Magic Angle Spinning (MAS) Solid state NMR spectroscopy

spectroscopy

2.9.1 Solid state NMR experimental procedure

13_{C cross polarisation (CP) magic angle spinning (MAS) NMR spectra were}

recorded on a wide bore Bruker Avance III 500 MHz solid state NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 500.1 MHz for 1_{H, 125.7 MHz for}13_{C and 50.68}

MHz for 15_{N. The spectrometer was equipped with a 4 mm triple resonance magic angle}

spinning (MAS) probe (Bruker) running in double resonance mode for 1_H/13_C

experiments and in triple resonance mode for 1_H/13_C/15_{N experiments. Samples were}

cooled with a Bruker BCU Xtreme unit to 258 K (-15°C) to reduce internal lipid motions, and the spinning frequency set to 8.5 kHz ± 5 Hz maintained by a Bruker BVT 3000 MAS controller unit. External 13_{C chemical shift referencing was carried out with respect to the}

carbonyl peak of natural abundance alanine (177.9 ppm) on the DSS scale (Morcombe and Zilm 2003; Harris, Becker et al. 2008). 15_{N referencing was carried out with respect to}

external 15_{N labelled histidine (46.4 ppm). Samples containing DMPC lipid vesicles}

containing 0 - 0.6 molar ratio cholesterol were prepared in order to study the lipid phase transition at experimental temperatures.

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2.9.2 1D

_P/

_{H lipid NMR experiments}

Static wide line 1D 31_{P and MAS}1_{H spectra were recorded on a Bruker Avance II+}

solid state NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 599.40 MHz for

1_{H and 242.64 for}31_{P and equipped with a 4 mm triple resonance magic angle spinning}

(MAS) probe running in double resonance mode. For 1_{H MAS experiments the spinning}

speed was set to 5 kHz ± 5 Hz maintained by a Bruker MAS controller unit. External 31_P

chemical shift referencing was carried out with respect to the phosphorous peak of

adenosine di-hydrogen phosphate (ADP) (0.9 ppm) on the DSS scale and 1_{H referencing}

to Hα of natural abundance alanine (4.2 ppm). For 1D 31_{P experiments a standard Hahn}

Echo pulse sequence (Rance and Byrd 1983) was used with an echo delay of 50 µs and 80 kHz two pulse-phase modulated (TPPM) proton decoupling during the 40 ms acquisition, and a recycle delay of 5 seconds for 256 co-added transients. Spectra were acquired using a π/2 (90º) pulse for excitation of 1_{H and}31_{P of 2.5 µs and 4 µs}

respectively. 31_{P spectra were acquired with 8k complex data points in F}

1 with a spectral

window of 412 ppm, and data were Fourier transformed into 16k complex data points. 1D

1_{H spectra were recorded for 20k complex points in F}

1 using a single proton pulse for 128

co-added transients with a 3 sec recycle delay with a spectral window of 834 ppm, data were Fourier transformed into 65k complex data points and EM line broadening of -1.0 Hz was applied during processing. Spectra were recorded at temperatures ranging from 253-298 K (20 - 25°C).

2.9.3 1D

_{C NMR experiments}

1D 13_{C spectra were recorded with a 10 ms acquisition time, with 1k complex data}

points in F1 for 1024 co-added transients, and a spectral window of 397 ppm. A 1_H-13_C

cross polarisation (CP) at 73 kHz with a contact time of 1 ms was used, along with 100 kHz proton decoupling during acquisition. Data were Fourier transformed into 65k complex data points and GM line broadening of 20 Hz was applied during processing. Unless otherwise stated, spectra were acquired using a 3 s recycle delay and a π/2 (90º) pulse for excitation of 1_{H and}13_{C of 2.5 µs and 4 µs respectively.}

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2.9.4 2D

_C-

_{C DARR NMR experiments}

Dipolar-assisted rotational recouping (DARR) spectroscopy (Takegoshi, Imaizumi et al. 2000) assists the spin diffusion process by using a combination of physical rotation of the sample and the application of continuous pulses on the proton channel during a mixing time (mix) to reintroduce homonuclear dipolar coupling that is normally

averaged out by magic angle spinning, thereby allowing the observation of long range

13_C-13_{C contacts at long mixing times. In the DARR experiments, carbon magnetisation}

was produced by using a (50-100%) ramped proton CP pulse of 1 ms with RF field strengths of 100 kHz for 1_{H and 80 kHz for}13_{C and transferred using dipolar-assisted}

rotational resonance (DARR). Proton decoupling was performed during signal acquisition at ~100 kHz by using the SPINAL-64 (Fung, Khitrin et al. 2000) (small phase incremental alteration with 64 steps) method employed during signal acquisition periods of 10 ms in the direct dimension and 5 ms in the indirect dimension, with decoupling field strengths of approximately 83 -100 Hz. Experiments were conducted with a range of mixing times (typically 10 - 600 ms) in order to probe short and long distance spin interactions. All 2D 13_C-13_{C DARR spectra were acquired with 994 complex}

data points in F2. Measurements of the mixture of GpA peptides (GpAV & GpAG) were

performed with 339 F1 increments and 308 co-added transients, while measurements of

the doubly-labelled GpA (GpAVG) were performed with 120 increments in F1 and 112 co-

added transients. For all 2D DARR experiments the spectral window for F2 and F1 was set

to 397 and 270 ppm respectively. The data were Fourier transformed to 4k (F2) × 2k (F1)

and GM (Lorentz-Gauss) line broadening of 1.0 Hz in F2 and QSINE line broadening

(SSB=0.3) applied. Spectra obtained were processed and analysed using Bruker Topspin 2.1 software. Cross peak integrals (obtained from cross peak volumes in 2D spectra) were plotted against mix to obtain build-up curves of cross peak intensity for intra- and

inter-helical correlations.

2.9.5 2D

_C-

_{N NMR experiments}

In order to probe through-space carbon and nitrogen coupling between labelled amino acids at the GpA dimer interface, Transfer Echo DOuble Resonance (TEDOR) experiments were used (Fyfe, Mueller et al. 1992; Jaroniec, Filip et al. 2002). Experiments were conducted with a range of mixing times (typically 5-10 ms) in order to probe both

P a g e| 57 short and long-range spin interactions. All 2D 13_C-15_{N TEDOR spectra were acquired with}

3k complex data points in F2 and 20 points in F1. The spectral windows for F2 and F1

were set to 397 and 49 ppm, respectively. Measurements of the mixture of GpA peptides (GpAV & GpAG) were acquired with 3840 co-added transients. The data were Fourier

transformed to 4k (F2) × 128 (F1) and GM (Lorentz-Gauss) line broadening of -1.0 Hz in F2

and QSINE (SSB =0.3) line broadening of 0.3 Hz applied.

In document Development of nuclear magnetic resonance methods for determination of membrane protein structure (Page 76-79)