Is Pro51 essential for subunit exchange between alpha B and alpha A crystallins?

Abstract

Abstract: : Purpose: Previously, using peptide scan method we have identified that residues 42–57 in alpha B crystallin (named as Recognition Sequence 1 or RS1) are involved in interaction with alpha A (IOVS 2001, 42(4) p.s292). In the present study, we mutated alpha B at R50 with G and P51 with A and studied the effect of mutations on structural and functional properties of the proteins. Methods: Mutations were introduced using site–directed mutagenesis, the mutant proteins expressed in E.coli BL21(DE3)pLysS cells were purified by ion–exchange and gel filtration chromatography. The purity was checked by SDS–PAGE and the mutations were confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone–like activity of wild type and mutant proteins was compared using citrate synthase. Subunit exchange rate was measured by FRET (fluorescence resonance energy transfer). For this purpose, purified alpha B, alpha B R50G, alpha B P51A were labeled with Alexa fluor 350 and Alexa fluor 488 was used to label alpha A. Results: Both wild type and mutant alpha B crystallins showed similar elution profiles on gel filtration chromatography. Mutant proteins showed marginal decrease in bis–ANS binding. Tryptophan fluorescence intensities decreased by 30% in alpha B P51A and by 45% in alpha B R50G as compared to wild type. There was no difference in the chaperone–like activity of alpha B and mutant proteins. The secondary structure of alpha B and alpha B R50G was similar, where as, alpha B P51A showed increased α–helical content. The rate of subunit exchange between alpha B, alpha B R50G and alpha A was similar. On the other hand, alpha B P51A exchanged subunits with alpha A at a 2–fold slower rate. RS1 could effectively compete with alpha A for interaction with alpha B P51A. Similarly, wild type alpha B competed with alpha B P51A for interaction with alpha A. Conclusions:Our results confirm our earlier observation that RS1 in alpha B is the interacting region for alpha A. The results indicate that P⁵¹ in alpha B may be important for normal subunit exchange with alpha A. Altered secondary structure of alpha B P51A may contribute to its slow subunit exchange with alpha A.

Keywords: crystallins • protein structure/function • cataract