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 Ca(2+)-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 Ca(2+)-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 100ns rotational correlation time (τ(c)); 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.

Figure 1

Amino acid sequences of PLB (A) WT-PLB (B) TOAC-10 PLB (C) TOAC-20 PLB (D) TOAC-42 PLB (E) TOAC-45 WT-PLB.

Figure 2

CW-EPR spectra of TOAC-42 PLB [(A), (B), (C)], and TOAC-45 PLB [(D), (E), (F)] in DMPC/DHPC bilcelles at 318 K. (A) and (D) represent parallel aligned (n || B_o) spectra, (B) and (E) represent perpendicular aligned (n ⊥ B_o) spectra, and (C) and (F) represent randomly dispersed spectra. The black traces represent experimental EPR spectra, and red traces are the corresponding fits from NLSL simulations.

Figure 3

CW-EPR spectra of TOAC-10 PLB in DMPC/DHPC bilcelles at 318 K. The black traces represent experimental EPR spectra and red traces are the fits from MOMD simulations.

Figure 4

CW-EPR spectra of 20-TOAC PLB in DMPC/DHPC bilcelles at 318 K. The black trace represents experimental EPR spectra and red trace is the fit from MOMD simulations.