the i-2 sample.

These data verify that 13C can be used successfully as the NMR active nuclei for this ESEEM approach to determine local secondary structure. However, it is clear from these samples that the signal intensities and signal to noise ratios when using 13C is much less than that of 2H-labeled amino acids. This is because 13C has nuclear spin (I) =1/2, whereas 2H has a nuclear spin (I) =1. This means that 13C is approximately three times less sensitive than 2H which results in lower signal intensity and poorer signal to noise. Since the sensitivity of ESEEM experiments is mainly dictated by the modulation depth of these experiments (k), for I=1/2, nuclei the FT peak amplitude is lowered significantly, resulting in a loss of sensitivity when using 13C compared to 2H [31–33]. Taking into account the nuclear spin and the gyromagnetic ratio of 13C, the maximum distance detectable is a maximum of 7 Å, whereas for 2H the detection limit is up to 8 Å. For 13C, however, the experimental modulation depth depends upon the choice of amino acid site and the nature of the sample preparation. Thus, the detection limit may slightly vary from the theoretical distance estimated using the detectable 2H ESEEM modulation. Additionally, there are fewer 13C nuclei in amino acids than there are 2H atoms when using these labeled amino acids, so there are fewer NMR active nuclei to interact with the electron spin when using 13C compared to 2H. Taken together, 13C-labeled amino acids are not as sensitive when compared to 2H labeled residues; however, this technique still provides pertinent local secondary structural information while overcoming the limitations of comparable solid-state NMR experiments. This also provides an alternative approach that could be useful for studying more challenging recombinant membrane proteins.

4. Conclusions

In summary, while the I=1/2 spin system suffers from lower sensitivity for this three-pulse ESEEM technique when compared to the I=1 for 2H, 13C has been able to be successfully used as the NMR-active nuclei in order to determine local secondary structure of a model membrane peptide for the first time. This technique using 13C-labeled amino acids is comparable to REDOR solid-state NMR spectroscopy techniques; however, it requires less sample concentration (μM) and shorter data acquisition times (~30 minutes) making it ideal for studying membrane protein secondary structure [34,35]. This method provides an alternative approach to this biophysical technique in order to provide more versatility and options when used to study a variety of secondary structures.

Supplementary Material

supplement
NIHMS963387-supplement.docx (124.8KB, docx)

Highlights.

  • ESEEM technique was expanded for the utilization of 13C-labeled amino acid for studying membrane proteins.

  • 13C-Labeled Leucine and Valine were used to probe α-helical local secondary structure of a M2δ (AChR M2δ) peptide using ESEEM spectroscopy.

  • ESEEM spectroscopy in combination with 13C-labeled amino acids can provide alternative options to study various membrane protein secondary structures.

Acknowledgments

This work was generously supported by the NIGMS/NIH Maximizing Investigator’s Research Award (MIRA) R35 GM126935 Award. The pulsed EPR spectrometer was purchased through funding provided by the NSF (MRI-1725502), the Ohio Board of Reagents and Miami University. Gary A. Lorigan would also like to acknowledge support from the John W. Steube Professorship.

Abbreviations

EPR

Electron Paramagnetic Resonance

ESEEM

Electron Spin Echo Envelope Modulation

SDSL

Site-Directed Spin Labeling

Footnotes

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