We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO-CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO-CA transfer block can be incorporated in a set of threedimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for resonance assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous resonance assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO-CA transfer schemes in well-established 3D ssNMR approaches for resonance assignment of solid biomolecules.

Previously introduced for highly deuterated proteins, band-selective magnetization transfer between CO and CA spins by dipolar-based homonuclear cross polarization is applied here to a protonated protein. Robust and efficient recoupling is achieved when the sum of effective radio-frequency fields on CO and CA resonances equals two times the spinning rate, yielding up to 33% of magnetization transfer efficiency in protonated ubiquitin. The approach is designed for moderate magic-angle spinning rates and high external magnetic fields when the isotropic chemical shift difference of CO and CA considerably exceeds the spinning rate. This method has been implemented in NiCOi-1CAi-1 and CAi(Ni)COi-1CAi-1 two-dimensional interresidual correlation experiments for fast and efficient resonance assignment of ubiquitin by solid-state NMR spectroscopy.

By applying [1-13 C]-and [2-13 C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 Å to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region b1-b2 but also for b-strands b1, b2, b3, and b5 as well as for the loop connecting a1 and b3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.

Scrambling, but not the Humpty Dumpty way! The crystallographically confirmed mixed lithium organic [ t BuLi]4·4[Me2NC6H4Li]4 (1) consists of independent alkyl- and aryllithium tetramers in a 1:4 ratio. It has been employed to study the interconversion in solution. 7Li-DOSY and 7Li-EXSY NMR spectroscopy was utilized to establish the nature of that exchange and to determine related rate constants along the equilibria to give [Me2NC6H4Li]4 (A), [(Me2NC6H4Li)3( t BuLi)] (B), [(Me2NC6H4Li)2( t BuLi)2] (C), [(Me2NC6H4Li)( t BuLi)3] (D), and [ t BuLi]4 (E).

The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-(13)C]Glucose ([2-(13)C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-(13)C]pyruvate produced mainly by glycolysis and [3-(13)C]/[1,3-(13)C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs (13)Cβ-(13)Cγ2 and (13)Cα-(13)Cγ1 in Val, and (13)Cγ-(13)Cδ2 and (13)Cβ-(13)Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.

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Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.