Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin. Biochemistry 2002 Nov 26;41(47):14076-84
Date
11/20/2002Pubmed ID
12437365DOI
10.1021/bi026288hScopus ID
2-s2.0-0037180391 (requires institutional sign-in at Scopus site) 79 CitationsAbstract
Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.
Author List
Kim YH, Kapfer DM, Boekhorst J, Lubsen NH, Bächinger HP, Shearer TR, David LL, Feix JB, Lampi KJAuthor
Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SubstitutionCircular Dichroism
Cloning, Molecular
Crystallins
Drug Stability
Electron Spin Resonance Spectroscopy
Escherichia coli
Lens, Crystalline
Mutagenesis, Site-Directed
Protein Conformation
Protein Denaturation
Protein Structure, Secondary
Sequence Deletion
Urea
