Ajay Pande

Crystal cataracts: Human genetic cataract caused by protein crystallization

Several human genetic cataracts have been linked recently to point mutations in the γD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of γD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

High-resolution X-ray Crystal Structures of Human γD Crystallin (1.25 Å) and the R58H Mutant (1.15 Å) Associated with Aculeiform Cataract

Several human cataracts have been linked to mutations in the gamma crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in gammaD crystallin. We have shown previously that this cataract is caused by crystallization of the mutant protein, which is an order of magnitude less soluble than the wild-type. Here, we report the very high-resolution crystal structures of the mutant and wild-type proteins. Both proteins crystallize in the same space group and lattice. Thus, a strict comparison of the protein-protein and protein-water intermolecular interactions in the two crystal lattices is possible. Overall, the differences between the mutant and wild-type structures are small. At position 58, the mutant protein loses the direct ion-pair intermolecular interaction present in the wild-type, due to the differences between histidine and arginine at the atomic level; the interaction in the mutant is mediated by water molecules. Away from the mutation site, the mutant and wild-type lattice structures differ in the identity of side-chains that occupy alternate conformations. Since the interactions in the crystal phase are very similar for the two proteins, we conclude that the reduction in the solubility of the mutant is mainly due to the effect of the R58H mutation in the solution phase. The results presented here are also important as they are the first high-resolution X-ray structures of human gamma crystallins.

Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-elec tro spray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidyleth a nol amine (A2-DHP-PE). The second pigment, A2-dihydropyridine-eth a nol amine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at ∼490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidyleth a nol amine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

Altered phase diagram due to a single point mutation in human gammaD-crystallin.

The P23T mutant of human γD-crystallin (HGD) is associated with cataract. We have previously investigated the solution properties of this mutant, as well as those of the closely related P23V and P23S mutants, and shown that although mutations at site 23 of HGD do not produce a significant structural change in the protein, they nevertheless profoundly alter the solubility of the protein. Remarkably, the solubility of the mutants decreases with increasing temperature, in sharp contrast to the behavior of the native protein. This inverted solubility corresponds to a strong increase in the binding energy with temperature. Here we have investigated the liquid–liquid coexistence curve and the diffusivity of the P23V mutant and find that these solution properties are unaffected by the mutation. This means that the chemical potentials in the solution phase are essentially unaltered. The apparent discrepancy between the interaction energies in the solution phase, as compared with the solid phase, is explicable in terms of highly anisotropic interprotein interactions, which are averaged out in the solution phase but are fully engaged in the solid phase.

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ–γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD—namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid–liquid phase separation behavior of E107A–α-crystallin mixtures compared to HGD–α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A–α mixtures differ much more in protein density than those that occur in HGD–α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD–α mixtures, the demixing of phases occurs primarily by protein type while in E107A–α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins—i.e., heterologous γ–α crystallin interactions—primarily due to the change in surface electrostatic potential in the mutant protein.

Increase in Surface Hydrophobicity of the Cataract-Associated, P23T Mutant of Human GammaD-Crystallin is Responsible for Its Dramatically Lower, Retrograde Solubility †

The cataract-associated Pro23 to Thr (P23T) mutation in human gammaD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore, there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, A., et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solubility change occurs even as the structure of the mutant protein remains essentially unchanged in vitro. We proposed that the retrograde solubility and aggregation of P23T were mediated by net hydrophobic, protein-protein interactions. On the basis of these initial findings for P23T and related mutants, and the subsequent finding that they show atypical phase behavior [McManus, J. J., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 16856-16861], we concluded that the protein clusters formed in solutions of the mutant proteins were held together by net hydrophobic, anisotropic interactions. Here we show, using chemical probes, that the surface hydrophobicities of these mutants are inversely related to their solubility. Furthermore, by probing the isolated N-terminal domains of HGD and P23T directly, we find that the increase in the surface hydrophobicity of P23T is localized in the N-terminal domain. Modeling studies suggest the presence of sticky patches on the surface of the N-terminal domain that could be engaged in the formation of protein clusters via hydrophobic protein-protein interactions. This work thus provides direct evidence of the dominant role played by net hydrophobic and anisotropic protein-protein interactions in the aggregation of P23T.

The cataract-associated R14C mutant of human gamma D-crystallin shows a variety of intermolecular disulfide cross-links: a Raman spectroscopic study.

The Arg14 to Cys (R14C) mutation in the human gammaD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed previously [Pande, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1993-1998] that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-cross-linked aggregates at physiological pH. Here we present a Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide cross-links with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein cross-links through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the cross-linking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide cross-linking, although such cross-linking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.

Resonance Raman study of the primary photochemistry of visual pigments. Hypsorhodopsin

We report here the first resonance Raman results of octopus hypsorhodopsin, a species formed photochemically at very low temperatures from visual pigments. A pump-probe technique was used to obtain Raman spectra from samples at 12 degrees K whose photostationary state mixtures were either hypsorhodopsin rich or hysorhodopsin poor. The data strongly suggest that the Schiff-base linkage between the chromophore of hysorhodopsin and apoprotein is protonated. Further, the results suggest that hypsorhodopsins chromophore is in some torsionally distorted conformation, possibly having torsional departures from an all-trans isomeric form.

Single-Walled Carbon Nanotubes Probing the Denaturation of Lysozyme

Resonance Raman spectroscopy measurements of lysozyme-bound single-walled carbon nanotubes have been made during different stages of the chemically and thermally induced misfolding and of the denaturation process of nanotube-bound lysozymes. Changes to the Raman intensity of single-walled carbon nanotubes (SWNTs) have been observed during the denaturation of lysozyme. The Raman intensity changes are attributed to excitonic transition energy (Eii ) shifts of the SWNTs during the denaturation of lysozyme. The Eii shift of SWNTs was confirmed by photoluminescence measurements.

Binding of γ-crystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone α-crystallin.

α-Crystallin is a small heat shock protein and molecular chaperone. Binding of Cu2+ and Zn2+ ions to α-crystallin leads to enhanced chaperone function. Sequestration of Cu2+ by α-crystallin prevents metal-ion mediated oxidation. Here we show that binding of human γD-crystallin (HGD, a natural substrate) to human αA-crystallin (HAA) is inversely related to the binding of Cu2+/Zn2+ ions: The higher the amount of bound HGD, the lower the amount of bound metal ions. Thus, in the aging lens, depletion of free HAA will not only lower chaperone capacity but also lower Cu2+ sequestration, thereby promoting oxidation and cataract.