No abstract

Nucleocapsid (N) encoded by SARS-CoV-2 plays key roles in the replication cycle and is a critical serological marker. Here we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high affinity RNA binding platform. We also map the RNA binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose-coils, showing how N/RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based upon antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.

Molecular dynamics (MD) simulations are used to model changes in the conformational preferences of a model peptide during the transition from a hydrated environment (charged nanodroplet generated by electrospray ionization) to the solvent-free peptide ion. The charged droplet consists of ∼2400 water molecules, 22 hydronium ions, and 10 chloride and contains a single Substance P (SP) [SP + 3H] ion (SP; amino acid sequence RPKPQQFFGLM-NH). Initially, droplet shrinkage involves a combination of solvent evaporation and ejection of excess charge, primarily hydronium ions. Further droplet shrinkage leads to a series of fission events, which includes the loss of some Cl ions. SP ions adapt to the smaller size droplet through small conformational changes that result in coiling of the hydrophobic C-terminus of the peptide on or near the droplet surface, intramolecular interactions involving the hydrophilic N-terminus of the peptide, and water-mediated interactions between the SP ion and HO and Cl ions. Calculated collision cross sections (CCS) for SP ions at various stages of desolvation are consistent with the results obtained from cryogenic ion mobility-mass spectrometry (cryo-IM-MS) measurements. Specifically, early in the decay of the charged droplet SP ions favor an extended conformation, whereas a compact conformer is favored during the final stages of dehydration.

Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biological functionality, and metal ion specificity. Structures of coordinatively saturated metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd, Zn, and Ag are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd-MT, Zn-MT, Ag-MT, and Ag-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addition of each Cd and Zn ion results in the incremental stabilization of unique unfolding intermediates.

Electrospray ionization (ESI) of ubiquitin from acidified (0.1%) aqueous solution produces abundant ubiquitin-chloride adduct ions, [M + nH + xCl]((n - x)+), that upon mild heating react via elimination of neutral HCl. Ion mobility collision cross section (CCS) measurements show that ubiquitin ions retaining chloride adducts exhibit CCS values similar to those of the "native-state" of the protein. Coupled with results from recent molecular dynamics (MD) simulations for the evolution of a salt-containing electrospray droplet, this study provides a more complete picture for how the presence of salts affects the evolution of protein conformers in the final stages of dehydration of the ESI process and within the instrument.