The combination of the fastest MAS and highest magnetic field allows to spectrally resolve the side-chain protons of proteins. It makes ¹H-detected MAS NMR a key player in the study of protein interactions with other macromolecules or nucleic acids.

The proper folding of outer membrane proteins in Gram-negative bacteria relies on their delivery to the β-barrel assembly machinery (BAM) complex. The mechanism by which survival protein A (SurA), the major periplasmic chaperone, facilitates this process is not well understood. We determine the structure of the holo insertase complex, where SurA binds BAM for substrate delivery. High-resolution cryo–electron microscopy structures of four different states and a three-dimensional variability analysis show that the holo insertase complex has a large motional spectrum. SurA bound to BAM can undergo a large swinging motion between two states. This motion is uncoupled from the conformational flexibility of the BamA barrel, which can open and close without affecting SurA binding. Notably, we observed conformational coupling of the SurA swing state and the carboxyl-terminal helix grip domain of BamC. Substrate delivery by SurA to BAM appears to follow a concerted motion that encodes a gated delivery pathway through the BAM accessory proteins to the membrane entry site.

SARS-CoV-2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus–host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV-2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly α-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR. We also show that ORF7b forms heterogeneous higher-order multimers. We determined ORF7b interactions with cellular TM leucine zipper proteins using both biochemical and NMR approaches, providing evidence for ORF7b interaction with the TM domains of E-cadherin, as well as phospholamban. Our results place ORF7b as a hypothetical interferer in cellular processes that utilize leucine zipper motifs in transmembrane multimerization domains.

Experimentally determined protein structures often feature missing domains. One example is the C‐terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic‐acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton‐detected fast magic‐angle‐spinning solid‐state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid‐state NMR observations to residues characterized by large‐amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Experimentally determined protein structures often feature missing domains. One example is the C‐terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic‐acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton‐detected fast magic‐angle‐spinning solid‐state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid‐state NMR observations to residues characterized by large‐amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.