Solid‐State NMR H–N–(C)–H and H–N–C–C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins

Abstract

Solid‐state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid‐state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1H‐detected assignment approaches, which correlate a given amide pair either to the two adjacent CO–CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross‐polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide‐protonated proteins from approximately 20 to 60 kDa monomer, at magic‐angle spinning (MAS) frequencies from approximately 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.

ssNMRlib: a comprehensive library and tool box for acquisition of solid-state NMR experiments on Bruker spectrometers

“…Figure 7B shows exemplary sensitivity comparisons of different experiments, for two different protein assemblies. The data are in good qualitative agreement with reported sensitivity comparisons presented elsewhere (Barbet-Massin et al, 2013;Fraga et al, 2017). It is interesting to compare e.g.…”

“…For 13 C-13 C transfers, we have implemented several options, including DREAM (Verel et al, 2001), DARR (Takegoshi et al, 2001), RFDR (Bennett et al, 1992) and BSH-CP (Chevelkov et al, 2013) transfers. Figure 6 shows one backbone pulse sequence from the library, a 3D h-CO-CA-CB experiment with BSH-CP and DREAM transfer steps, which, to our knowledge, has not been proposed before, and its application to a 50 kDa protein that assembles to tube-like structures (Fraga et al, 2017).…”

“…The largest part of the current ssNMRlib implementation comprises 1 H-detected experiments, which are ideally used in combination with deuteration and reprotonation of e.g. amide, methyl or aromatic sites (Barbet-Massin et al, 2013;Fricke et al, 2017;Fraga et al, 2017;Gauto et al, 2019;Xiang et al, 2015;Linser et al, 2010;Zhou et al, 2007). When used at MAS frequencies above 60-100 kHz, fully protonated systems can also yield comparably good 1 H line widths (Stanek et al, 2016).…”

“…Quadrature detection is achieved with phases Φ1, Φ2 and Φ4, whereby the phase is changed by either +90 or -90 degrees, as indicated. (B) Sensitivity comparison of diverse pulse programs with a tube-like protein assembly formed by 50 kDa-large subunits (Fraga et al, 2017) and decameric assembly of 10x21 kDa, in orange and blue respectively. The data were obtained by integration of one-dimensional variants of the pulse sequences.…”

Protein−Protein Interfaces Probed by Methyl Labeling and Proton‐Detected Solid‐State NMR Spectroscopy

“…Recently, protein deuteration and magic angle spinning (MAS) at frequencies at or above 40 kHz have enabled proton‐detected experiments that offer a higher sensitivity and dimensionality than their traditional carbon‐detected counterparts . While these methodologies allow for unambiguous spectral assignments (via a “backbone walk”) of large proteins, structural data such as long‐distance restraints, which are required for 3D structure determination, are still collected in a highly ambiguous and/or insensitive manner. To this date, most restraints are extracted from carbon‐detected experiments (e. g. 2D carbon‐carbon correlations with homonuclear mixing through space), or proton‐detected 4D HNhhNH experiments with radio frequency driven recoupling mixing (RFDR mixing) between protons (correlating amide groups through space) ,.…”