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

Abstract

Obtaining unambiguous resonance assignments remains a major bottleneck in solid‐state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three‐dimensional (3D) spectra are used. Here, we present a proton‐detected 4D solid‐state NMR assignment procedure that is tailored for large assemblies. The 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 low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail‐tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

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

“…To check the significance of this newly discovered interface, we removed the last 7 and 14 C‐terminal residues of gp17.1 (1–170 and 1–163) and observed that both mutants are incapable of polymerization, but remain monomeric (see size‐exclusion chromatography data in Figure S5 and solution NMR data in Figure S6 in the Supporting Information) . This emphasizes the proposition that the C‐terminus forms a molecular bridge that embraces the next monomer within the tube, similar to what was described for the N‐terminus of the T4 phage tail tube protein gp19 …”

Assessment of Non‐Uniform Sampling Schemes in Solid State NMR of Bacteriophage Viruses

“…The ratio between the first contour level and the noise in the projections are set to be the same in every projection, but is different in the uniformly sampled experiments shown in Figure . The goal of performing a lower‐dimensionality uniformly sampled experiment is to confirm that no artifacts are accidentally used in the analysis (or presented above the height of the first contour as defined). We chose to compare the nine schedules by using a similar number of FID scans, emphasizing the difference in time saving when reducing the coverage schedule.…”

Architecture of the flexible tail tube of bacteriophage SPP1

“…After dialysis gp17.1 was left for 3 weeks at room temperature to polymerize. The gp17.1 filaments were sedimented by ultracentrifugation at 90,000 × g for 2 h. 100 mg of gp17.1 protein pellet could be isolated 16 . For methyl-labeling, 12 C 6 ,D 7 -glucose was used instead of 13 C 6 ,D 7 -glucose and specific precursor molecules were supplied to the bacterial cultures 1 h prior to induction (Table S1).…”

“…However, virions only containing gp17.1 are still viable and infectious, indicating that the additional C-terminal FN3 domain is dispensable for phage assembly and infection 15 . gp17.1 monomers are unstable in solution and spontaneously self-polymerize into long tubes in vitro which are indistinguishable from native tubes 6,16 . Previously, we presented the proton-detected solid-state NMR (ssNMR) assignment of deuterated, 100% back-exchanged gp17.1 tubes and deduced secondary structure information from the assigned chemical shifts, which confirmed and extended an existing homology model of a polymerized gp17.1 subunit 6,16 .…”

“…gp17.1 monomers are unstable in solution and spontaneously self-polymerize into long tubes in vitro which are indistinguishable from native tubes 6,16 . Previously, we presented the proton-detected solid-state NMR (ssNMR) assignment of deuterated, 100% back-exchanged gp17.1 tubes and deduced secondary structure information from the assigned chemical shifts, which confirmed and extended an existing homology model of a polymerized gp17.1 subunit 6,16 . Additionally, we introduced new concepts based on the use of specifically labeled isoleucine-methyl groups to simplify ssNMR data, inspired by earlier progress based on methyl-labeling in solution state NMR 17,18 .…”