Bicelle-enabled structural studies on a membrane-associated cytochrome B5 by solid-state MAS NMR spectroscopy - PubMed (original) (raw)

Bicelle-enabled structural studies on a membrane-associated cytochrome B5 by solid-state MAS NMR spectroscopy

Jiadi Xu et al. Angew Chem Int Ed Engl. 2008.

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Figures

Figure 1

Figure 1

15N isotropic chemical shift spectra of 3.5:1 DMPC:DHPC (1,2-Dimyristoyl-_sn-_Glycero-3-Phosphocholine: 1,2-Diheptanoyl-_sn_-Glycero-3-Phosphocholine) bicelles (a and b), well-hydrated DMPC MLVs (multilamellar vesicles) (c and d) and DMPC MLVs with less hydration (e and f) containing a 268 nmol of 15N-labeled rabbit cyt b5 (protein:DMPC ratio is 1:220) under a 5kHz MAS at room temperature. The intensity and resolution of spectra obtained using two different preparation schemes, namely RINEPT (refocused insensitive nuclei enhanced by polarization transfer) (b, d and f) and RampCP (ramped-cross-polarization) (a, c and e), are different. In the RINEPT sequence, 2.66 and 1.5 ms were used in the first (before the pair of 90° pulses) and second (after the pair of 90° pulses) delays, respectively. A 2 ms contact time was used in RampCP experiments. Observation of isotropic 31P chemical shifts (data not shown) from bicelles under MAS suggests that the magnetic alignment of the bicelle sample was lost and therefore the spectral resolution in 15N MAS spectra was rendered by the sample spinning.

Figure 2

Figure 2

2D 1H/15N chemical shift correlation spectrum of DMPC:DHPC bicelles containing 15N-labeled rabbit cyt b5 under 5kHz MAS. The pulse sequence consisted of a 90° pulse to prepare 1H transverse magnetization, a 180° pulse during t1 to refocus 1H-15N dipolar couplings, a RINEPT sequence to transfer 1H magnetization to 15N, and acquisition of 15N magnetization under 1H decoupling. 128 t1 increments with 64 scans and a recycle delay of 2s were used.

Figure 3

Figure 3

13C chemical shift spectra of DMPC:DHPC bicelles containing 13C&15N-labeled rabbit cyt b5 under 5kHz MAS obtained using (a) RINEPT, (b) RampCP and (c) NOE (nuclear Overhauser effect) sequences. The peaks from DMPC and DHPC molecules are marked as *. Use of deuterated DMPC and DHPC would significantly suppress these signals and further improve the resolution of RINEPT and NOE spectra.

Figure 4

Figure 4

2D 13C isotropic chemical shift correlation spectra of DMPC:DHPC bicelles containing 13C&15N-labeled rabbit cyt b5 under 5kHz MAS obtained using the (CTUC) (constant-time uniform-sign cross-peak) COSY (correlation spectroscopy) (a) and DARR (dipolar-assisted rotational resonance) or RAD (radiofrequency-assisted diffusion)-mixing (b) sequences. Cross peaks correspond to 13C nuclei that are scalar coupled and dipolar coupled appear in the CTUC (a) and DARR (b) spectra, respectively. Cross peaks correspond to specific amino acids are labeled in the spectrum (a).

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