Figure 6

Intra- and interprotomer interactions in DGK. (a) 2D ¹³C-¹³C DARR spectrum of U-¹³C,¹⁵N-DGK-ILV with 800 ms mixing time. Crosspeaks occur between Cα of Ser61 and the sidechain carbons of Trp112, which demonstrates an intraprotomer contact between helices 2 (Ser61) and 3 (Trp112). (b) 2D NCOCX spectrum of U-¹³C,¹⁵N-DGK with a 400 ms DARR mixing step. Crosspeaks between 32ArgNη1/2 and 32ArgNε with 25TrpC’, Glu28Cβ and 29AlaCα/Cβ/C’ are detected, caused by an intraprotomer contact between these residues in helix 1 and the surface helix. (c) DNP enhancement illustrated for a ¹³C-CP spectrum of DGK incubated with 20 mM AMUPol. Upon microwave irradiation, a 45-fold sensitivity enhancement is gained. (d) Activity of DGK with (+) and without (−) AMUPol, demonstrating that the presence of the biradical has no influence on the activity. 100% activity corresponds to the rate recorded with wtDGK in 90 mol% DMPC/10 mol% DMPA of 90 (±9.9) µmol min⁻¹ mg⁻¹. Experiments were repeated three times. The activity was calculated as the mean value. Error bars correspond to standard deviations. (e) DNP-enhanced ¹⁵N-¹³C-TEDOR spectra of mixed-labelled [CN(Arg)]-DGK trimers (right) and of a U-¹³C-labelled control sample ([CC]-DGK) (left). Both spectra show natural abundance crosspeaks (grey box): intra-protomer N-C’ (peak 1), intra-protomer N-Cα (peak 2), intra-residue Arg-Nε,η-Arg-Cζ (peak 3). An additional crosspeak (green box) is observed in [CN(Arg)]-DGK (peak 4). It can be assigned to an interprotomer contact, representing a through-space correlation between Arg and Asn/Asp/Glu. (f) This cross-peak demonstrates that salt bridges or H-bonds between Asp/Glu/Asn and Arg must exist at the protomer interfaces. Mutations R9A, R81A and R92A cause a reduction in intensity of peak (4) (inset in (e)) demonstrating their involvement in these cross-protomer contacts (see Fig. S8d for further details). The TEDOR spectra were recorded with a mixing time of 6.25 ms.