Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

During the COVID-19 pandemic, an at-home enzyme assay was developed for a biochemistry laboratory course at Arizona State University. The experiment was designed to use items that could be easily obtained and safe to use. The experiment focused on the analysis of salivary amylase using starch from food as a substrate, black tea as an inhibitor, and tincture of iodine to quantify the amount of starch present. In this article, we describe the design of the experiment and an analysis of the student performance with specific mention of the common problems that the students experienced. While this experiment was specifically designed as an at-home experiment, it could easily be adapted to use in a typical undergraduate biochemistry laboratory.

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Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combating the 2019 SARS-CoV-2 pandemic. Following emergency use authorization, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination. To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation. We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT.

The photochemical reaction center (RC) features a dimeric architecture for charge separation across the membrane. In green sulfur bacteria (GSB), the trimeric Fenna-Matthews-Olson (FMO) complex mediates the transfer of light energy from the chlorosome antenna complex to the RC. Here we determine the structure of the photosynthetic supercomplex from the GSB Chlorobaculum tepidum using single-particle cryogenic electron microscopy (cryo-EM) and identify the cytochrome c subunit (PscC), two accessory protein subunits (PscE and PscF), a second FMO trimeric complex, and a linker pigment between FMO and the RC core. The protein subunits that are assembled with the symmetric RC core generate an asymmetric photosynthetic supercomplex. One linker bacteriochlorophyll (BChl) is located in one of the two FMO-PscA interfaces, leading to differential efficiencies of the two energy transfer branches. The two FMO trimeric complexes establish two different binding interfaces with the RC cytoplasmic surface, driven by the associated accessory subunits. This structure of the GSB photosynthetic supercomplex provides mechanistic insight into the light excitation energy transfer routes and a possible evolutionary transition intermediate of the bacterial photosynthetic supercomplex from the primitive homodimeric RC.

Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. All available structures from the genus Borrelia represent proteins expressed without a membrane-targeting signal peptide, thus avoiding conserved pathways that modify, fold and assemble membrane protein complexes. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. P66 was also expressed using the PelB signal sequence fused to maltose binding protein. Membrane-associated BBA57 lipoprotein was solubilized by non-ionic and zwitterionic detergents. We show BBA57 translocation to the outer membrane, purification at a level sufficient for structural studies, and evidence for an α-helical multimer. Previous studies showed multiple critical roles of BBA57 in transmission, joint arthritis, carditis, weakening immune responses, and regulating other Bb outer surface proteins. In describing the first purification of membrane-translocated BBA57, this work will support subsequent studies that reveal the precise mechanisms of this important Lyme disease virulence factor.

Infrared spectroscopy is a powerful technique for characterising protein structure. It is now possible to record energy losses corresponding to the infrared region in the electron microscope and to avoid damage by positioning the probe in the region adjacent to the structure being studied. Spectra from bacteriorhodopsin, a protein that is predominately a α helix, and OmpF porin, a protein that is mainly β sheet show significant differences over a spectral range from ∼0.1 to 0.25 eV (∼1000 to 1800 cm -1 ). Although the energy resolution equivalent to 60 cm -1 is inferior to Fourier Transform InfraRed Spectroscopy (FTIR) the spectra are very sensitive to molecular orientation. Polar bonds aligned parallel to the specimen grid make particularly strong contributions to the energy loss spectra.Ultra-high-resolution energy loss spectroscopy in the electron microscope can potentially add useful information to imaging and diffraction for determining the secondary structure misfolding believed to be responsible for dementia diseases such as Alzheimer's.

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