Abstract
Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.
Highlights
Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and waterrelated stress
We seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection– fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations
We used imaging total internal reflection–fluorescence correlation spectroscopy (ITIR-FCS)4 in combination with atomic-scale explicitly solvated MD simulations to study the effect of Lti30 on membrane lipid diffusion and membrane organization
Summary
Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and waterrelated stress. Lti is one such dehydrin found in Arabidopsis thaliana that shows very high expression at cold temperatures and during desiccation [21] It is composed of six conserved K-segments flanked with pairs of histidines that have been found to be responsible for membrane binding [6]. ITIR-FCS is a multiplexed camerabased imaging FCS modality that provides spatially resolved diffusion coefficient maps [23] This method employs total internal reflection illumination, which excites molecules close to the surface, and allows measurements primarily at the membrane and avoids background signal contributions by the bulk solution. MD simulation is a theoretical approach that enables the study of the conformational dynamics of single molecules or complexes thereof in atomic resolution, which provides information complementary to single molecule fluorescence experiments [24]
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