DSC and FCS Studies Reveal the Mechanism of Thermal and Chemical Unfolding of CIA17, a Polydisperse Oligomeric Protein from Coccinia Indica.

The mechanism of thermal and chemical unfolding of Coccinia indica agglutinin (CIA17), a chitooligosacharide-specific phloem exudate lectin, was investigated by biophysical approaches. DSC studies revealed that the unfolding thermogram of CIA17 consists of three components (Tm ∼ 98, 106, and 109 °C), which could be attributed to the dissociation of protein oligomers into constituent dimers, dissociation of the dimers into monomers, and unfolding of the monomers. Intrinsic fluorescence studies on the chemical denaturation by guanidinium thiocyanate and guanidinium chloride indicated the presence of two distinct steps in the unfolding pathway, which could be assigned to dissociation of the dimeric protein into monomers and unfolding of the monomers. Results of fluorescence correlation spectroscopic studies could be interpreted in terms of the following model: CIA17 forms oligomeric structures in a concentration dependent manner, with the protein existing as a monomer below 1 nM concentration but associating to form dimers at higher concentrations (KD ≈ 2.9 nM). The dimers associate to yield tetramers with a KD of ∼50 μM, which further associate to form higher oligomers with further increase in concentration. These results are consistent with the proposed role of CIA17 as a key player in the defense response of the plant against microbes and insects.