Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein−metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γScrystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged β- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state NMR and solution NMR, as well as cryo-electron microscopy. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

βγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET.

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