To assess the extent to which PDSD is suppressed with alternately labelled samples, experiments were conducted at 60 kHz MAS (using a Bruker 1.3 mm probe) on both uniformly and alternately labelled samples of fully protonated GB1 at 600 MHz field. For each sample, aliphatic carbon-detected 2D experiments were run with a “mixing” period (see experimental details) consisting of a typical R1 relaxation delay. Any cross-peaks
observed in such experiments would be evidence of magnetisation transfer between 13C
sites by PDSD. Figure 9.2 shows the spectra resulting from these experiments, with mixing times of 1 s and 3 s. As the bulk T1 for the 13Cα sites in fully protonated GB1
(measured first in 1D) is ~8 s (some individual resonances will be shorter), these times should easily allow sufficient time for any potential polarisation transfer via PDSD, whilst at the same time ensuring that cross-peaks are not unobservable simply because they have decayed beyond detection.
Even at 60 kHz MAS, the difference between the uniformly and alternately labelled samples is stark: after 1 s, numerous cross-peaks are observed for the uniformly labelled sample across the entire aliphatic region (Figure 9.2a). In this case, the total integrated intensity of all aliphatic cross peaks is 47% of that of the diagonal. After 3 s many cross peaks are still seen, with the ratio of cross-peak integrals to diagonal peak integrals even larger at 77%, although most of the peaks originating from CH2 and CH3
sites are missing due to their shorter T1 times. For the [1,3-13C,15N]-labelled sample,
xv While the majority of assignments can be taken as correct with a high degree of
certainty, the assignments for I6, T11, T16, T44, T49, T53 and T55 should be taken as markedly less reliable (and therefore used for proof-of-concept purposes only).
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almost no cross-peaks are observed at either mixing time (Figure 9.2b). At 1 s (3 s), the total integral of all cross-peak intensity is just 9% (11%) of the total auto-peak integral, representing an 81% (86%) reduction in relative peak integral. These are likely to be reduced even further at the higher MAS frequencies available with the 0.8 mm probe. Figures 9.2c-e show comparative slices of the spectra in Figures 9.2a and 9.2b (at the chemical shifts indicated with dotted lines). The most significant cross-peaks that remain
Figure 9.2. 2D 13C-13C correlation spectra obtained from experiments at 600 MHz field
and 60 kHz MAS using a pulse sequence in which the mixing period consisted of a typical R1 delay of 1 s (left) and 3 s (right), for (a) fully protonated [U-13C,15N]GB1 (blue)
and (b) fully protonated [1,3-13C,15N]GB1 (red). Comparisons of example slices (taken at
the chemical shifts shown with dotted lines in (a) and (b)) are shown in (c-e). In each case, the slices are scaled such that the intensities of the autopeaks of the red and blue spectra are matched.
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in the spectrum of the alternately labelled sample appear between ~59 ppm and ~75 ppm in either dimension,xvi which correspond to dipolar transfer between the 13Cα and 13Cβ sites of threonine residues. According to the labelling pattern given by LeMaster et al.
(illustrated in Figure 3.1), only the Cα sites should be 13C-enriched, although the presence
of both 13Cα-1H peaks (<67 ppm in the 13C dimension) and 13Cβ-1H peaks (>67 ppm in
the 13 dimension) for threonine residues in Figure 9.1 attests otherwise. In this respect,
the labelling pattern is, then, more alike to that given by Castellani et al.49, where both the
Cα and Cβ sites in threonine are fractionally 13C-labelled. This would explain the presence
of 13Cα-13Cβ cross peaks in Figure 9.2b – whilst suppressed to an extent, a certain fraction
of the threonine residues in the protein will be enriched at both sites, leading to efficient PDSD. This is likely to be exacerbated in threonine residues where the chemical shift differences between 13Cα and 13Cβ sites are relatively small. Note, however, that neither
suggested labelling pattern predicts the presence of alanine 13C resonances as are
observed (albeit relatively weakly) in Figure 9.1 (and Figure C.5).
Similar experiments were conducted to test for the occurrence of r.f.-driven spin diffusion in aliphatic 13C R
1ρ experiments. These again consisted of 2D 13C-detected
experiments, but with a 13C spin-lock pulse of typical nutation frequency (17 kHz) during
each “mixing” period. Spin-lock times of 10 ms and 100 ms were chosen based on the measured bulk 13Cα relaxation time of ~100 ms. The results of these experiments are
shown in Figure 9.3, for both the uniformly labelled sample (Figure 9.3a) and the [1,3-
13C,15N]-labelled sample (Figure 9.3b). Example slices are given in Figures 9.3c-e. Once
again, even at 60 kHz, the use of alternate carbon labelling significantly suppresses polarisation transfer, with the spectra in Figure 9.3b virtually devoid of cross-peaks. The ratios of total cross-peak integrated intensities to the total diagonal integrated intensities are 14% and 30% for the uniformly labelled sample at 10 ms and 100 ms (respectively), which drop to just 3% and 2% for the alternately labelled sample. Interestingly, cross peaks appear predominantly within a distinctive band running perpendicular to the diagonal and centred at the r.f. frequency. Because of this, the threonine 13Cα-13Cβ cross
peaks that were relatively intense in the R1-like experiments above (Figure 9.2) are much
weaker here. With alternate labelling, it is instead cross-peaks nearer the centre of the spectrum that apparently are of more concern, namely between ~25 ppm and ~37 ppm in either dimension. The cause of these is likely again to be fractional labelling of carbon
xvi Note that for both samples at 1 s, the horizontal rows of weak cross-peaks that appear
at 32.2 ppm and 52.6 ppm in the F1 dimension – a separation of 10.2 ppm either side of
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sites (of for example lysine residues) that are neighbouring and/or are close in chemical shift. There are no cross peaks for 13Cα sites at all.
It can therefore be concluded that, under ≥60 kHz (and potentially slower) spinning conditions, spin diffusion for Cα sites (as well as the majority of other aliphatic
sites) is sufficiently inhibited in alternately labelled proteins for both R1 and R1ρ
experiments to be conducted reliably, with the caveat that for certain residues it can only
Figure 9.3. 2D 13C-13C correlation spectra obtained from experiments at 600 MHz field
and 60 kHz MAS using a pulse sequence in which the mixing period consisted of a typical R1ρ spin-lock pulses of 10 ms (left) and 100 ms (right), for (a) fully protonated [U- 13C,15N]GB1 (blue) and (b) fully protonated [1,3-13C,15N]GB1 (red). Comparisons of
example slices (taken at the chemical shifts shown with dotted lines in (a) and (b)) are shown in (c-e). In each case, the slices are scaled such that the intensities of the autopeaks of the red and blue spectra are matched.
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be significantly reduced rather than eliminated entirely unless more sparsely enriched samples can be used. Spinning the sample even faster will reduce these effects still further for an even smaller contribution to measured relaxation decay rates. This is confirmed for the R1 case (no 13Cα cross peaks were seen for the R
1ρ case even at 60 kHz)
by the spectra shown in Figures 9.4b,c, which are the results of aliphatic 1H-detected 13C- 1H experiments at 86 kHz MAS, which, as above, were performed with typical R
1 delay
periods of 1 s and 3 s. Within the pulse sequence, these elements were inserted after the
13C evolution period. Magnetisation transfer from carbon “A” to carbon “B” would thus
be observed as a cross peak with an identical 13C chemical shift to carbon A, and with the
same 1H chemical shift as carbon B’s directly-bonded protons. In the context of PDSD
between threonine Cα and Cβ sites, these would appear within the box (blue dashed line)
indicated in the figures. Comparing Figures 9.4b,c to a reference 1H spectrum (with no
extra delay or spin-lock pulse, and therefore no spin diffusion; Figure 9.4a), it appears
Figure 9.4. 2D 13C-1H spectra of crystalline fully protonated [1,3-13C,15N]GB1 obtained
at 850 MHz field and 86 kHz MAS using a proton-detected heteronuclear correlation pulse sequence with an “R1-like” delay after 13C evolution of (a) 0 s, (b) 1 s and (c) 3 s.
The presence of additional cross-peaks at longer delay times would be an indication of spin-diffusion effects. The region outlined by the blue dashed line indicates that in which threonine 13Cα-1Hβ and/or 13Cβ-1Hα cross-peaks arising from spin diffusion would appear.
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that no cross-peaks of this sort are present above the level of the noise. Peaks are seen only to disappear (compared to the reference spectrum) due to relaxation effects.