Abstract
The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ∼75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a “molten-globule” like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.
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Abbreviations
- ANS:
-
8-anilino-1-naphthalene-sulfonic acid
- A-state:
-
acid induced state
- CTA:
-
Clitoria ternatea agglutinin
- GdnHCl:
-
guanidine hydrochloride
- MRE:
-
mean residue ellipticity
- SEC:
-
size-exclusion chromatography
- UA:
-
acid unfolded state
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Published in Russian in Biokhimiya, 2009, Vol. 74, No. 10, pp. 1336–1345.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-002, July 5, 2009.
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Naeem, A., Saleemuddin, M. & Khan, R.H. Compact acid-induced state of Clitoria ternatea agglutinin retains its biological activity. Biochemistry Moscow 74, 1088–1095 (2009). https://doi.org/10.1134/S0006297909100046
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DOI: https://doi.org/10.1134/S0006297909100046