Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin.
The P23T mutation in the human gammaD-crystallin gene has in recent years been associated with a number of well known cataract phenotypes. To understand the molecular mechanism of lens opacity caused by this mutation, we expressed human gammaD-crystallin (HGD), the P23T mutant, and other related mutant proteins in Escherichia coli and compared the structures and thermodynamic properties of these proteins in vitro. The results show that the cataract-causing mutation P23T does not exhibit any significant structural change relative to the native protein. However, in marked contrast to the native protein, the mutant shows a dramatically lowered solubility. The reduced solubility results from the association of the P23T mutant to form a new condensed phase that contains clusters of the mutant protein. The monomer-cluster equilibrium is represented by a solubility curve in the phase diagram. When the solubility limit is exceeded, the mutant protein forms the condensed phase after a nucleation time of 10-20 min. We found that the solubility of the P23T mutant exhibits an inverse dependence on temperature, i.e., the protein clusters are increasingly soluble as the temperature of the solution decreases. The solubility of P23T can be substantially altered by the introduction of specific mutations at or in the immediate vicinity of residue 23. We examined the mutants P23S, P23V, P23TInsP24, and P23TN24K and found that the latter two mutations can restore the solubility of the P23T mutant. These findings may help develop a strategy for the rational design of small molecule inhibitors of this type of condensed phase.
Pande
Ajay
A
Materials Processing Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Annunziata
Onofrio
O
Asherie
Neer
N
Ogun
Olutayo
O
Benedek
George B
GB
Pande
Jayanti
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002386:Cataract / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D008027:Light; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011392:Proline / Q000235:genetics*; D011487:Protein Conformation; D051381:Rats; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013816:Thermodynamics; D013912:Threonine / Q000235:genetics*; D038222:gamma-Crystallins / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
D038222:gamma-Crystallins; D013912:Threonine; D011392:Proline