Kaylee R. Troxel

DEER EPR Measurements for Membrane Protein Structures via Bifunctional Spin Labels and Lipodisq Nanoparticles

Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy.

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the α‐helical M2δ subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine‐mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the α‐helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the 2H nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an α‐helix, the i + 3 and i + 4 samples reveal a strong signal from the 2H nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest 2H‐labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (μg).

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).

Probing the Membrane Bound KCNE1 Protein with Solid State NMR Spectroscopy

KCNE1, also known as MinK, is a membrane protein that associates with the KCNQ1 channel protein to form a voltage-gated potassium channel. This ion channel is essential to the cardiac action potential that mediates heartbeat and is also critical for potassium ion homeostasis in the inner ear. Dominant mutations in KCNE1 lead to congenital long-QT syndrome and congenital deafness. KCNE1 has been over expressed in E. coli, purified into micelles using his-tag affinity chromatography, and reconstituted into POPC/POPG vesicles. 31P NMR powder spectra results confirm vesicle formation. Different KCNE1 mutants have been labeled using MTSL, one mutant outside the membrane and the other inside the membrane. By measuring 31P relaxation times of the lipids, we can determine the depth that at which KCNE1 is buried inside the vesicles. We also introduced a bicelle system to study the topology of uniform 15N labeled KCNE1 with respect to the lipid bilayer. By measuring the 15N NMR signal, we are able to figure out the structural topology of KCNE1 within the lipid bilayer.

Utilizing Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy to Probe the Structure of Membrane Proteins

New approaches are needed to more efficiently probe the structural properties of membrane proteins. A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed Electron Paramagnetic Resonance (EPR) technique of Electron Spin Echo Envelope Modulation (ESEEM). This technique can measure short-range distances between a nitroxide spin label and a 2H nucleus out to approximately 8A. For this study a model membrane peptide M2δ, was constructed by solid phase peptide synthesis and inserted into a DMPC/DHPC bicelle membrane. We report for the first time, the direct detection of 2H modulation between a 2H-labeled d8 Val residue and a nitroxide spin label three and four residues away that is characteristic of an alpha-helical secondary structure. Simulations of the ESEEM data reveal a distance of approximately 6.4 +/- 0.5A that agrees well with molecular modeling studies. ESEEM spectra in this work yielded high-quality data in less than an hour with as little as 35μg of protein sample.

Structural Studies on the Conformation of Human KCNEL1 Membrane Protein via Electron Paramagnetic Resonance Spectroscopy

Multi-frequency CW-EPR, Electron Spin Echo Envelope Modulation (ESEEM), and Double Electron Electron Resonance (DEER) coupled with site-directed spin labeling (SDSL), molecular dynamics modeling, and rigorous data analysis can be used to report both qualitative and quantitative information about structure and dynamics of a complex biological system. The short range distances can be measured between isotopically coupled nuclear spins and nitroxide electronic spin labels up to a distance of about 8A using ESEEM and long range distances of 20–70A between two nitroxide electronic spin labels using DEER. The transmembrane domain (TMD) of KCNE1 membrane protein plays a key role in the modulation of voltage gated channel activity. In order to describe the conformation of TMD of KCNE1, cysteine mutants were generated along the TMD and extracellular region of KCNE1 and further modified by MTSL nitroxide spin labels. The purified proteins were reconstituted into model membranes: Fos-Choline, LMPG micelles and POPC/POPG bilayer vesicles. CW-EPR experiments were performed on the mutants at X and Q-bands in the rigid limit and motional regime. A simultaneous multi-frequency EPR data analysis was employed to obtain the dynamic behavior of spin labels along the protein sequence. The isotropic motion of spin probe was found to decrease towards the interior region of the TMD of the protein and reaches a minimum at the G60C position indicating that the motion of the probe is hindered by the nearby overlapped hydrophobic residues and membrane environment. Additional structural information was revealed by performing ESEEM experiments on i+1 to i+5 sites, where i represents the deuterium position V502H on the TMD, and DEER was on sites V47C-I66C and V50C-S68C. The distances extracted from ESEEM and DEER are in good agreement with NAMD/ VMD and MMM modeling results.