Abstract
Purpose: :
We have shown earlier that the mechanism of light scattering and opacity caused by the R14C mutant of human gamma-D crystallin (HGD) under physiological conditions, is due to the ready formation of disulfide-crosslinked aggregates that precipitate (1). We suggested that (a) Cys 14, which is highly reactive, and (b) a second reactive Cys normally present in HGD, can form a variety of such protein aggregates. The work presented here was undertaken to substantiate the suggestions (a) and (b).
Methods: :
Ionization of Cys-thiols as a function of pH, was monitored by the absorption of the thiolate anion at 240nm. Molecular modeling studies were based on the high-resolution x-ray crystal structure of HGD [PDB ID: 1hk0]. Raman spectra were measured using 785 nm laser excitation. The Raman frequency of Cys 14 was determined by the difference-Raman spectra in the -SH region of R14C and HGD at about pH 4. Reaction with DTNB (5,5’-dithiobis-2-nitrobenzoic acid) was carried out at pH 7.4 to form the protein-thionitrobenzoic acid disulfide (PS-TNB) derivatives, followed by reaction with KCN to form the protein-thiocyanate (PS-CN) derivatives.
Results: :
Compared to the normal cysteine pKa of 8.5, Cys 14 in R14C has a pKa close to 5. The lowered pKa implies that at physiological pH, Cys14 is present primarily as a thiolate anion. Molecular modeling studies based on the crystal structure of HGD, show the presence of a neighboring Glutamic acid residue with which the Cys14-thiolate could form a H-bond. This putative interaction could lead to a lowering of the pKa of Cys14. However, despite its ease of ionization to the thiolate form, the molecular environment of the parent Cys14 thiol appears to be similar to that of other cysteine thiols in HGD. This is shown by the Raman frequency of the -SH group of Cys14 (2575 cm-1), which is comparable to that of other Cys residues in HGD. We also observe that at pH 7, one Cys residue reacts with DTNB in HGD, while two Cys residues react in R14C. In addition, the thiocyanate derivatives of these proteins show distinct Raman and IR bands at the same frequency (2160 cm-1), for both R14C and HGD, but with an intensity ratio of 2:1, consistent with 2 reactive cysteines in R14C.
Conclusions: :
The reactivity of R14C mutant is largely due to the lower pKa of Cys14. Therefore R14C forms large intermolecular S-S linked aggregates and precipitates, under physiological conditions in vitro, while the aggregation of HGD does not proceed beyond dimers.1. Pande A. et al (2000) Proc Natl Acad Sci U S A. 97(5):1993-8
Keywords: cataract • crystallins • proteins encoded by disease genes