Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5

Abstract

Cytochrome b5 (cyt b5) is a membrane-anchored electron-carrier protein containing a heme in its soluble domain. It enhances the enzymatic turnover of selected members of the cytochrome P450 superfamily of catabolic enzymes, localized in the endoplasmic reticulum of liver cells. Remarkably, its alpha-helical membrane-anchoring domain is indispensable for the cyt b5/cyt P450 interaction. Here, we present the first solid-state NMR studies on holo-cyt b5 in a membrane environment, namely, macroscopically oriented DMPC:DHPC bicelles. We have presented approaches to selectively investigate different domains of the protein using spectral editing NMR techniques that utilize the unique motional properties of each domain. Two-dimensional 1H-15N HIMSELF spectra showed PISA-wheel patterns reporting on the structure and dynamics of the membrane anchor of the protein.

Figure 1

Molecular model and ¹⁵N-NMR spectra of uniformly aligned DMPC:DHPC bicelles containing ¹⁵N-labeled cyt b5. The model (A) shows cyt b5 in the context of a DMPC bilayer; the transmembrane, linker, and heme-carrying soluble domains are evident. The ¹⁵N-RINEPT spectrum (B) shows spectral intensity only in the 100-130 ppm region, consistent with a high mobility of the soluble domain. In the RINEPT sequence, 2.6 and 1.3 ms were used in the first (before the pair of 90° pulses) and second (after the pair of 90° pulses) delays, respectively. This spectral region (100-130 ppm) also shows peaks in cross-polarization (CP) spectra at 3.0 ms (C) and 0.8 ms (D) contact times. At a short contact time of 0.1 ms (E), however, spectral intensity is mostly observed in the range from 40 to 90 ppm. This spectral component is most likely from the relatively immobile transmembrane domain. A D₂O-exchange experiment (F) with a 0.8 ms contact time confirms this spectral assignment.

Figure 2

¹⁵N-HIMSELF spectra of U-¹⁵N-cyt b5, recorded with a CP contact time of 0.8 ms (A) and 0.1 ms (B). (C) Recorded after the water in the bicelle sample was replaced by D₂O, suppressing the spectral contributions of the soluble domain.