RESUMO
Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.
RESUMO
A small series of copoly(α,l-glutamic acid/dl-allylglycine)s with the same chain length and allylglycine content (â¼10 mol %) but different spatial distribution of allylglycine units was synthesized and subsequently glycosylated via thiol-ene chemistry. Dilute aqueous copolypeptide solutions (0.1 wt %, physiological saline) were analyzed by circular dichroism spectroscopy, dynamic light scattering, and cryogenic transmission electron microscopy. The copolypeptides adopted a random coil or α-helix conformation, depending on solution pH, and the glycosylated residues either distorted or enhanced the folding into an α-helix depending on their location and spatial distribution along the chain. However, regardless of their secondary structure and degree of charging, all partially glycosylated copolypeptides self-assembled into 3D spherical structures, supposedly driven by a hydrophilic effect promoting microphase separation into glucose-rich and glutamate-rich domains.
Assuntos
Solução Salina , Solução Salina/química , Ácido Glutâmico/química , Glicosilação , Dicroísmo Circular , Soluções , Glicina/química , Concentração de Íons de HidrogênioRESUMO
To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review, we summarize current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, post-translational modifications, or water deprivation, we provide a detailed summary of current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil to helix transitions, self-assembly, and their recently discovered ability to undergo liquid-liquid phase separation. We point out the promising potential of LEA proteins in biotechnological and agronomic applications, and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.
Assuntos
Desidratação , Desenvolvimento Embrionário , Desidratação/metabolismo , Água/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (RS). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions.
Assuntos
Descoberta de Drogas/métodos , Exenatida/química , Exenatida/farmacologia , Ácidos Graxos Voláteis/química , Peptídeo 1 Semelhante ao Glucagon/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptores de Glucagon/agonistas , Acilação , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Difusão Dinâmica da Luz , Interações Hidrofóbicas e Hidrofílicas , Lagartos/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Estrutura Secundária de ProteínaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Biologia Computacional/métodos , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Dobramento de Proteína , Estresse FisiológicoRESUMO
The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an α-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity. Coarse-grained ensemble modelling allowed a structure-based interpretation of the SAXS data. Our results demonstrate that COR15A behaves as a biomacromolecule with polymer-like properties which strongly depend on solution osmolarity. Biomacromolecular self-assembly occurring at high solvent osmolarity is initiated by the occurrence of two specific structural subpopulations of the COR15A monomer. The osmolarity dependent structural selection mechanism is an elegant way for conformational regulation and assembly of COR15A. It highlights the importance of the polymer-like properties of IDPs for their associated biological function.
Assuntos
Proteínas de Arabidopsis/química , Proteínas Intrinsicamente Desordenadas/química , Concentração Osmolar , Conformação Proteica , Espalhamento a Baixo Ângulo , Solventes/química , Raios XRESUMO
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 α-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.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Dobramento de Proteína , Arabidopsis , Glicolipídeos/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. Current potato varieties are highly susceptible to drought stress. In view of global climate change, selection of cultivars with improved drought tolerance and high yield potential is of paramount importance. Drought tolerance breeding of potato is currently based on direct selection according to yield and phenotypic traits and requires multiple trials under drought conditions. Marker-assisted selection (MAS) is cheaper, faster and reduces classification errors caused by noncontrolled environmental effects. We analysed 31 potato cultivars grown under optimal and reduced water supply in six independent field trials. Drought tolerance was determined as tuber starch yield. Leaf samples from young plants were screened for preselected transcript and nontargeted metabolite abundance using qRT-PCR and GC-MS profiling, respectively. Transcript marker candidates were selected from a published RNA-Seq data set. A Random Forest machine learning approach extracted metabolite and transcript markers for drought tolerance prediction with low error rates of 6% and 9%, respectively. Moreover, by combining transcript and metabolite markers, the prediction error was reduced to 4.3%. Feature selection from Random Forest models allowed model minimization, yielding a minimal combination of only 20 metabolite and transcript markers that were successfully tested for their reproducibility in 16 independent agronomic field trials. We demonstrate that a minimum combination of transcript and metabolite markers sampled at early cultivation stages predicts potato yield stability under drought largely independent of seasonal and regional agronomic conditions.
Assuntos
Biomarcadores , Secas , Marcadores Genéticos , Solanum tuberosum/fisiologia , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Aprendizado de Máquina , Modelos Genéticos , Melhoramento Vegetal/métodos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Tubérculos/genética , Tubérculos/metabolismo , Reprodutibilidade dos Testes , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Amido/genética , Amido/metabolismo , Estresse FisiológicoRESUMO
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 α-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 α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°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 α-helix.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Água/metabolismo , Fosfatidilcolinas/metabolismo , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Propriedades de SuperfícieRESUMO
PURPOSE: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. METHODS: Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. RESULTS: Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. CONCLUSION: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Assuntos
Hipoglicemiantes/química , Insulina Regular Humana/química , Humanos , Injeções Subcutâneas , Insulina/análogos & derivados , Insulina/química , Insulina Aspart/química , Insulina Lispro/química , Insulina de Ação Curta , Cinética , Peso Molecular , Fenóis/química , Agregados Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade , Zinco/químicaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Sequência de Aminoácidos , Dicroísmo Circular , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Extratos Vegetais/química , Folhas de Planta/química , Estrutura Secundária de Proteína , Desdobramento de ProteínaRESUMO
Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins.
Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Congelamento , Membranas Intracelulares/metabolismo , Cloroplastos/enzimologia , LipossomosRESUMO
Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tilacoides/metabolismo , Prótons , Antiporters/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Cloroplastos/metabolismo , LuzRESUMO
Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrinsicamente Desordenadas , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Congelamento , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
With an estimate of hundred thousands of protein molecules per cell and the number of metabolites several orders of magnitude higher, protein-metabolite interactions are omnipresent. In vitro analyses are one of the main pillars on the way to establish a solid understanding of how these interactions contribute to maintaining cellular homeostasis. A repertoire of biophysical techniques is available by which protein-metabolite interactions can be quantitatively characterized in terms of affinity, specificity, and kinetics in a broad variety of solution environments. Several of those provide information on local or global conformational changes of the protein partner in response to ligand binding. This review chapter gives an overview of the state-of-the-art biophysical toolbox for the study of protein-metabolite interactions. It briefly introduces basic principles, highlights recent examples from the literature, and pinpoints promising future directions.
Assuntos
Proteínas , Fenômenos Biofísicos , Biofísica/métodos , Cinética , Ligantes , Ligação Proteica , Proteínas/químicaRESUMO
LEA (late embryogenesis abundant) proteins were originally described almost 30 years ago as accumulating late in plant seed development. They were later found to be induced in vegetative plant tissues under environmental stress conditions and also in desiccation-tolerant micro-organisms and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Most LEA proteins are predicted to be intrinsically disordered and this has been experimentally verified in several cases. In addition, some LEA proteins partially fold, mainly into α-helices, during drying or in the presence of membranes. Recent studies have concentrated on the potential roles of LEA proteins in stabilizing membranes or sensitive enzymes during freezing or drying, and the present review concentrates on these two possible functions of LEA proteins in cellular dehydration tolerance.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Osmose , Adaptação Fisiológica , Arabidopsis/química , Proteínas de Arabidopsis/química , Água/química , Água/metabolismoRESUMO
COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly alpha-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic alpha-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Lipídeos de Membrana/química , Sequência de Aminoácidos , Dicroísmo Circular , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.
Assuntos
Sequência Conservada , Exenatida/química , Interações Hidrofóbicas e Hidrofílicas , Incretinas/química , Sequência de Aminoácidos , Ácidos Graxos/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT50 values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.
Assuntos
Aclimatação , Clorofila/metabolismo , Eletrólitos/metabolismo , Congelamento , Folhas de Planta/fisiologia , Fenômenos Fisiológicos Vegetais , Arabidopsis/fisiologia , Bioensaio , Fluorescência , Imagem Molecular/métodos , Fotossíntese , Desenvolvimento VegetalRESUMO
Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coilâ»helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freezeâ»thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of α-helical structure and the increased α-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coilâ»helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of α-helical structure during freezing induced dehydration.