Structural characterisation of recombinant LEA protein originated from Ramonda serbica

Desiccation, an extreme form of dehydration, reduces the cellular water content to below 5% and thus, presents a significant challenge for most plants. Ramonda serbica, a resurrection plant and tertiary relict is an excellent model for studying vegetative desiccation tolerance. Late Embryogenesis Abundant (LEA) proteins are mostly Intrinsically Disordered Proteins (IDPs) and have an important role in this adaptive trait. However, their precise molecular function still remains unclear. The majority of the LEA4 protein family group exhibited a high propensity to form α-helical structure, particularly A-type of α-helix, with differentiated charged and hydrophobic faces. In this study, for the first time, we report the secondary structure analysis of the recombinantly produced desiccation-induced LEA 4 protein family member from a dicotyledonous resurrection plant species. Through immobilized metal affinity and size-exclusion chromatography, we purified this LEA protein to a purity exceeding 95%, demonstrating a robust and scalable method for the production of other LEA proteins. Secondary structure predictions using various algorithms suggest that more than 95% of the amino acid sequence of this protein has a strong tendency to form α-helices. However, disorder prediction tools reveal that approximately 85% of the sequence is intrinsically disordered, showing its structural flexibility under varying conditions. Structural characterisation via circular dichroism (CD) spectroscopy revealed that LEA protein is predominantly disordered. In the presence of 2,2,2-trifluoroethanol and lipid mimetic detergent, the protein mostly folds into an α-helical structure, implying the importance of structural plasticity. Structural disorder-to-order transitions may be a key feature enabling LEA protein protective function in the desiccation tolerance mechanism. We suggest that this structural duality underpins the protective function of LEA protein in chloroplasts, likely through interactions with thylakoid membrane and desiccation-sensitive proteins associated with photosynthesis. This function may be essential for the rapid recovery of photosynthetic components upon rehydration. Our study provides new insights into the structure-function relationship of LEA proteins in resurrection plants. It lays the groundwork for the development of future bioengineering strategies aimed at enhancing drought tolerance in crops.