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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Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens.

Cataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single- to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of γS; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of γS-crystallin in the human lens.