TY - JOUR A1 - Bremer, Anne A1 - Kent, Ben A1 - Hauss, Thomas A1 - Thalhammer, Anja A1 - Yepuri, Nageshwar R. A1 - Darwish, Tamim A. A1 - Garvey, Christopher J. A1 - Bryant, Gary A1 - Hincha, Dirk K. T1 - Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration JF - Biophysical journal N2 - Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic a-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into a-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3 degrees C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we. could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein alpha-helix. Y1 - 2017 U6 - https://doi.org/10.1016/j.bpj.2017.06.027 SN - 0006-3495 SN - 1542-0086 VL - 113 SP - 572 EP - 579 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Bremer, Anne A1 - Wolff, Martin A1 - Thalhammer, Anja A1 - Hincha, Dirk K. T1 - Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes JF - The FEBS journal N2 - 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 alpha-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. KW - intrinsically disordered proteins KW - late embryogenesis abundant proteins KW - osmolytes KW - protein folding KW - protein-membrane interaction Y1 - 2017 U6 - https://doi.org/10.1111/febs.14023 SN - 1742-464X SN - 1742-4658 VL - 284 SP - 919 EP - 936 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Hilker, Monika A1 - Schwachtje, Jens A1 - Baier, Margarete A1 - Balazadeh, Salma A1 - Bäurle, Isabel A1 - Geiselhardt, Sven A1 - Hincha, Dirk K. A1 - Kunze, Reinhard A1 - Mueller-Roeber, Bernd A1 - Rillig, Matthias G. A1 - Rolff, Jens A1 - Schmülling, Thomas A1 - Steppuhn, Anke A1 - van Dongen, Joost A1 - Whitcomb, Sarah J. A1 - Wurst, Susanne A1 - Zuther, Ellen A1 - Kopka, Joachim T1 - Priming and memory of stress responses in organisms lacking a nervous system JF - Biological reviews KW - priming KW - stress signalling KW - epigenetics KW - memory KW - fitness KW - stress tolerance KW - defence KW - bet hedging Y1 - 2016 U6 - https://doi.org/10.1111/brv.12215 SN - 1464-7931 SN - 1469-185X VL - 91 SP - 1118 EP - 1133 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Hundertmark, Michaela A1 - Dimova, Rumiana A1 - Lengefeld, Jan A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding. N2 - Intrinsically disordered proteins (IDPs) constitute a substantial part of cellular proteomes. Late embryogenesis abundant (LEA) proteins are mostly predicted to be IDPs associated with dehydration tolerance in many plant, animal and bacterial species. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been experimentally investigated in a small number of proteins. Here, we report on the structure and interactions with membranes of the Pfam LEA_1 protein LEA18 from the higher plant Arabidopsis thaliana. This functionally uncharacterized positively charged protein specifically aggregated and destabilized negatively charged liposomes. Isothermal titration calorimetry showed binding of the protein to both charged and uncharged membranes. LEA18 alone was largely unstructured in solution. While uncharged membranes had no influence on the secondary structure of LEA18, the protein partially folded into ;-sheet structure in the presence of negatively charged liposomes. These data suggest that LEA18 does not function as a membrane stabilizing protein, as suggested for other LEA proteins. Instead, a possible function of LEA18 could be the composition-dependent modulation of membrane stability, e.g., during signaling or vesicle-mediated transport. Research Highlights Y1 - 2011 SN - 0006-3002 ER - TY - JOUR A1 - Hundertmark, Michaela A1 - Dimova, Rumiana A1 - Lengefeld, Jan A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding JF - Biochimica et biophysica acta : Biomembranes N2 - Intrinsically disordered proteins (IDPs) constitute a substantial part of cellular proteomes. late embryogenesis abundant (LEA) proteins are mostly predicted to be IDPs associated with dehydration tolerance in many plant, animal and bacterial species. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been experimentally investigated in a small number of proteins. Here, we report on the structure and interactions with membranes of the Pfam LEA_1 protein LEA18 from the higher plant Arabidopsis thaliana. This functionally uncharacterized positively charged protein specifically aggregated and destabilized negatively charged liposomes. Isothermal titration calorimetry showed binding of the protein to both charged and uncharged membranes. LEA18 alone was largely unstructured in solution. While uncharged membranes had no influence on the secondary structure of LEA18, the protein partially folded into beta-sheet structure in the presence of negatively charged liposomes. These data suggest that LEA18 does not function as a membrane stabilizing protein, as suggested for other LEA proteins. Instead, a possible function of LEA18 could be the composition-dependent modulation of membrane stability, e.g., during signaling or vesicle-mediated transport. KW - Intrinsically disordered protein KW - Late embryogenesis abundant protein KW - Membrane stability KW - Protein-membrane interaction KW - Protein folding Y1 - 2011 U6 - https://doi.org/10.1016/j.bbamem.2010.09.010 SN - 0005-2736 VL - 1808 IS - 1 SP - 446 EP - 453 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Hundertmark, Michaela A1 - Popova, Antoaneta V. A1 - Rausch, Saskia A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - Influence of drying on the secondary structure of intrinsically disordered and globular proteins JF - Biochemical and biophysical research communications N2 - Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying. KW - Desiccation KW - CD spectroscopy KW - FTIR spectroscopy KW - Intrinsically disordered proteins KW - LEA proteins KW - Protein secondary structure Y1 - 2012 U6 - https://doi.org/10.1016/j.bbrc.2011.11.067 SN - 0006-291X VL - 417 IS - 1 SP - 122 EP - 128 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Knox-Brown, Patrick A1 - Rindfleisch, Tobias A1 - Günther, Anne A1 - Balow, Kim A1 - Bremer, Anne A1 - Walther, Dirk A1 - Miettinen, Markus S. A1 - Hincha, Dirk K. A1 - Thalhammer, Anja T1 - Similar Yet Different BT - Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins JF - International Journal of Molecular Sciences N2 - The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics. KW - IDP KW - LEA protein KW - abiotic stress KW - dehydration KW - conformational rearrangement KW - membrane stabilization KW - sequence-structure-function relationship Y1 - 2020 U6 - https://doi.org/10.3390/ijms21082794 SN - 1422-0067 VL - 21 IS - 8 PB - Molecular Diversity Preservation International CY - Basel ER - TY - JOUR A1 - Navarro-Retamal, Carlos A1 - Bremer, Anne A1 - Alzate-Morales, Jans H. A1 - Caballero, Julio A1 - Hincha, Dirk K. A1 - Gonzalez, Wendy A1 - Thalhammer, Anja T1 - Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana JF - Physical chemistry, chemical physics : a journal of European Chemical Societies N2 - The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain alpha-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state. Y1 - 2016 U6 - https://doi.org/10.1039/c6cp02272c SN - 1463-9076 SN - 1463-9084 VL - 18 SP - 25806 EP - 25816 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Navarro-Retamal, Carlos A1 - Bremer, Anne A1 - Ingolfsson, Helgi I. A1 - Alzate-Morales, Jans A1 - Caballero, Julio A1 - Thalhammer, Anja A1 - Gonzalez, Wendy A1 - Hincha, Dirk K. T1 - Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A JF - Biophysical journal N2 - Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic alpha-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results. Y1 - 2018 U6 - https://doi.org/10.1016/j.bpj.2018.08.014 SN - 0006-3495 SN - 1542-0086 VL - 115 IS - 6 SP - 968 EP - 980 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Popova, Antoaneta V. A1 - Hundertmark, Michaela A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes JF - Biochimica et biophysica acta : Biomembranes N2 - Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic a-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% alpha-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% alpha-helical in the dry state. However, the dry protein contained 15% beta-sheets. FTIR spectroscopy revealed the (beta-sheets to be largely due to aggregation. beta-Sheet content was reduced and alpha-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates. KW - Desiccation KW - CD spectroscopy KW - FTIR spectroscopy KW - LEA protein KW - Protein-membrane interactions KW - Protein secondary structure Y1 - 2011 U6 - https://doi.org/10.1016/j.bbamem.2011.03.009 SN - 0005-2736 VL - 1808 IS - 7 SP - 1879 EP - 1887 PB - Elsevier CY - Amsterdam ER -