Ratna Gupta1, Om P Srivastava. 1. Department of Physiological Optics, University of Alabama at Birmingham, Birmingham, Alabama 35294-4390, USA.
Abstract
PURPOSE: To elucidate the effect of deamidation on the structural and functional properties of human alphaB-crystallin. METHODS: Site-directed mutagenesis was used to generate three deamidated mutants of alphaB-crystallin: N78D, N146D, and N78D/N146D. The mutations were confirmed by DNA sequencing and matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Recombinant native alphaB-crystallin (wild type [WT]) and the three mutated alphaB species were expressed, and each species was purified to homogeneity by ion-exchange chromatography followed by hydrophobic interaction chromatography. The structural and functional properties compared with WT protein were investigated, respectively, by static light scattering (SLS), circular dichroism (CD), and fluorescence spectroscopy and by determining chaperone activity with the use of three substrates. RESULTS: Native WT and the N78D mutant showed relatively higher chaperone activity compared with the N146D and N78D/N146D mutants with all the substrates. Further, during binding experiments with 1-anilino-8-naphthalenesulfonate (ANS), the WT and N78D mutant showed relatively more solvent-exposed hydrophobic residues than the N146D and N78D/N146D mutants. On determining far-UV circular dichroism and tryptophan (Trp) fluorescence spectra, significant secondary and tertiary structural changes were observed in the N146D and N78D/N146D mutants compared with WT and the N78D mutant. The static light scattering data showed a high order of oligomerization in all the three mutants. N146D and N78D/N146D formed the largest oligomers of 750 and 770 kDa, respectively, compared with WT (580 kDa). CONCLUSIONS: The results show that the deamidation of N146 but not of N78 have profound effects on the structural and functional properties of alphaB-crystallin.
PURPOSE: To elucidate the effect of deamidation on the structural and functional properties of humanalphaB-crystallin. METHODS: Site-directed mutagenesis was used to generate three deamidated mutants of alphaB-crystallin: N78D, N146D, and N78D/N146D. The mutations were confirmed by DNA sequencing and matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Recombinant native alphaB-crystallin (wild type [WT]) and the three mutated alphaB species were expressed, and each species was purified to homogeneity by ion-exchange chromatography followed by hydrophobic interaction chromatography. The structural and functional properties compared with WT protein were investigated, respectively, by static light scattering (SLS), circular dichroism (CD), and fluorescence spectroscopy and by determining chaperone activity with the use of three substrates. RESULTS: Native WT and the N78D mutant showed relatively higher chaperone activity compared with the N146D and N78D/N146D mutants with all the substrates. Further, during binding experiments with 1-anilino-8-naphthalenesulfonate (ANS), the WT and N78D mutant showed relatively more solvent-exposed hydrophobic residues than the N146D and N78D/N146D mutants. On determining far-UV circular dichroism and tryptophan (Trp) fluorescence spectra, significant secondary and tertiary structural changes were observed in the N146D and N78D/N146D mutants compared with WT and the N78D mutant. The static light scattering data showed a high order of oligomerization in all the three mutants. N146D and N78D/N146D formed the largest oligomers of 750 and 770 kDa, respectively, compared with WT (580 kDa). CONCLUSIONS: The results show that the deamidation of N146 but not of N78 have profound effects on the structural and functional properties of alphaB-crystallin.