Decrease in Protein Solubility and Cataract Formation Caused by the Pro23 to Thr Mutation in Human γD-Crystallin^,

Abstract

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.

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

“…As the temperature changes from 5°to 35°C, the binding energy of the mutant increases by Ϸ2.4 kT. Comparison with data on other mutants (1) suggests that the underlying source of these binding energy changes is associated with the presence or absence of proline in the vicinity of position 23.…”

“…The solubility line of mutants of HGD at site 23 shows a retrograde temperature dependence. The replacement of Thr-23 with a Ser or Val residue shifts the location of the inverted solubility line to higher concentrations (1). On the other hand, LLPS for P23V appears to be the same as for HGD, and the CD, Raman, and IR spectra show that no major secondary or tertiary structural changes take place upon mutagenesis (1).…”

“…Mutations in HGD in particular have been associated with a number of childhood cataracts (1)(2)(3)(4). Recently, the P23T mutant of HGD has been associated with coralliform, cerulean, and fasciculiform cataract phenotypes (refs.…”

“…A more detailed investigation of P23T and other mutants of HGD at site 23 (i.e., P23S and P23V) has shown that unusual phase behavior is displayed by all three of these mutants, implying that the region around site 23 of HGD is important in maintaining the solubility of the protein (1). Although substituting a single amino acid residue at site 23 does not alter the regular form of the coexistence curve, it results in decreased protein solubility and remarkably inverts the form of the solubility line.…”

Amino Acid Contribution to Protein Solubility: Asp, Glu, and Ser Contribute more Favorably than the other Hydrophilic Amino Acids in RNase Sa

“…Also, low protein solubility is a factor in several types of disease. Therefore, methods that can be used to increase protein solubility are of great interest for high resolution structural studies 1; 2 , crystallization of membrane proteins [3][4][5][6][7][8] , pharmaceutical applications [9][10][11] , and treatment of human disease [12][13][14][15][16] . Protein solubility as a thermodynamic parameter is defined as the concentration of soluble protein that is in equilibrium with a crystalline solid phase under given conditions of pH, temperature, buffer concentration, and various additives 1 .…”