Novel unfolding sequence of banana lectin: Folded, unfolded and natively unfolded-like monomeric states in guanidine hydrochloride

• Published in: Biochimie 2014-04, Vol.99, p.138-145
• Main Authors: Ghosh, Goutam, Mandal, Dipak K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Banana lectin; Circular Dichroism; Guanidine - chemistry; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Musa - chemistry; Natively unfolded state; Plant Lectins - chemistry; Protein Denaturation; Protein Structure, Secondary; Random coil; Structured intermediate; Unfolding sequence
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2013.11.022

The sequence of unfolding events of dimeric banana lectin (Banlec), as induced by guanidine hydrochloride (GdnHCl), has been investigated by size-exclusion HPLC, fluorescence, far-UV CD, low temperature phosphorescence and selective chemical modification. 8-Anilino-1-naphthalenesulfonate (ANS) binding indicates a structured unfolding intermediate which has been characterized as dissociated monomer by size-exclusion chromatography. Interestingly, the unfolding elution pattern reveals two distinct unfolded states. One is a usual random coil. The other represents a novel species having elution behavior and structural compactness (Stokes radius) similar to dissociated monomer but showing no regular secondary structure as determined by far-UV CD, thus resembling a natively unfolded state. N-Bromosuccinimide (NBS) oxidation shows that single tryptophan residue remains unmodified in dissociated monomer intermediate while the same is oxidized in natively unfolded-like species. Such difference in tryptophan environment in these species is supported by acrylamide quenching studies, and phosphorescence results at 77 K which show a blue-shift of (0,0) band from 414.8 nm to 409.2 nm. The present results reveal subtlety of structural characteristics of unfolded states of Banlec in GdnHCl, which provide important insight in protein unfolding reaction. •Unfolding of banana lectin in guanidine–HCl involves folded monomer intermediate.•Unfolding sequence reveals two different unfolded states.•One unfolded state is a randomly coiled monomer.•Final unfolded state is a compact, natively unfolded-like species.