Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy

Abstract

Wild-type Phospholamban (WT-PLB), a Ca2+-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca2+-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13° ± 4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100 ns rotational correlation time (τc); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.

Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions

“…It is reversibly inhibited by PLN, a 52 amino acid single-pass membrane protein [11]. PLN exists as a pinwheel-shaped pentamer, with the inhibitory region comprised of the transmembrane domain II and more hydrophilic domain Ib crossing the lipid membrane with a ~15-20° tilt angle [12–14]. The transmembrane domains of the protomers are arranged in a left-handed coiled-coil of approximately 40 Ǻ in length.…”

Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes

“…Bicelles were prepared based on methods previously described [18]. Briefly, 5-DOXYL-stearic acid (5-DSA, Sigma Aldrich, St. Louis, MO) was combined with lipids (DMPC, POPC, DHexPC, Avanti Polar Lipids, Alabaster, AL), and cholesterol (Sigma Aldrich), all dissolved in chloroform.…”

“…Briefly, 5-DOXYL-stearic acid (5-DSA, Sigma Aldrich, St. Louis, MO) was combined with lipids (DMPC, POPC, DHexPC, Avanti Polar Lipids, Alabaster, AL), and cholesterol (Sigma Aldrich), all dissolved in chloroform. This produced bicelles with 700 long-chain lipids per 5-DSA [10], 13% cholesterol (mol cholesterol / mol long-chain lipid) [12, 18], and q (molar ratio of long- to short-chain lipids [4]) = 4. Organic solvent was evaporated under N 2 gas and then completely removed by storage in a vacuum desiccator overnight.…”

“…100 mM HEPES buffer (pH 7.0) was added up to 25% (lipid mass / buffer volume), then the sample was vortexed for 2 min and placed on ice for 5 min. After repeating this process twice, samples were sonicated in an ice bath for 30 min [18]. Prepared lanthanide stock (TmCl 3 *6H 2 O (Sigma Aldrich, St. Louis, MO) in 100 mM HEPES) was added to the sample up to 20% (mol lanthanide / mol long-chain lipid), then vortexed for 2 min [18].…”

“…After repeating this process twice, samples were sonicated in an ice bath for 30 min [18]. Prepared lanthanide stock (TmCl 3 *6H 2 O (Sigma Aldrich, St. Louis, MO) in 100 mM HEPES) was added to the sample up to 20% (mol lanthanide / mol long-chain lipid), then vortexed for 2 min [18]. Samples were then flash-frozen in liquid N 2 three times to further homogenize, yielding a clear, viscous fluid.…”