Chemical Properties Determine Solubility and Stability in βγ-Crystallins of the Eye Lens

Abstract

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.

Keywords: beta-gamma-crystallin; cataracts; long-lived proteins; protein aggregation; protein solubility.

Figure 1:

HγS displayed to highlight the conserved structure among γ-crystallins. Protein structures are rendered using UCSF Chimera.^[17] The N-terminus is circled and the C-terminus is boxed. Greek-key motifs are numbered from the N-terminus to the C-terminus. Conserved tryptophan side-chains are shown as sticks. (A) HγS (PDB ID: 2M3T) with individual Greek key motifs highlighted in red and blue. The left and right side are two wedge-like β-sandwich domains, where one Greek key (red) binds to the other (blue). The hydrophobic interdomain interface holds the two blue Greek key motifs together. (B) HγS rotated 90°. (C) Schematic of the Greek key motif. Each Greek key is formed by antiperiplanar β-sheets, which are continuously connected via linker sequences.

Figure 2:. Types of βγ-crystallins.

(A) Tunicate βγ-crystallin (PDB ID: 2BV2)^[20] is monomeric and has a single domain. This protein can bind two calcium ions (shown in red). (B) HγS (PDB ID:2M3T)^[22] in monomeric but has two double Greek key domains. (C) β-crystallins have the same domain organization, but are typically dimeric. Human βB2-crystallin can form domain-swapped dimers (PDB ID: 1YTQ).^[23]

Figure 3:

ANS binding can be used to probe hydrophobic surface area. (A) ANS is a mostly hydrophobic small molecule with a negatively charged sulfate group. This allows the molecule to bind to exposed hydrophobic pockets on a protein, as shown in this docking simulation of a predicted structure for the G18A variant of γS-crystallin binding to ANS. The protein structure is color coded from blue (hydrophobic) to orange (hydrophilic). (B) When ANS binds to a protein, it will fluoresce more strongly; hydrophobic surface exposure correlates with intensity, as shown in this simulation of a γS-G18A (green) compared to γS-WT (blue) suggesting that the hydrophobic core of the protein is more exposed in the variant. (C) This florescence in the bound state is caused by the absorption of a photon, causing electronic excitation; when the electron relaxes back to the ground state, energy is released as a lower-energy photon.

Figure 4:. Transition metal Förster resonance energy transfer (tmFRET) and an example of a crystallin protein where it is useful.

(A) A model of one of the Cu²⁺-binding sites of HγS, showing the Cys residues that coordinate the metal ion FRET donor, and the nearby Trp 47, which acts as a FRET acceptor. (B) Schematic depiction of the interactions in (A), with key distances labeled. Both (A) and (B) are based on data from.^[58] (C) Drawing showing the approximate positions of relevant spectral peaks. Trp in a protein has a strong absorption peak centered at around 280 nm, and fluorescence at around 350 nm (although this can vary considerably depending on the local environment.) Transition metals such as Cu²⁺ often have a relatively broad absorbance peak at around 400 nm, allowing for substantial overlap with the Trp fluorescence, and hence FRET. (D) Jablonski diagram indicating the various energy transfer mechanisms, including excitation, fluorescence, FRET, and vibrational relaxation (small red arrows.)

Figure 5:. Deamidation Mechanisms

Deamidation of asparagine and glutamine residues yield their acidic counterparts, altering the biophysical properties of the protein. Backbone amide attack of asparagine or glutamine residues result in succinimide or glutarimide intermediates, respectively. Hydrolysis results in either conversion of the starting amide to an acidic residue (top) or breaking of backbone amide bonds to form iso-aspartic acid (γ or iso-glutamic acid (bottom), respectively.

Figure 6:

The formation of tryptophan metabolites through the kynurenine pathway. Enzymes are indicated adjacent to each arrow. At the beginning and the end of the pathway are drawings illustrating the healthy lens and the development of brunescent cataract as a result of tryptophan metabolite production.^[193,194]

Figure 7:. Disulfide bond schematic view

(A) Cu²⁺ oxidization of the intramolecular Cys108-Cys110 disulfide bond on HγD (PDB: 1HK0). (B) Oxidization of the Cys32-Cys41 disulfide bond leading to aggregation of HγD (PDB: 1HK0).

Figure 8:

Schematic representation of several possible aggregation pathways for γ-crystallins. Rectangular shapes indicate stable states, whereas rounded shapes denote transient species. Species shown on blue backgrounds are in solution; pink backgrounds indicate insoluble aggregates. Red lines indicate modifications to the protein structure. This schematic is not a comprehensive representation of all potential HγS- aggregation pathways, but instead illustrates currently known mechanisms.

Figure 9:. Schematic view of liquid-liquid phase separation (LLPS).

(A) Drawing of the human eye, showing the position of the lens. (B) A γ-crystallin protein in a homogeneous phase. (C) In LLPS, the solution partitions into protein-rich and protein-poor phases, forming droplets and producing reversible opacification.