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.
Pande
Ajay
A
Department of Chemistry, Life Sciences Research Building, University at Albany, State University of New York, Albany, New York 12222, USA.
Ghosh
Kalyan S
KS
Banerjee
Priya R
PR
Pande
Jayanti
J
D002386:Cataract / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011392:Proline / Q000235:genetics; D017434:Protein Structure, Tertiary; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000235:genetics; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics
C504903:CRYGD protein, human; D038222:gamma-Crystallins; D013912:Threonine; D011392:Proline