Function and Aggregation in Structural Eye Lens Crystallins

Abstract

Conspectus Crystallins are transparent, refractive proteins that contribute to the focusing power of the vertebrate eye lens. These proteins are extremely soluble and resist aggregation for decades, even under crowded conditions. Crystallins have evolved to avoid strong interprotein interactions and have unusual hydration properties. Crystallin aggregation resulting from mutation, damage, or aging can lead to cataract, a disease state characterized by opacity of the lens. Different aggregation mechanisms can occur, following multiple pathways and leading to aggregates with varied morphologies. Studies of variant proteins found in individuals with childhood-onset cataract have provided insight into the molecular factors underlying crystallin stability and solubility. Modulation of exposed hydrophobic surface is critical, as is preventing specific intermolecular interactions that could provide nucleation sites for aggregation. Biophysical measurements and structural biology techniques are beginning to provide a detailed picture of how crystallins crowd into the lens, providing high refractivity while avoiding excessively tight binding that would lead to aggregation. Despite the central biological importance of refractivity, relatively few experimental measurements have been made for lens crystallins. Our work and that of others have shown that hydration is important to the high refractive index of crystallin proteins, as are interactions between pairs of aromatic residues and potentially other specific structural features. This Account describes our efforts to understand both the functional and disease states of vertebrate eye lens crystallins, particularly the γ-crystallins. We use a variety of biophysical techniques, notably NMR spectroscopy, to investigate crystallin stability and solubility. In the first section, we describe efforts to understand the relative stability and aggregation propensity of different γS-crystallin variants. The second section focuses on interactions of these proteins with the holdase chaperone αB-crystallin. The third, fourth, and fifth sections explore different modes of aggregation available to crystallin proteins, and the final section highlights the importance of refractive index and the sometimes conflicting demands of selection for refractivity and solubility.

The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study

“…In some cases, such as for γ-crystallin, pressures of several hundred bar were shown to be able to induce the transformation from the phase-separated to the homogeneous solution state 9 – 11 . Here, we applied, to our knowledge for the first time, the pressure-perturbation approach to study also the kinetics of the LLPS process of a protein from the family of crystallins, which belong to the family of mammalian lens proteins and are particularly concentrated in the cytoplasm of the eye lens cells 12 , 13 . Perturbation by high pressure has become increasingly popular in the field of protein folding due to the specific effects of pressure on non-covalent bonds, mainly hydrophobic and electrostatic interactions, and on protein systems displaying a significant amount of packing defects or void volume 14 – 17 .…”

“…We chose the protein γD-crystallin, which is a major component of the mammalian lens proteins and a good model system for globular proteins undergoing LLPS 22 . The concentrations of lens proteins reach values as high as several hundred mg mL −1 , and their dense and homogeneous packing defines the refractive index of the eye lens, which needs to be transparent 12 , 13 . Density and concentration fluctuations imposed by LLPS and/or protein crystallization induce light scattering and hence loss of lens opacity, which is the cause of cold cataract.…”

“…Density and concentration fluctuations imposed by LLPS and/or protein crystallization induce light scattering and hence loss of lens opacity, which is the cause of cold cataract. In fact, highly concentrated solutions of crystallins are known to undergo phase separation, however at low temperatures only, but their UCST depends on the protein’s specific amino acid sequence and can change significantly through mutations 12 , 13 , 22 , 23 . In view of the charged and hydrophobic residues on the folded γD-crystallin surface, the phase-separated condensed phase at low temperature is stabilized by transient electrostatic, hydrophobic, and van der Waals interactions among neighboring protein molecules 11 .…”

Replica Exchange Molecular Dynamics Simulations Reveal Self-association Sites in M- Crystallin Caused by Mutations Provide Insights of Cataract

“…The dominant protein crystallins in the eye lens constitute 90% of the total soluble proteins 2 . High concentration of soluble crystallins in the cytoplasm of the lens ber cells provide transparency and high refractive index to the eye-lens 3,4 .…”

“…Cataract is caused due to aggregation of eye lens crystallins which causes scattering of the light and prevent it from reaching the retina [4][5][6] . In several studies the molecular insights of the cause of cataract is elusive.…”