Lishan Liu

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Determining α-helical and β-sheet secondary structures via pulsed electron spin resonance spectroscopy.

A new method has been developed to determine α-helical and β-sheet secondary structural components of aqueous and membrane-bound proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) technique was used to detect weakly coupled (2)H-labeled nuclei on side chains in the proximity of a strategically placed nitroxide spin-label up to 8 Å away. Changes in the ESEEM spectra for different samples correlate directly to periodic structural differences between α-helical and β-sheet motifs. These distinct trends were demonstrated with α-helical (M2δ subunit of the acetylcholine receptor) and β-sheet (ubiquitin) peptides in biologically relevant sample environments.

Enhancement of Electron Spin Echo Envelope Modulation Spectroscopic Methods to Investigate the Secondary Structure of Membrane Proteins

This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a (2)H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. (2)H-SL distance distributions from the MD results indicated that (2)H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the α-helical M2δ peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with (2)H-labeled Leu d(10) at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an α-helix (3.6 residues per turn). Strong (2)H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The (2)H modulation depth observed for (2)H-labeled d(10) Leu was significantly enhanced (×4) when compared to previous ESEEM measurements that used (2)H-labeled d(8) Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of (2)H modulation and the corresponding Fourier Transform (FT) peak intensity for (2)H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different (2)H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (~50 μM).

Solid-state NMR (31)P paramagnetic relaxation enhancement membrane protein immersion depth measurements.

Paramagnetic relaxation enhancement (PRE) is a widely used approach for measuring long-range distance constraints in biomolecular solution NMR spectroscopy. In this paper, we show that 31P PRE solid-state NMR spectroscopy can be utilized to determine the immersion depth of spin-labeled membrane peptides and proteins. Changes in the 31P NMR PRE times coupled with modeling studies can be used to describe the spin-label position/amino acid within the lipid bilayer and the corresponding helical tilt. This method provides valuable insight on protein–lipid interactions and membrane protein structural topology. Solid-state 31P NMR data on the 23 amino acid α-helical nicotinic acetylcholine receptor nAChR M2δ transmembrane domain model peptide followed predicted behavior of 31P PRE rates of the phospholipid headgroup as the spin-label moves from the membrane surface toward the center of the membrane. Residue 11 showed the smallest changes in 31P PRE (center of the membrane), while residue 22 shows the largest 31P PRE change (near the membrane surface), when compared to the diamagnetic control M2δ sample. This PRE SS-NMR technique can be used as a molecular ruler to measure membrane immersion depth.

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides. This chapter introduces a novel approach established and developed in the Lorigan lab to investigate membrane protein and peptide local secondary structures utilizing the pulsed EPR technique electron spin echo envelope modulation (ESEEM) spectroscopy. Detailed sample preparation strategies in model membrane protein systems and the experimental setup are described. Also, the ability of this approach to identify local secondary structure of membrane proteins and peptides with unprecedented efficiency is demonstrated in model systems. Finally, applications and further developments of this ESEEM approach for probing larger size membrane proteins produced by overexpression systems are discussed.

Probing the Secondary Structure of Membrane Peptides Using 2H-Labeled d10-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing (2)H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was (2)H-labeled, such as (2)H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with (2)H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique (2)H-ESEEM patterns were observed with the (2)H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the (2)H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, (2)H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.

Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane‐bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a 2H‐labeled d8‐Val overexpressed membrane protein in a membrane mimetic environment. The membrane‐bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α‐helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α‐helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α‐helical in a more native‐like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X‐ray crystallography and/or NMR spectroscopy.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

Solid-state NMR investigations of peptide-lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106.

The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro-Gly hinge, an amphipathic α-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, (31)P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide-lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid.

Investigating the Secondary Structure of Membrane Peptides Utilizing Multiple 2H-Labeled Hydrophobic Amino Acids via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

An electron spin echo envelope modulation (ESEEM) approach was used to probe local secondary structures of membrane proteins and peptides. This ESEEM method detects dipolar couplings between 2H-labeled nuclei on the side chains of an amino acid (Leu or Val) and a strategically placed nitroxide spin-label in the proximity up to 8 Å. ESEEM spectra patterns for different samples correlate directly to the periodic structural feature of different secondary structures. Since this pattern can be affected by the side chain length and flexibility of the 2H-labeled amino acid used in the experiment, it is important to examine several different hydrophobic amino acids (d3 Ala, d8 Val, d8 Phe) utilizing this ESEEM approach. In this work, a series of ESEEM data were collected on the AChR M2δ membrane peptide to build a reference for the future application of this approach for various biological systems. The results indicate that, despite the relative intensity and signal-to-noise level, all amino acids share a similar ESEEM modulation pattern for α-helical structures. Thus, all commercially available 2H-labeled hydrophobic amino acids can be utilized as probes for the further application of this ESEEM approach. Also, the ESEEM signal intensities increase as the side chain length gets longer or less rigid. In addition, longer side chain amino acids had a larger 2H ESEEM FT peak centered at the 2H Larmor frequency for the i ± 4 sample when compared to the corresponding i ± 3 sample. For shorter side chain amino acids, the 2H ESEEM FT peak intensity ratio between i ± 4 and i ± 3 was not well-defined.