Abstract
Understanding triacylglycerol (TAG) metabolism is crucial for developing algae as a source of biodiesel. TAGs are the main reservoir of energy in most eukaryotes. The final, rate-limiting step in the formation of TAGs is catalyzed by 1,2-diacylglycerol acyltransferases (DGATs). In the green alga Chlamydomonas reinhardtii, DGAT3 is phylogenetically related to plant DGAT3 but unrelated to other DGATs from eukaryotes, such as DGAT1 and DGAT2. In this study, we described the conformational preferences and the lipid-binding features of the DGAT3 from C. reinhardtii. To characterize its conformational stability and structural features, we used several biophysical probes, namely, fluorescence, circular dichroism (CD), and differential scanning calorimetry (DSC). Our results showed that the protein was mainly disordered, containing a small population of folded conformations in a narrow pH range (pH 8 to 10). The conformational stability of the folded structure of DGAT3 was very low, as shown by urea or guanidinium denaturations. Thermal denaturation, followed by fluorescence or CD, as well as calorimetric denaturation, followed by DSC, did not yield any transition in the pH range where DGAT3 acquired a "native-like" conformation. Furthermore, we used two approaches to demonstrate the interaction of DGAT3 with lipid membranes at the pH at which it had acquired a "native-like" conformation. The first involved the measurement of anisotropy and fluorescence quenching of the protein. The second approach focused on examining possible modifications of the biophysical properties of lipids due to their interaction with DGAT3, through anisotropy measurements and leakage assays. Both methods produced consistent results, suggesting that DGAT3 preferentially interacted with negatively charged membranes. These results will allow the design of a more efficient and stable DGAT3, as well as an in-depth understanding of how the metabolism of TAGs is accomplished in C. reinhardtii.