1,720,990 research outputs found
The K2 Dehydrin: An Intrinsically Disordered Membrane Protector
No full textDehydrins are disordered plant proteins which are expressed when the plant is exposed to stresses such as drought, high salinity, and low temperatures. Cell membranes appear to be the primary site of injury, and dehydrins interact with membranes in vitro and in vivo, often exerting a protective effect. K2 dehydrin from Vitis riparia provides an opportunity to study the protective effect of K-segment binding on membrane surfaces. Liposome fusion assays indicate that K2 attenuates freeze-thaw-induced damage in PC/PA membranes. This effect is not a result of significant coverage of the membrane surface, since merocyanine 540 accessibility is unaffected. Protection is also not a result of microviscosity change in the hydrophobic interior or the water-phospholipid interface of PC/PA membranes. Differential scanning calorimetry experiments indicate that K2 reduces the thermotropic phase transition temperature of 1,2-dimyristoyl-sn-glycero-3-phosphate/1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes by 3°C. This effect might be due to increased hydration of phosphate head groups by K2
Probing the potential of a charge-based stabilizing interaction between the vitis riparia YSK2 dehydrin and yeast frataxin homologue 1
Full text linkDehydrins are intrinsically disordered proteins expressed throughout the plant kingdom in response to low intracellular water. They likely act as macromolecule cryoprotectants. The impact of the charge distribution of a YSK2 dehydrin from Vitis riparia on the stability of yeast frataxin homologue 1 (Yfh1) at 1˚C was examined using circular dichroism spectroscopy. Three mutant YSK2-constructs were created: YSK2-SpaceK (evenly distributed positive charge), YSK2-Neut (locally neutralized charge), and YSK2-K→R (lysine substituted for arginine). The cryoprotective efficiency of these constructs in addition to a fourth, YSK2-φ→T (specific hydrophobic residues substituted with threonine) were also compared in a lactate dehydrogenase (LDH) activity assay. Charge distribution appears to be an important part of the cryoprotective mechanism of dehydrins with respect to Yfh1 but not LDH, where hydrodynamic radius is a more significant factor. This is suggestive of a charge-based interaction between YSK2 and Yfh1 that should be studied in greater detail in future work
Structural and Biochemical Investigation into Arabidopsis thaliana LEA3 proteins
Full text linkLEA3 proteins are intrinsically disordered proteins expressed during seed development and in response to abiotic stress. Limited studies have shown they can confer stress tolerance in plants, yet their mechanism remains elusive. This thesis aims to further our understanding of the LEA3 group. First, a comprehensive bioinformatics analysis revealed two previously undiscovered C-terminal motifs containing conserved acidic and hydrophobic residues and four N-terminal motifs. Five general architectures were proposed for LEA3 and the physiochemical properties of the different architectures showed clustering in a relatively narrow range compared to the previously studied dehydrins. The evolutionary analysis revealed that the proteins grouped into clades based on their architecture, and that there appears to be at least two distinct groups of LEA3 proteins based on their architectures and physiochemical properties. The presence of LEA3 proteins in non-vascular plants but their absence in algae suggests that LEA3 may have arose in the evolution of land plants. A protocol to express and purify AtLEA3-2 with 15N and 13C isotopes in E. coli is described, although the protocol can be adapted for any LEA3 with or without isotopic labeling. The AtLEA 3-2 gene was cloned into the pET-SUMO vector, which allowed for the SUMO-AtLEA 3-2 fusion protein to be purified using Ni-affinity chromatography and, through the use of Ulp1, a SUMO protease, resulted in an AtLEA 3-2 with a native N-terminus. Lastly, several biochemical experiments were performed to elucidate the function of LEA3 proteins. I show that the LEA3 proteins are disordered in solution, have regions with propensity for order, and are more hydrophobic than other LEA groups. One member, LEA3-4, bound Cu2+ and Fe3+ ions with micromolar affinity. All LEA3 proteins were effective cryoprotectants of LDH and showed a gain in α-helicity in the presence of SDS, while only LEA3-4, showed a gain in α-helicity in the presence of the membrane mimic DPC. I used 15N-HSQC NMR to show that the additional W- and DAELR motifs present in LEA3-4 are involved in the interaction with DPC. I conclude that the LEA3 group could have multiple functions in protecting cells during stress
The roles of residue position and charge in the cryoprotective behaviour of the Vitis riparia K2 and YSK2 dehydrins
No full textDehydrins are group II Late Embryogenesis Abundant proteins that accumulate in plants during seed development and in response to abiotic stresses. The biological function of dehydrins remain elusive, as they are multi-functional in vitro. The contribution of sequence and charge to the cryoprotective behaviour of dehydrins was analyzed using synthetic variants of the Vitis riparia YSK2 and K2 proteins: ScYSK2, a scrambled version of YSK2; and AntiK2, a charge-reversed version of K2. Circular dichroism (CD) spectroscopy analysis revealed that dehydrin-induced changes in the structure of YFH1 was dependent on sequence and independent of residue charge. An LDH cryoprotection assay showed that the efficiency of YSK2 was unrelated to its sequence, whereas the efficiency of K2 depended on its residue charge type. CD spectroscopy analysis also suggested that the mechanism of protein stabilization by dehydrins may be more like glycerol than polyethylene glycol. Identifying functionally important residues in dehydrins would provide insight into the potential mechanism(s) of their protective behaviour
Exploring the Role of Pseudophosphorylation in the Nuclear Localization and Function of VrDHN1
No full textDehydrins are intrinsically disordered proteins known for their ability to confer desiccation tolerance to plants, potentially through the protection of DNA, membranes, and proteins. With the Vitis riparia dehydrin VrDHN1, I explore potential mechanisms for nuclear localization through phosphorylation of the S-segment and/or the proposed nuclear localization signal called the basic domain. Using pseudophosphorylated constructs, I show that the nuclear to cytoplasmic ratio did not increase proportionally to the level of pseudophosphorylation in the S-segment, but that the S70E mutation caused a 27% increase in nuclear localization. Blocking phosphorylation and basic domain binding did not prevent nuclear localization, suggesting that phosphorylation may result in a shift from passive to active transport. In membrane binding and protein cryoprotection assays, pseudophosphorylation resulted in increased α-helicity in the presence of SDS, and a reduced PD50 in the LDH assay, indicative of improved cryoprotective efficiency. Further work investigating the potential phosphorylation-mediated active-to-passive switch is required
The role of a Vitis riparia dehydrin in protecting DNA from oxidative stress
Full text linkDehydrins are intrinsically-disordered proteins expressed during seed development and during abiotic stress. They have been shown to confer stress tolerance to plants, and many different methods of action have been proposed for these proteins. Here, I first show using both circular dichroism and NMR that the long-proposed nucleotide binding role of the Y-segment of dehydrins is incorrect. I then examine the expression, subcellular localization, and role of the YSK2-type Vitis riparia dehydrin, VrDHN1. Through phenol protein extraction studies, it was found that VrDHN1 was expressed during cold and dehydration stresses, as well as in response to the plant stress hormone ABA. Fluorescence-localization, confirmed by subcellular fractionation, revealed that VrDHN1 was found in the nucleus and in the cytoplasm of plant cells. The ability of VrDHN1 to bind to DNA in vitro was then investigated. VrDHN1 was shown to bind to DNA in a manner that was dependent on the sequence of VrDHN1, but not on the sequence of the DNA. This binding was not stimulated by metal ions, and was somewhat reduced by the pseudophosphorylation of the protein. Furthermore, the binding of VrDHN1 to DNA was shown to protect the DNA from oxidative damage caused by hydroxyl radicals, a function that was unhindered by the pseudophosphorylation of VrDHN1. In vivo experiments to confirm this protection of DNA were attempted, but were limited by difficulties in generating stable VrDHN1 transformants. I propose a model of DNA protection against oxidative damage, where VrDHN1 binds to DNA in a low-affinity and non-DNA sequence specific manner to reduce damage caused by an ROS attack.Natural Sciences and Engineering Research Council of CanadaOntario Graduate Scholarshi
The role of the K2 dehydrin from Vitis riparia in the cryoprotection of lactate dehydrogenase
Full text linkThis thesis focuses on the K2 dehydrin from 'Vitis riparia' (wild grape) and how it functions to protect the enzyme, lactate dehydrogenase (LDH) from freeze-thaw damage. Dehydrins are proteins which protect select plants from cold and dehydrative stress. The biochemical mechanism by which they protect proteins from dehydrative damage is not well characterized. These proteins are currently proposed to function by binding water and protecting cellular proteins and membranes. Initial research involved measuring the cryoprotective effect of K2 on LDH, which was found to be greater than that of our positive control, bovine serum albumin. Nuclear magnetic resonance was performed to establish which residues in K2 may be involved in these interactions; however, no interactions were observed. 1-anilinonaphthalene-8-sulfonic acid, gel filtration and light scattering were used to determine how K2 protected LDH from denaturation. The results showed that aggregation was a major cause of LDH denaturation. Current studies focus on dilution experiments to establish whether K 2 acts as a molecular shield to protect LDH during freeze-thaw, and salt cryoprotection assays to observe the importance of electrostatic interactions during protection
Codon Bias in Human Genes
No full textCodon bias describes the tendency to use certain synonymous codons to encode amino acids. It is well established that codon bias varies between different organisms and plays a role in gene expression and co-translational folding. It is important to understand codon bias because a better understanding of gene expression and translation mechanics may allow for more efficient recombinant protein production, and could ultimately improve the ability to create synthetic genes. Human genes were investigated to elucidate the connection between their codon bias and the subsequent impact on structure, function, and tissue specific expression levels. Analysis was performed by representing human genes according to their codon bias, then clustering genes together that have a similar codon bias. Gene clusters were studied to see if genes that use similar codons are statistically more likely to share other properties. Clustering was performed using a novel data driven approach to a simple clustering algorithm called anchor clustering. Anchor clustering was used because it is fast and deterministic; two qualities that other approaches can struggle with when clustering data in high dimensional spaces. To study the connection between gene product structure and codon bias, clusters were analysed according to their likelihood to contain intrinsically disordered proteins. Because structure and function are so closely related, clusters were also analysed for GO term overrepresentation. Last, clusters were examined through the lens of tissue specific gene expression by incorporating expression information at the mRNA and protein levels. The analyses revealed an association between codon usage and the propensity of a gene product to be intrinsically disordered, while the functional analyses revealed that codon bias is associated with cell cycle regulation and cell type differentiation. Expression analysis revealed that in humans there may be a codon bias associated with highly expressed genes indiscriminate of tissue, as well as tissue specific codon biases in the cortex, testis, and liver. Some of the tissue specific findings have been found by other groups, but this investigation distinguishes between an organism-wide codon bias associated with high expression and particular codon biases associated with high expression in individual tissues. In addition, this work builds on the current knowledge of codon bias, determining if these findings previously only evaluated using mRNA levels also appear at the protein concentration level. The results suggest that codon harmonization can be improved further by seeking to replicate the tissue codon bias in which a gene could be highly expressed
Bioprospecting and Biotechnology of Extremophiles
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
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