Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

Abstract

Obtaining unambiguous resonance assignments remains am ajor bottlenecki ns olid-state NMR studies of protein structure and dynamics.P articularly for supramolecular assemblies with large subunits (> 150 residues), the analysis of crowded spectral data presents ac hallenge,e ven if three-dimensional (3D) spectra are used. Here,w ep resent ap roton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies.T he key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as lowa s2%). As ap roof of principle,weacquired 4D (H)COCANH, (H)CACONH, and (H)CBCANHs pectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in at otal time of two and ah alf weeks.T hese spectra were sufficient to obtain complete resonance assignments in as traightforwardm anner without use of previous solution NMR data.

Protons as Versatile Reporters in Solid-State NMR Spectroscopy

“…(See an elaborate comparison of efficiencies for a deuterated protein spun at 100 kHz by Penzel et al) Figure A and C displays some of those experiments, here employed as purely dipolar versions for assignments of hCAII. These experiments have also been expanded to four dimensions by us and others. ,− More sophisticated magnetization transfer pathways enable correlation between one amide group and either one (3D) or both shifts (4D) of the next one (Figure A (fourth scheme) and D) . At around 60 kHz MAS, the exclusively dipolar transfer pathway of this hNcacoNH (or HNcacoNH) is highly efficient and yields better sensitivity than an hcaCBcaNH under comparable conditions.…”

Dynamics and Interactions of a 29 kDa Human Enzyme Studied by Solid-State NMR

“…Solid-state NMR overcomes critical molecular-weight limitations of solution NMR and thus is amenable for characterization of large proteins or protein assemblies. For example, it has been used extensively for structural elucidation of amyloid proteins, which are poorly accessible for more standard structural-biology techniques. − Similarly, detailed insights for supramolecular assemblies like bacterial needle proteins, virus capsid structures, and structural components of bacteriophages are obtained more and more routinely. − Based on partial protein deuteration and/or fast spinning, the detection of protons has recently been used to increase sensitivity and resolution for residue assignment. − Additional advantages include chemical shifts of protons for resonance assignment, − their high gyromagnetic ratio for proton–proton distance restraints, − and assessment of the amide HN pair for characterization of protein backbone dynamics. − Most solid-state NMR’s target proteins comprise large polymeric networks; however, apart from some larger exceptions in the 30 kDa range, − the monomer size of the structures successfully elucidated has rarely been above 15 kDa. This is because the difficulty in assigning proteins with increasing size scales steeply with the molecular weight due to challenges from sensitivity, spectral overlap, and resolution.…”