Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

The COVID-19 Community Research Partnership (CCRP) is a multisite surveillance platform designed to characterize the epidemiology of the SARS-CoV-2 pandemic. This manuscript describes the CCRP study design and methodology. The CCRP includes two prospective cohorts, one with six health systems in the mid-Atlantic and southern United States, and the other with six health systems in North Carolina. With enrollment beginning April 2020, sites invited persons within their healthcare systems as well as community members to participate in daily surveillance for symptoms of COVID-like illnesses, testing and risk behaviors. Participants with electronic health records were also asked to volunteer data access. Subsets of participants, representative of the general population and including oversampling of populations of interest, were selected for repeated at home serology testing. By October 2021, 65,739 participants (62,261 adult and 3,478 pediatric) were enrolled, with 89% providing syndromic data, 74% providing EHR data, and 70% participating in one of two serology sub-studies. An average of 62% of participants completed a daily survey at least once a week, and 55% of serology kits were returned. The CCRP provides rich regional epidemiologic data and the opportunity to more fully characterize the risks and sequelae of SARS-CoV-2 infection.

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