Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure.
The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.
Gupta
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R
Department of Vision Sciences, University of Alabama, Birmingham, Alabama 35294-4390, USA.
Srivastava
K
K
Srivastava
O P
OP
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea / Q000494:pharmacology; D038205:beta-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D038204:beta-Crystallins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C489108:CRYBA1 protein, human; D038205:beta-Crystallin A Chain; D038204:beta-Crystallins; D014508:Urea