Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G²FT NMR experiments

A suite of novel (5,3)D G²FT triple resonance NMR experiments encoding highly resolved 5D spectral information is presented for sequential resonance assignment of proteins exhibiting high chemical shift degeneracy. Efficient resonance assignment is achieved by separate joint sampling of (i) chemical shifts which solely serve to provide increased resolution and (ii) shifts which also provide sequential connectivities. In these G²FT experiments, two G-matrix transformations are employed. Peaks are resolved along a first GFT dimension at both Ω(¹⁵N) ⁺ Ω(¹³C′) and Ω(¹⁵N) - Ω(¹³C′), or at Ω(¹⁵N) ⁺ Ω(¹³C^α) and Ω(¹⁵N) - Ω(¹³C^α), to break backbone ¹⁵N,¹H^N chemical shift degeneracy. Sequential connectivities are established along a second GFT dimension by measuring intraresidue and sequential correlations at 2Ω(¹³C^α), Ω(¹³C^{α + 13}Cβ), and Ω(¹³C^α - ¹³Cβ), or at Ω(¹³C^{α + 1}H^α) and Ω(¹³C^α - ¹H^α), to resolve ¹³C^α/β,¹H^α chemical shift degeneracy. It is demonstrated that longitudinal proton relaxation optimization of out-and-back implementations suitable for deuterated proteins and nonlinear data sampling combined with maximum entropy reconstruction further accelerate G²FT NMR data acquisition speed. As a result, the spectral information can be obtained within hours, so that (5,3)D G²FT experiments are viable options for high-throughput structure determination in structural genomics. Applications are presented for 17 kDa α-helical protein YqbG and 13.5 kDa protein rps24e, targets of the Northeast Structural Genomics consortium, as well as for 9 kDa protein Z-domain. The high resolving power of the G²FT NMR experiments makes them attractive choices to study α-helical globular/membrane or (partially) unfolded proteins, thus promising to pave the way for NMR-based structural genomics of membrane proteins.