Application of paramagnetic solid-state NMR to amyloids is demonstrated, using Y145Stop human prion protein modified with nitroxide spin-label or EDTA-Cu2+ tags as a model. By using sample preparation protocols based on seeding with pre-formed fibrils we show that paramagnetic protein analogs can be induced into adopting the wild-type amyloid structure. Measurements of residue-specific intramolecular and intermolecular paramagnetic relaxation enhancements enable determination of protein fold within the fibril core and protofilament assembly. These methods are expected to be widely applicable to other amyloids and protein assemblies.

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using 15N rotating frame (R1ρ) relaxation dispersion solid‐state nuclear magnetic resonance spectroscopy over a wide range of spin‐lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two‐state exchange process with a common exchange rate of 1000 s−1, corresponding to protein backbone motion on the timescale of 1 ms, and an excited‐state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited‐state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.

Multidimensional magic-angle spinning solid-state NMR experiments are described that permit cis and trans peptide bonds in uniformly 13C,15N-labeled peptides and proteins to be unambiguously distinguished in residue-specific manner by determining the relative orientations of the amide 13C’ CSA and 1H-15N dipolar coupling tensors. The experiments are demonstrated for model peptides glycylglycine and 2,5-diketopiperazine containing trans and cis peptide bonds, respectively. Subsequently, the measurements are extended to two representative proteins that contain exclusively trans peptide bonds, microcrystalline B3 immunoglobulin domain of protein G and Y145Stop human prion protein amyloid fibrils, to illustrate their applicability to a wide range of protein systems.

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ~10–20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ~60 kHz for samples containing ~10–50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.

The rate of alcoholic hepatitis (AH) has risen in recent years. AH can cause as much as 40–50% mortality in severe cases. Successful abstinence has been the only therapy associated with long-term survival in patients with AH. Thus, it is crucial to be able to identify at-risk individuals in order to implement preventative measures. From the patient database, adult patients (age 18 and above) with AH were identified using the ICD-10 classification from November 2017 to October 2019. Liver biopsies are not routinely performed at our institution. Therefore, patients were diagnosed with AH based on clinical parameters and were divided into “probable” and “possible” AH. Logistic regression analysis was performed to determine risk factors associated with AH. A sub-analysis was performed to determine variables associated with mortality in AH patients. Among the 192 patients with alcohol dependence, there were 100 patients with AH and 92 patients without AH. The mean age was 49.3 years in the AH cohort, compared to 54.5 years in the non-AH cohort. Binge drinking (OR 2.698; 95% CI 1.079, 6.745; p = 0.03), heavy drinking (OR 3.169; 95% CI 1.348, 7.452; p = 0.01), and the presence of cirrhosis (OR 3.392; 95% CI 1.306, 8.811; p = 0.01) were identified as characteristics more commonly found in the AH cohort. Further, a higher inpatient mortality was seen in those with a probable AH diagnosis (OR 6.79; 95% CI 1.38, 44.9; p = 0.03) and hypertension (OR 6.51; 95% CI 9.49, 35.7; p = 0.02). A higher incidence of mortality was also noted among the non-Caucasian race (OR 2.72; 95% CI 4.92; 22.3; p = 0.29). A higher mortality rate despite a lower incidence of alcohol use among non-Caucasian patients may indicate healthcare disparities.