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

Abstract

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 (T m ∼ 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 (K D ≈ 2.9 nM). The dimers associate to yield tetramers with a K D 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.

Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability

“…46 A blue shift in the λ max to 335 nm upon binding of chitooligosaccharides indicates a more compact structure on ligand binding, whereas in the presence of 4 M GdmSCN, the emission λ max shifted to 350 nm, indicating complete unfolding of the protein as reported in our previous study. 46 Fluorescence intensity decreases gradually with a decrease in pH from 8.5 to 2.0, although the λ max remains essentially unaltered as shown in Figure S1.…”

“…The cross-correlation data for the labeled protein were better fitted to eq 5 which includes the contribution of the conformational relaxation term along with the simple diffusive component to the fluorescence intensity fluctuation as discussed earlier. 46 The correlation data for free Alexa 488 (A488) could be fitted well to the single diffusion model (eq 4) irrespective of the pH of the buffer (shown in Figure S6).…”

“…43 The concentration of CIA17 was estimated using its molar extinction coefficient of 53 700 M −1 .cm −1 at 280 nm. 46 2.3. Circular Dichroism (CD) Spectroscopy.…”

“…The lectin was labeled with the Alexa fluor 488 carboxylic acid succinimide ester (A488) following the procedure described earlier. 46 A pulsed diode laser (λ ex = 485 nm with a full-width at half-maximum of 144 ps) was used as the excitation source.…”

“…CIA17 is a polydisperse oligomeric protein and self assembles to form higher oligomeric structures in a concentration-dependent manner. 46 In the present study, pH-induced changes in the conformation and/or structural dynamics of the lectin have been investigated by circular dichroism (CD) spectroscopy, steady-state and time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). Thermal stability of the lectin at different pHs was investigated by differential scanning calorimetry (DSC).…”