J. Am. Chem. Soc., 129 (21), 6670 -6671, 2007.
Solid-State NMR Reveals Structural and Dynamical Properties of a Membrane-Anchored Electron-Carrier Protein, Cytochrome b5
Ulrich H. N. Dürr, Kazutoshi Yamamoto, Sang-Choul Im, Lucy Waskell, and Ayyalusamy Ramamoorthy
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 -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.
J. Am. Chem. Soc., 129 (21), 6682 -6683, 2007.
J-Deconvolution Using Maximum Entropy Reconstruction Applied to 13C-13C Solid-State Cross-Polarization Magic-Angle-Spinning NMR of Proteins
Ingo Scholz, Stefan Jehle, Peter Schmieder, Matthias Hiller, Frank Eisenmenger, Hartmut Oschkinat, and Barth-Jan van Rossum
Scalar couplings between 13C spins can impair both resolution and sensitivity in 13C-labeled preparations. It is demonstrated that deconvolution of magic-angle-spinning NMR data with maximum entropy (MaxEnt) reconstruction allows the removal of splittings due to J-couplings without expenses in sensitivity. A combination of MaxEnt reconstruction in t2 with selective pulses in t1 produces fully J-resolved data in both dimensions. The possibility to obtain J-resolved 13C-13C data without compromising the sensitivity is particularly important for solid-state NMR of "difficult" biological samples, like membrane proteins, where sacrifices in signal-to-noise are fatal. The method is demonstrated using preparations of -spectrin SH3 domain (62 residues) as small test system and of outermembrane protein G as example of a membrane protein with higher molecular weight (281 residues). Both preparations were obtained using [2-13C]-glycerol as the carbon source during the bacterial growth.