@article{NavarroRetamalBremerAlzateMoralesetal.2016, author = {Navarro-Retamal, Carlos and Bremer, Anne and Alzate-Morales, Jans H. and Caballero, Julio and Hincha, Dirk K. and Gonzalez, Wendy and Thalhammer, Anja}, title = {Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana}, series = {Physical chemistry, chemical physics : a journal of European Chemical Societies}, volume = {18}, journal = {Physical chemistry, chemical physics : a journal of European Chemical Societies}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1463-9076}, doi = {10.1039/c6cp02272c}, pages = {25806 -- 25816}, year = {2016}, abstract = {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.}, language = {en} } @misc{NavarroRetamalBremerAlzateMoralesetal.2016, author = {Navarro-Retamal, Carlos and Bremer, Anne and Alzate-Morales, Jans H. and Caballero, Julio and Hincha, Dirk K. and Gonz{\´a}lez, Wendy and Thalhammer, Anja}, title = {Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-394503}, pages = {25806 -- 25816}, year = {2016}, abstract = {The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-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.}, language = {en} } @article{BremerKentHaussetal.2017, author = {Bremer, Anne and Kent, Ben and Hauss, Thomas and Thalhammer, Anja and Yepuri, Nageshwar R. and Darwish, Tamim A. and Garvey, Christopher J. and Bryant, Gary and Hincha, Dirk K.}, title = {Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration}, series = {Biophysical journal}, volume = {113}, journal = {Biophysical journal}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2017.06.027}, pages = {572 -- 579}, year = {2017}, abstract = {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.}, language = {en} } @article{BremerWolffThalhammeretal.2017, author = {Bremer, Anne and Wolff, Martin and Thalhammer, Anja and Hincha, Dirk K.}, title = {Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes}, series = {The FEBS journal}, volume = {284}, journal = {The FEBS journal}, publisher = {Wiley}, address = {Hoboken}, issn = {1742-464X}, doi = {10.1111/febs.14023}, pages = {919 -- 936}, year = {2017}, 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 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.}, language = {en} } @phdthesis{Bremer2017, author = {Bremer, Anne}, title = {Structural and functional characterization of three closely related intrinsically disordered proteins from the model plant Arabidopsiis thaliana}, school = {Universit{\"a}t Potsdam}, pages = {86}, year = {2017}, language = {en} } @article{NavarroRetamalBremerIngolfssonetal.2018, author = {Navarro-Retamal, Carlos and Bremer, Anne and Ingolfsson, Helgi I. and Alzate-Morales, Jans and Caballero, Julio and Thalhammer, Anja and Gonzalez, Wendy and Hincha, Dirk K.}, title = {Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A}, series = {Biophysical journal}, volume = {115}, journal = {Biophysical journal}, number = {6}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2018.08.014}, pages = {968 -- 980}, year = {2018}, abstract = {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.}, language = {en} }