Fast magic angle spinning NMR with heteronucleus detection for resonance assignments and structural characterization of fully protonated proteins

Abstract

Heteronucleus-detected dipolar based correlation spectroscopy is established for assignments of 1H, 13C, and 15N resonances and structural analysis in fully protonated proteins. We demonstrate that 13C detected 3D experiments are highly efficient and permit assignments of the majority of backbone resonances, as shown in an 89-residue dynein light chain 8, LC8 protein. With these experiments, we have resolved many ambiguities that were persistent in our previous studies using moderate MAS frequencies and lacking the 1H dimension. The availability of 1H isotropic chemical shifts measured with the heteronucleus-detected fast-MAS experiments presented here is essential for the accurate determination of the 1H CSA tensors, which provide very useful structural probe. Finally, our results indicate that 13C detection in fast-MAS HETCOR experiments may be advantageous compared with 1H detection as it yields datasets of significantly higher resolution in the 13C dimension than the 1H detected HETCOR versions.

Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations

“…We have reported the residue-specific assignments of the aliphatic and carbonyl carbons of LC8 previously. 51,52 However, in those earlier data sets aromatic sidechain assignments could not be obtained. As shown in Fig.…”

“…We have reported the residue-specific assignments of the aliphatic and carbonyl carbons of LC8 previously. 51,52 However, in those earlier data sets aromatic sidechain assignments could not be obtained. As shown in Figure 6, the quality of the 2D RFDR spectrum is such that the assignments of the majority of the aromatic sidechains were readily accomplished.…”

Magic Angle Spinning NMR Spectroscopy: A Versatile Technique for Structural and Dynamic Analysis of Solid-Phase Systems

“…The corresponding NCA and NCOCX experiments recorded with conventional 3.2 mm probes at MAS frequencies of 24 kHz and below require 15–30 mg of sample. Panels a–d and e of the figure and the caption are reproduced with permission from refs 19,52,55,56 , respectively.…”

Recent Advances in Theoretical and Physical Aspects of NMR Chemical Shifts

“…Calculated values from plane wave calculations are made to agree with experiment only upon incorporation of motional averaging caused by the fast rotation of the water molecule about its C2 axis (Carnevale et al, 2014). With a magnetic field of 19.9 T and MAS frequency of 62 kHz, three dimensional heteronucleus-detected dipolar based solid state NMR experiments have been used to observe and assign backbone resonances for the 89-residue dynein light chain 8, LC8 protein (Guo et al, 2014). In this scheme, the fast spinning allows for 1 H resonances to be included, which aids greatly in making assignments, while direct observation of 13 C provides greater resolution.…”