Aaron T. Coey

Solid-state 2H and 15N NMR studies of side chain and backbone dynamics of phospholamban in lipid bilayers: investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca(2+) transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca(2+) and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with (2)H and (15)N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). (2)H and (15)N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD(3)-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca(2+)-ATPase; thus, showing increased inhibition of Ca(2+)-ATPase.

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).

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.