2.10.1
Bicelle preparation
Isotropic bicelles (Sanders and Schwonek 1992; Vold, Prosser et al. 1997) prepared from long chain phospholipids and short chain detergents were prepared for
solution NMR studies as follows; 20 mg of 1,2-dihexanoyl-sn-glycero-3-phosphocholine
(DHPC) was dissolved in 2 mL of TFE and 10 mg of DMPC in 1 mL of TFE to form 10 mg/mL stock solutions for producing bicelles. For q = 0.25 bicelles, where q=[DMPC]/[DHPC – 15mM] (Vold, Prosser et al. 1997), 681 µL of DHPC in TFE was mixed with 380 µL of DMPC to prepare a lipid bicelle suspension. Bicelles with a q = 0.33 were prepared by combining 681 µL of DHPC and 500 µL of DMPC. Once the two components were mixed, TFE was evaporated off using a stream of nitrogen and samples were placed in a desiccator under high vacuum overnight in order to remove all traces of organic solvent. The resulting lipid film was then rehydrated in 1 mL of HEPES/TRIS buffer.
For BPV E5V2 samples, 321 µL of purified peptide (4 mg/ mL) in TFE was pipetted
into a glass vial. TFE was removed by a stream of nitrogen gas and the sample was placed in a desiccator under high vacuum overnight to remove all traces of organic solvent. To the resulting peptide film, 180 µL of q= 0.25 or q = 0.33 bicelles was added followed by brief sonication to resuspend the film and vortexing before being subjected to five freeze/thaw cycles (liquid nitrogen/40 °C water bath) to form bicelles with a final peptide concentration of 1 mM, a lipid to protein molar ratio of 30:1 (40:1 w/w) and a total amphiphile concentration of 2%. Samples were then transferred to 3 mm bore NMR tubes (Bruker) for analysis by NMR.
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2.10.2Solution NMR experimental procedure
Solution NMR spectra were recorded on a Bruker Avance II 700 MHz solution state NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 700.1 MHz for 1H,
70.94 MHz for 15N and 176.04 MHz for 13C. The spectrometer was equipped with a
cryoprobe (Bruker) running in triple resonance mode for 1H/13C/15N experiments.
Experiments were typically run at 313 K (40 °C) using samples in 3 mM bore NMR tubes (Bruker). 1H chemical shift referencing was carried out with respect to water (4.7 ppm) on
the DSS scale (Wishart, Bigam et al. 1995). Samples contained 10% D2O which was used
to lock onto the spectrometer field during acquisition.
2.10.31D
1H NMR spectroscopy
1D 1H spectra were recorded with a 400 ms acquisition time, 1.25 s recycle delay,
16 co-added transients, and a typically a 7.5 µs π/2 pulse for excitation of 1H with a power
level of 5.9 dB. Spectra were acquired with 32k complex data points in F1 and a spectral
window of 20 ppm. Data were Fourier transformed into 65k complex data points and GM line broadening of -0.2 Hz was applied during processing
2.10.42D heteronuclear NMR spectroscopy
2D 1H-15N heteronuclear single quantum coherence (HSQC) spectra (Schleucher,
Schwendinger et al. 1994) were recorded with 2k data complex data points in F2, 64
increments in F1, and 16 co-added transients per plane. A spectral width of 15 ppm in F2
and 40 ppm in F1 was used. Spectra were recorded at a range of temperatures (25-50
ºC), but typically 40 ºC was used. Spectra were acquired using a π/2 (90º) pulse for excitation of 15N of 2.5 µs and 4 µs with a power level of −1.30 dB, with 16.80 dB 13C
decoupling. The raw data were Fourier transformed (2k (F2) × 256 (F1) complex data
points) and QSINE line broadening (SSB =0.3) in both F2 and F1 applied before peak
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2.10.53D heteronuclear NMR spectroscopy
3D 15N edited 1H-1H-heteronuclear TOCSY-HSQC (Hwang and Shaka 1995) (TOtal
Correlation SpectroscopY) experiments with spin lock times of 40, 60 and 70 ms were recorded to aid assignment of the backbone amide chemical shifts in BPV E5V2. 15N
edited 1H-1H NOESY-HSQC (Nuclear Overhauser Effect SpectroscopY) experiments with
mixing times of 20-300 ms were also recorded to investigate intra- and intermolecular interactions between peptide monomers. Spectra were acquired as pseudo-3D spectra with 2k data complex data points in F3, 256 increments in F1, and only one increment in
F2. The spectral widths for F3, F2 and F1 were set to 15, 40 and 15 ppm, respectively. A
total of 256 co-added transients were acquired for HSQC-TOCSY experiments and 96 co-added transients for HSQC-NOESY experiments. HSQC-TOCSY raw data was Fourier transformed into 2k (F3) × 1 (F2) x 512 (F1) complex data points and QSINE line
broadening of SS1 Hz (F3) and 0.3 Hz (F2) applied. For HSQC-NOESY experiments, raw
data was Fourier transformed into 2k (F3) × 1 (F2) x 256 (F1) complex data points and
QSINE line broadening (SSB =1) (F3) and 0.3 Hz in F2 and F1 applied. Experiments were
typically acquired at 40 °C and all 3D experimental data were processed in Bruker Topspin 2.1.
2.11
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