Folding of intrinsically disordered plant LEA proteins is driven by glycerol‐induced crowding and the presence of membranes

Abstract

Late embryogenesis abundant (LEA) proteins are related to cellular dehydration tolerance. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., to be intrinsically disordered proteins (IDPs), but they may acquire α-helical structure upon drying. In the model plant Arabidopsis thaliana, the LEA proteins COR15A and COR15B are highly induced upon cold treatment and are necessary for the plants to attain full freezing tolerance. Freezing leads to increased intracellular crowding due to dehydration by extracellular ice crystals. In vitro, crowding by high glycerol concentrations induced partial folding of COR15 proteins. Here, we have extended these investigations to two related proteins, LEA11 and LEA25. LEA25 is much longer than LEA11 and COR15A, but shares a conserved central sequence domain with the other two proteins. We have created two truncated versions of LEA25 (2H and 4H) to elucidate the structural and functional significance of this domain. Light scattering and CD spectroscopy showed that all five proteins were largely unstructured and monomeric in dilute solution. They folded in the presence of increasing concentrations of trifluoroethanol and glycerol. Additional folding was observed in the presence of glycerol and membranes. Fourier transform infra red spectroscopy revealed an interaction of the LEA proteins with membranes in the dry state leading to a depression in the gel to liquid-crystalline phase transition temperature. Liposome stability assays revealed a cryoprotective function of the proteins. The C- and N-terminal extensions of LEA25 were important in cryoprotection, as the central domain itself (2H, 4H) only provided a low level of protection.

Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A

“…On average, over the 50 simulations in four different orientations, 20% of unfolded, 38% of partially folded, and 43% of folded protein molecules had adsorbed onto the ICMM bilayer at the end of the 200 ns simulations. This indicates that COR15A has to be at least partially folded to efficiently interact with membranes, in agreement with our previous experimental data (5,10). In addition, the orientation of the protein relative to the membrane surface also played an important role in adsorption, indicating that the fully folded protein interacted most efficiently when the hydrophobic faces of the helices were oriented toward the membrane surface (Fig.…”

“…This is not observed in the absence of osmolyte, indicating that a certain degree of helicity is necessary for the proteins to interact with membranes. In agreement with the subcellular localization of the proteins, liposomes containing a high fraction of chloroplast glycolipids were stabilized by COR15 proteins during a freeze-thaw cycle, in line with their physiological role as in vivo membrane stabilizers under such conditions (5,10).…”

“…Experimentally, different degrees of folding can be induced in COR15A by either glycerol-induced crowding (11) or by gradual rehydration of the fully folded, dry protein at different RHs (16). Indirect evidence for membrane binding is obtained by adding liposomes to the crowded system, which leads to an increase in protein folding (10). Following the same logic, we have shown here that the presence of ICMM liposomes increased COR15A folding under partially dehydrated but not under fully hydrated conditions.…”

“…Molecular dynamics (MD) simulations indicate that glycerol stabilizes the folded state because it is excluded from the proteins leading to backbone dehydration (11), as suggested previously for the folding of IDPs in the presence of other osmolytes (12)(13)(14). Additional folding is induced in COR15 proteins suspended in glycerol solutions by the addition of membranes (5,10). This is not observed in the absence of osmolyte, indicating that a certain degree of helicity is necessary for the proteins to interact with membranes.…”

“…Folding of the COR15 proteins can also be induced by trifluoroethanol and glycerol (5,10). Molecular dynamics (MD) simulations indicate that glycerol stabilizes the folded state because it is excluded from the proteins leading to backbone dehydration (11), as suggested previously for the folding of IDPs in the presence of other osmolytes (12)(13)(14).…”