Application of R70 to DGK

Sample dynamics pose an intrinsic complication for classical homo- and heteronuclear transfer steps. PDSD is an example for the former, as it relies on homo-nuclear dipolar couplings to achieve mixing. These can be highly perturbed, if the sample shows dynamics beyond a certain degree, as was seen for the spectra shown in Fig.

8.10.

Hetero-nuclear transfer, on the other hand, is mostly done via cross polarization or INEPT transfer steps, for rigid and fluid systems, respectively. The efficiency of

8.3. NMR

Figure 8.15: One dimensional spectra of Ile, Val selectively unlabeled DGK and the lipid DOPC. The spectra were recorded with 32, 2048 and 1024 scans for the blue, red and green spectra respectively, using 10kHz MAS at 273K in all cases. All spectra were processed with 20Hz exponential line broadening. Comparing the DP, INEPT and DOPC-DP reference spectra, it can be seen that some, but not all, of the narrow resonances stem from the lipid. This indicates the existence of highly flexible residues in the protein, that can be separated from the rest of the resonances via INEPT polarization transfer. For comparison the spectra have been scaled to the same height of the lipid chain resonances. This corresponds to a scaling factor of 0.214 and 0.3 for the red and green spectra, respectively. The sequences used, are depicted to the right of respective spectra.

Figure 8.16: One dimensional¹⁵N spectra of uniformly¹³C−¹⁵N labeled DGK. The spectra were recorded with 1024, 16 and 128 scans for the blue, red and green spectra respectively, using 8kHz MAS at 264K. The red spectrum was processed with 40Hz all other spectra with 20Hz exponential line broadening. Comparing the DP, CP and INEPT spectra, it can be seen that the narrow resonances that appear in the DP spectrum are selected in the INEPT spectrum, whereas the broad peaks appear in the CP spectrum. For comparison the spectra have been scaled to the same height. This corresponds to a scaling factor of 12 and 8 for the red and green spectra, respectively.

The sequences used are depicted to the right of the respective spectra.

Figure 8.17: ¹H −¹³C correlation spectrum of Ile, Val selectively unlabeled DGK via ¹H −¹³C HETCOR. The spectrum was recorded with 192 scans in the direct dimension, 192 increments in the indirect dimension with 10kHz MAS at 264K. The spectrum was processed with exponential line broadening of 5Hz in the direct dimen-sion and qsine (sine bell shift 3) in the indirect dimendimen-sion. The pulse sequence used is shown as an inset in the spectrum.

8.3. NMR

Figure 8.18: ¹H −¹⁵N correlation spectrum of Ile, Val selectively unlabeled DGK via ¹H −¹⁵N HETCOR. The spectrum was recorded with 192 scans in the direct dimension, 192 increments in the indirect dimension with 10kHz MAS at 264K. The spectrum was processed with exponential line broadening of 5Hz in the direct dimen-sion and qsine (sine bell shift 4) in the indirect dimendimen-sion. The pulse sequence used is shown as an inset in the spectrum.

both of these methods declines rapidly, if the dynamics of the sample is in between a rigid solid and an isotropic solution. CP, for instance, relies on the heteronuclear dipolar coupling to transfer magnetization between the nuclei and is perturbed by sample dynamics as seen in Fig. 8.8. INEPT, on the other hand, is susceptible to dipolar couplings during the transfer period, which lead to strong relaxation during free evolution.

Integral membrane proteins often feature both kinds of domains, rigid as well as solution like, in the form of transmembrane helices and flexible loops. Therefore methods, such as CP and INEPT, can be used as a filter to select certain regions of the protein, but cannot be employed to study the whole protein in a single experiment.

This filtering characteristic can be appreciated in the spectra shown in Figs. 8.15 and 8.16.

TheR70^16,16₆ sequence outlined in chapter 4 was designed to overcome these limi-tations, and to yield a more uniform excitation profile, with respect to motion. Two dimensional¹H−¹³C correlation spectra, with INEPT, R70 or CP as the heteronu-clear transfer step, were recorded of DGK reconstituted into 80:20 DOPC/DOPG (mol/mol), as shown in Fig. 8.19.

Here, it can be seen that no phase errors or artifacts are introduced into the spectrum for R70 based heteronuclear correlation. A striking feature of the R70 spectra of DGK are the resonances in the carbonyl region. These originate from polarization relayed through the homo-nuclearCα−COJ-couplings in the fully¹³Clabeled protein, a magnetization transfer pathway, which does not occur in the INEPT or CP spectra.

This can simplify the identification of genuine protein resonances in theCαregion in the presence of a lipid mixture, as the unlabeled lipids will generally not show transfer to their carbonyl groups, in contrast to the labeled protein.

It has to be noted, that no homo-nuclear decoupling was applied during the ¹H chemical shift evolution, which biases the observation towards mobile residues. This is most likely the cause for the limited amount of resonances observable in the spectra,

Figure 8.19: 2D ¹H-¹³C heteronuclear correlation spectra of reconstituted DGK recorded at 277K and 5kHz MAS. The heteronuclear transfer step was INEPT for the black spectrum,R70^16,16₆ for the red and CP for the blue spectrum (the red and blue spectra have been offset by -0.8ppm and -1.6ppm, respectively). The spectra are similar, with exception of the carbonyl region, whereR70^16,16₆ shows relayed trans-fer. This is due to homonuclear J-transfer active during mixing, leading to a relay of magnetization for the uniformly¹³Clabeled protein. The pulse sequence employed is shown as an inset in the upper left corner of the spectrum. Protein peak pairs, created by relayed transfer, are indicated with black dotted lines.

shift evolution.