Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility

Abstract

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.

FIGURE 1

Chemical structures of bis-ANS and Nile Red.

FIGURE 2

Relative fluorescence intensity at the emission maximum, 515 nm (excitation at 390 nm) for mixtures of bis-ANS with human γD-crystallin and its mutants (P23V, P23S and P23T) at various bis-ANS concentrations. Protein concentration kept constant at 0.1 mg/ml (~5 μM) in 0.1 M phosphate buffer, pH 7.0. Symbols represent the data and lines are merely visual guides. Inset shows the corresponding fluorescence emission spectra of the mixtures at a bis-ANS concentration of 250 μM. The dotted line is the emission spectrum of bis-ANS alone.

FIGURE 3

Relative fluorescence intensity at the emission maximum, 650 nm (excitation at 540 nm) for mixtures of Nile Red with human γD-crystallin and its mutants (P23V, P23S and P23T) at various Nile Red concentrations. Protein concentration kept constant at 0.1 mg/ml (~5 μM) in 0.1 M phosphate buffer, pH 7.0. Symbols represent the data and lines are merely visual guides. Inset shows the corresponding fluorescence emission spectra of the mixtures at a Nile-red concentration of 80 μM. The dotted line is the emission spectrum of Nile Red alone.

FIGURE 4

Fluorescence emission spectra of HGD, Nt-HGD, P23T, and Nt-P23T in mixtures with bis-ANS. Spectra are corrected for bis-ANS by subtracting its contribution. Protein concentration was about 5μM and bis-ANS concentration was 250μM in each case. Excitation wavelength was 390 nm.

FIGURE 5

(A) Far-UV and (B) near-UV CD spectra of Nt-HGD, Nt-P23T and HGD at pH4.5. Protein concentrations were 0.1 mg/ml in 5 mM acetate buffer for the far-UV CD measurements, and 1.2 mg/ml in 20 mM acetate buffer for the near-UV CD measurements.

FIGURE 6

Fluorescence emission spectra (excitation at 390 nm) for mixtures of bis-ANS with bovine serum albumin (BSA), albumin from chicken egg white (ovalbumin), lysozyme from chicken egg white, ribonuclease A from bovine pancreas (RNase A), and HGD and P23T at protein concentrations of ~ 5 μM and bis-ANS concentration of 250 μM in 0.1 M phosphate buffer pH 7.0. Dotted line shows the emission spectrum of 250 μM bis-ANS alone in the same buffer.

FIGURE 7

Temporal change in hydrodynamic radius (left-axis, solid line), light scattering intensity (left-axis, dashed line) and fluorescence emission, (emission maximum, 515 nm, excitation at 390 nm, right-axis, dotted line) for bis-ANS mixtures with Nt-HGD (■) and Nt-P23T (▼) in 0.1 M phosphate buffer pH 7.0. Bis-ANS and protein concentrations are 250 μM and 0.1 mg/ml (5 μM) respectively.

FIGURE 8

Surface representation of (A) the crystal structure of HGD (PDB ID: 1HK0) and (B) the NMR structure 3 of P23T (PDB ID: 2KFB). The potentially “sticky” patches on the surface of HGD (shown in red and yellow in (A) and described earlier (8)), appear to extend over a wider surface and become more hydrophobic in P23T (B). The models are depicted here according to the surface hydrophobicity color-scale. In (B), the small rectangle at the bottom of the smaller cyan patch marks the location of residue 23. Figures were made in PyMol (32).