Here we have examined the molecular effect of the G18V mutation on HγS- crystallin by comparing the conformation and stability of mutant with the wild type γS- crystallin under both normal and stressed conditions. G18, along with the adjacent two amino acids F16 and Q17, is conserved in all βγ-crystallins in all species from zebra fish to humans. One reason for this is that Glycine, which lacks a side chain, is the only amino acid able to achieve the dihedral angle required to stabilize the folded hairpin of the βγ motif (16
). The presence of a residue with a side chain would be expected to expand and to some extent destabilize the Greek key structure of the first motif, but not necessarily to alter the association or solubility of the γS-crystallin protein. However, examination of the normal and G18V mutant under mild conditions shows little difference. The secondary structure of the G18V mutant protein, judged by CD spectra, indicates it has a native-like conformation similar to that of wild type HγS-crystallin, and its association properties are identical to those of normal γS-crystallin. The native-state fluorescence emission maximum of 328nm and an unfolded maximum of 355nm displayed in this study by Wild-type and mutant HγS- crystallin displayed are similar to those previously reported (14
). Analytical sedimentation equilibrium demonstrates that wild type and mutant HγS-crystallin are monomeric species in their native states, once more in agreement with previous findings (14
). The fluorescence emission intensity increased upon unfolding, indicating that the tryptophans were quenched in the native fold. This phenomenon has been described previously for several of the γ-crystallins (15
), and is proposed to relate to charge transfers from the buried tryptophans to the backbone of the polypeptide chain.
While the structures of the G18V mutant HγS-crystallin under mild conditions appear to be similar to wild type, there is strong evidence that the G18V mutation decreases the stability of HγS-crystallin. Although the CD spectra of the native and G18V mutant in aqueous buffer are indistinguishable and similar to previously reported results (14
), as are the spectra under completely denaturing conditions, in 1M GuHCl the fluorescence emission maximum of G18V mutant but not the wild type is red shifted. This suggests that the microenvironment of aromatic amino acids was altered and is consistent with partial denaturation of the G18V mutant. This is similar to the fluorescence emission spectra of the wild type and G18V mutant proteins under increasing concentrations of GuHCl. Decreased stability of the mutant relative to the wild type is further supported by the ANS binding results. ANS is an anionic fluorescence probe that binds to the apolar interface and exhibits a shift in the emission maxima due to changes in hydrophobic patches on protein misfolding. In the conventional model aggregation is dominated by hydrophobic interactions of side chains that are normally buried in the native state but that are more exposed in an unfolded, non-native state (20
). Thus, sensitivity to aggregation can derive from the ability of a mutation either to facilitate the accumulation of a non-native state that is prone to aggregation, or to increase the intrinsic tendency of the mutant protein to aggregate.
The thermodynamic studies provide additional information about the effects of the G18V mutation on stability. The fluorescence emission at 328nm and 355nm were recorded and their ratio was used to estimate the molar fractions of folded and unfolded protein in aqueous solutions as a function of temperature, allowing quantitative estimation of the stability of the wild type and mutant proteins (21
). It has previously been shown that the N-terminal domain of γS-crystallin (γSN
=69.1°C) is less stable than the C-terminal domain (γSC
=75.1°C), and the TM
was similar to the TM
of the full length γS (14
). However, when the intact γS-crystallin was examined a two state model was found to be sufficient to fit the experimental data (17
) even though there are suggestions of more complex behavior, and we confirmed this as well (HγSwt
Tm = 75.6°C).
The G18V mutation dramatically decreases the thermal stability of γS-crystallin as estimated by both tryptophan fluorescence and CD at 218 nm. When monitored by tryptophan fluorescence the G18V mutation converts the transition curve into a three state model, beginning just above 42°C and continuing the transition until approximately 70°C with two apparent steps. However, when monitored by CD at 218 nm only a single transition is apparent, with a sharp transition at 65°C, a decrease of 10 degrees relative to the wild type. This suggests that the G18V mutation causes mild changes in the structure at temperatures between 42° and 60°C, altering the microenvironment of the normally buried tryptophans although the basic protein fold remains intact. Then at 65°C the amino and carboxy domains denature in quick succession as reflected by the sharp transition in the CD curve. Subsequently, the unfolded species were aggregation prone, consistent with the progressive cataract formation seen clinically (15
Dynamic stabilities of the wild type and mutant proteins were examined in greater detail by equilibrium unfolding/refolding in GuHCl. Wild type HγS-crystallin demonstrates a two state transition, suggesting that the two domains of γS-crystallins have similar stabilities, as has been previously described (17
). The NMR solved structure of murine γS-crystallin also demonstrated high structural similarity among domains (22
). Inefficiency in refolding γ-crystallins below 1 M GuHCl also has been noted previously (19
). In contrast to wild type HγS-crystallin the G18V mutant follows a biphasic transition, similar to the transitions seen with substitutions of alanine adjacent to the interface of γD-crystallin, R79A and M147A (19
). While the alanine substitutions are proposed to destabilize the amino terminal domain by increasing exposure of hydrophobic interface residues to solvent, the G18V mutation is predicted to destabilize the first Greek key motif directly (2
). However as with the R79A and M147A changes, the most likely explanation for our results is that the G18V substitution destabilizes the N-terminal domain selectively, so that a stable intermediate in the form of partially unfolded protein exists at intermediate levels of GuHCl. The second transition, presumably between the intermediate and unfolded state, occurs very close to that of the wild type molecule, suggesting that stability of the C-terminal domain is minimally affected by the G18V mutation.
The free energies of partial unfolding, ΔG°NI (2.58Kcal/mol for the mutant) are less than those required for unfolding to a totally denatured protein (4.83Kcal/mol), although the G18V mutant requires almost the same energy for the second step of unfolding as the wild-type. There was a little difference in transition midpoints estimated here from previous studies, which can be interpreted with caution as stability measures are dependent upon the experimental conditions (pH, ionic strength, temperature, and protein concentration) which may differ between laboratories.
The G18V mutation occurring at the terminal end of the first strand in first Greek motif of HγS-crystallin has been associated with autosomal dominant progressive cortical cataracts (7
). The cortical location of this cataract is consistent with the known expression pattern of γS-crystallin, which tends to be most highly expressed in cortical fiber cells (23
). CRYGS mutations have also been associated with a lamellar cataract (24
) also consistent with the CRYGS expression pattern and a total cataract with denser opacity in the nucleus (25
), suggesting the possibility of a more severe mutation. As discussed more fully elsewhere (8
), mutations in crystallins that are sufficient in and of themselves to cause immediate protein aggregation or denaturation usually result in congenital cataract, while less severe mutations that merely increase susceptibility to environmental insults tend to contribute to age related cataract. The results of these studies indicate normal association and structural properties of the G18V mutant γS-crystallin under mild conditions, but increased sensitivity stress, which are thus consistent with the progressive nature of the cataracts in this family. Mutations in several additional genes are associated with progressive cataract, including CRYGD, CRYBB2, GJA8, MIP and BFSP2 (26
). However, the molecular basis of most of these progressive cataracts is unclear. The molecular R14C mutation in γD-crystallin, which is also associated with progressive juvenile-onset hereditary cataracts, results in a mutant protein with nearly identical secondary and tertiary structures and stabilities, but appears to have increased susceptibility to oxidative formation of disulfide mediated oligomers and subsequent aggregation (31
Isolated cortical cataracts with Mendelian inheritance are relatively rare, but secondary and age related cortical cataracts are common. Diabetes mellitus and hyperglycemia are major modifiable risk factors for the development of cortical cataract (32
). The potential role of mutations in age related cortical cataracts is emphasized by estimates from a Twin study in the United Kingdom that two thirds of age related cortical cataracts can be explained by genetic factors (33
). The Beaver Dam Eye Study has reported that a single major gene can account for 58% of the variability of age- and sex-adjusted measures of cortical cataract (34
) and identified a major locus on chromosome 6p12-q12 (35
Our present study further confirms the high stability of wild type HγS-crystallin and demonstrates that the G18V mutation leads to destabilization of HγS-crystallin when subjected to heat and GuHCl -induced stress. These findings are consistent with the progressive nature of the cataracts in individuals carrying this mutation. In this family the lenses of affected individuals are initially clear or mildly opaque, but over time environmental stress results in progression to fully developed cortical cataracts (7
). We are currently addressing the further pathogenesis of cataracts resulting from this mutation by studying the effects of G18V HγS-crystallin in the lenses of transgenic mice.