Supporting Information for: “13C- and 1H-detection under fast MAS for the study of
poorly available proteins: application to sub-milligram quantities of a 7 transmembrane protein”.
Hugh R. W. Dannatta,1, Garrick F. Taylora,1, Krisztina Vargaa, Victoria A. Higmana, MarcPhilipp Pfeila, Lubica Asilmovskaa, Peter J. Judgea, Anthony Wattsa,2
aDepartment
1H.R.W.D.
2To
of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU (UK)
and G.F.T. contributed equally to this work.
whom correspondence should be addressed. E-mail: anthony.watts@bioch.ox.ac.uk.
Materials & Methods
Sample Preparation
Bacteriorhodopsin (bR) was expressed in H. salinarum strain S9 using 15N,13C-labelled
synthetic media (Celtone 13C,15N 98%), and purified in the purple membrane as described
previously (Varga et al. 2008). Briefly, H. salinarum was grown over 2 weeks in 1 litre of
labelled media under oxygen-limited conditions and the cells harvested. The cell pellet was
dialysed against 2 l of 0.1 M NaCl and then centrifuged at 20,000 x g for 1 hour to pellet the
purple membrane. It was then resuspended in 30 ml of milliQ water and loaded onto sucrose
gradients of 60% w/v (7 ml), 40% w/v (9 ml), 20% w/v (10 ml). The gradients were ultracentrifuged in a swing-out rotor at 112,000 x g for a minimum of 14 hours. The purple layer
was extracted from the gradients and washed by repeated centrifugation and resuspension in
milliQ water. Finally, the purified purple membrane was resuspended in 20 mM sodium citrate
at pH 6.0 containing 0.01% w/v NaN3.
For the 60 kHz MAS ssNMR data, the purple membranes were then centrifuged into a 1.3
mm NMR rotor using a custom rotor-filling tool (Böckmann et al. 2009) at 29,000 x g for 2 h,
rotating the tool by 180° every 30 mins to ensure even packing.
NMR Spectroscopy at Fast MAS
All fast spinning ssNMR measurements described here were performed on a 18.8 T (1H
larmor frequency of 800 MHz) Bruker Avance III spectrometer at 60 kHz MAS using a 1.3 mm
rotor. The temperature unit was set to 225 K (−48 °C) with a high gas flow rate, which gave a
sample temperature of approximately 20 °C as measured from the chemical shift of the free
H2O signal (Lesage et al. 2008). Lower temperatures could not be attained.
For the 1H-15N CP-HSQC (hNH) spectrum, CP from 1H to 15N was accomplished using a
linearly ramped pulse from 90–100% of a maximum power of 100 kHz on 1H and a
rectangular pulse on 15N at 40 kHz. The CP duration was 1750 μs. CP from
15N
to 1H was
achieved using the same powers, with the ramp on 1H reversed, and with a duration of 600
μs. For the 1H-15N J-HSQC spectrum, transfer was achieved assuming uniform 1JHN values of
90 Hz. Water suppression was carried out with the MISSISSIPPI scheme (Zhou and Rienstra
2008), by storing 15N magnetisation on +z while saturating the water resonance with constant
wave decoupling with alternating x/y phase at 10 kHz for 200 ms. Both dipolar coupling and
scalar coupling-based HSQC experiments were run using 128 15N increments (acquisition
time in 15N of 23 ms) with 96 scans per increment, and a total experiment time of 4 hours per
spectrum.
For the 13C radio frequency driven recoupling (RFDR) (Bennett et al. 1992) spectrum,
selective CP (Laage et al. 2008) from 1H to the 13C aliphatic region was performed using a
linearly ramped pulse from 90–100% of a maximum power of 100 kHz on 1H and a
rectangular pulse on 13C of 20 kHz for 1750 μs, with the 13C offset placed at 40 ppm. The
rotor-synchronised 180° 13C pulses for the 13C-13C transfers were performed with a B1 field of
90 kHz, as lower fields were found to give less efficient magnetisation transfer. The RFDR
mixing was performed for 2 ms. The indirect 13C dimension was recorded with 200 increments
(acquisition time in 13C of 6.2 ms) with 672 scans per increment. The selective CP allowed for
reduction of the indirect dimension to cover only the aliphatic regions without folding C’
signals, reducing total experiment time, which was 38 hrs. 13C carrier frequency was 45 ppm.
For the NCA and NCO spectra, CP from 1H to 15N was achieved as for the CP-HSQC
spectrum, and CP from
15N
to 13C was performed using a rectangular pulse on 13C at 25 kHz
and a tangentially ramped pulse centred at 35 kHz on
15N
for 8000 μs. 80 15N increments
were recorded, giving an acquisition time in 15N of 15 ms, with 1024 scans per increment.
Both NCA and NCO spectra were recorded for 24 hours. For the NCA, the
13C
receiver offset
was 60 ppm, whereas it was set to 176 ppm for the NCO. The 1D 13C CP spectrum was
recorded with the 13C offset set to 40 ppm, an acquisition time of 16 ms, and 96 scans.
For the HNCA spectrum, CP transfers from 1H to 13C and from
described above, whilst the transfer from
13C
13C
to 1H were done as
to 15N was done using identical conditions as for
the reverse transfer in the NCA/NCO spectra as above. 40
(acquisition time of 7.3 ms), whilst 38
15N
15N
increments were recorded
increments were recorded (acquisition time of 3.1
ms) with 176 scans per increment. Total experiment time was 90 hrs.
During 15N evolution periods, 1H decoupling was accomplished using swept low-power TPPM
(slTPPM) (Lewandowski et al. 2010) decoupling at 15 kHz, and a refocusing pulse was
applied on the 13C channel. During 13C evolution periods, 1H decoupling as above was used
while a refocusing pulse was applied on the
15N
channel. During 1H acquisition, WALTZ-16
(Shaka et al. 1983) decoupling at 10 kHz was applied on both the 15N and 13C channels.
During 13C signal acquisition, 1H decoupling was achieved using slTPPM at 15 kHz as above,
and 15N decoupling was achieved using WALTZ-16 decoupling at 10 kHz. No proton
decoupling was employed during transfers between
13C
and 15N spins. For all fast MAS
spectra, the inter-scan delay was 1 s.
NMR Spectroscopy at Slow MAS
Slow spinning ssNMR experiments were run on a 18.8 T (1H Larmor frequency of 800 MHz)
Varian/Magnex Infinity Plus spectrometer equipped with a 3.2 mm HCN Balun probe spinning
at 10.776 kHz in the case of the 13C dipolar assisted rotational resonance (DARR) (Takegoshi
et al. 2001) spectrum, and at 10.0 kHz for the NCA and NCO spectra. The temperature unit
was set to −10 °C, which with frictional heating corresponds to a sample temperature of
approx. 0 °C.
For the NCA spectrum, cross-polarisation (CP) from 1H to 15N was achieved using a
rectangular pulse at 45 kHz on 1H and a tangentially ramped pulse centred at 35 kHz on
for a duration of 1400 μs. CP from
15N
15N,
to 13C was performed using a linearly ramped pulse
from 100-80% of a maximum power of 15 kHz on 13C, and a rectangular pulse at 25 kHz on
15N.
The contact time was 6000 μs. 140 increments in
15N
were recorded, giving an
acquisition time of 14 ms. 256 scans per increment were recorded. The inter-scan delay was
3 s. Total experiment time was 30 hours.
For the NCO spectrum, CP from 1H to 15N was achieved as above. CP from
15N
to 13C was
performed using the same shaped pulses but with B1 fields of 35 kHz on 15N and 45 kHz on
13C.
The contact time was 3500 μs. 160 increments in
15N
were recorded, giving an
acquisition time of 16 ms. 256 scans per increment were recorded. The inter-scan delay was
3 s. Total experiment time was 36 hours.
In both cases, 1H decoupling was performed using a power of 75 kHz throughout, using the
TPPM (Bennett et al. 1995) scheme during acquisition or frequency labelling periods, and
constant wave during the CP from
15N
to 13C. 13C decoupling during 15N frequency labelling
periods was achieved with a single 180° pulse.
The 13C DARR spectrum has been previously published along with the relevant acquisition
parameters (Higman et al. 2011; Varga et al. 2008). Experiment time for the DARR spectrum
was 38 hours. The 1D 13C CP spectrum was recorded using the same CP conditions as the
DARR spectrum, with an acquisition time of 20 ms, and 32 scans.
NMR Spectral Processing & Plotting
All spectra were acquired and processed using the States-TPPI procedure (Marion et al.
1989). NMR data recorded under 60 kHz MAS were processed using Topspin 3.1. All slow
spinning NMR data had previously been processed in NMRPipe (Delaglio et al. 1995),
including the already published DARR spectrum (Higman et al. 2011; Varga et al. 2008), and
so in the interests of a fair comparison the same window functions were used for all 13Cdetected spectra recorded at both spinning speeds: sine bell functions shifted by 90° for both
dimensions. The CP-HSQC spectrum was processed using sine-squared functions shifted by
45° in both dimensions to maximise resolution. The J-HSQC was processed using sinesquared functions shifted by 90° in both dimensions. Finally, the HNCA spectrum was
processed using sine-squared functions shifted by 45°, 45°, and 60° for 1H, 13C, and 15N
respectively.
All 13C-detected spectra were plotted using an increment between levels of 1.2, with the
baseline adjusted to just above the noise. Where the slow spinning
13C-detected
spectra were
adjusted to compensate for longer acquisition times, the base level of the plots were
multiplied relatively by the square root of the ratio of acquisition times. Sensitivity
measurements were performed in NMRView.
Acceleration and Pressure Calculations
Acceleration (relative to earth gravity), a / g:
𝑎⁄𝑔 = 𝑟ω2 ⁄𝑔 = 𝑟(2π𝑓)2 ⁄𝑔
Hydrostatic pressure, p
𝑝 = ρ𝑎𝑟 = ρ(2𝜋𝑓𝑟)2
Where
r = radius (inner radius as given in Table 1), m.
ω = angular speed, rad/s.
g = acceleration due to gravity, 9.81 m/s2
f = oscillation frequency (i.e. MAS rate), Hz.
ρ = density, taken here as 50% sucrose (1230 kg/m3), reflecting the position on the sucrose
gradient used for purification where bR in purple membrane appears.
Results
Cross-Polarisation (CP) Conditions
To ensure maximum sensitivity, various CP matching conditions were tested for the initial
transfer from the 1H nuclei in the measurements at 60 kHz MAS, using the 1H to 13C CP
transfer to test CP efficiency. Initially we tested the low power second-order cross polarisation
(SOCP, n=0) condition (Lange et al. 2009). Although we optimised two conditions, with the B1
fields on both channels set to 9 or 12 kHz (both away from any resonance conditions), we
found this CP condition to give significantly less signal compared to other conditions. We
tested the zero quantum (ZQ, n=1) and double quantum (DQ, n=2) conditions at a range of B1
field combinations, and found those conditions with the largest B 1 fields applied on the 1H
channel (100 kHz) gave the best signal.
Although the ZQ condition with 40 kHz applied on the
13C
channel gave approximately 5%
more signal, it was necessary to use the DQ condition with only 20 kHz applied on
13C
to
ensure that the CP transfers were selective for either aliphatic or carbonyl regions on the
spectrum. 1H to 15N CP transfers were done using the 100/40 kHz combination, as selectivity
was not an issue. These initial CP schemes are relatively high power but make up a very
small proportion of the total length of the pulse sequences, and did not give rise to any
sample heating as monitored by the position of the water 1H signal.
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