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Molecular properties of disordered plant dehydrins: Membrane interaction and function in stress
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Pia Harryson)ORCID iD: 0000-0003-2749-2415
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2).

Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions.

Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18). 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2016. , p. 62
Keywords [en]
abiotic stress, dehydrin, intrinsically disordered proteins, Lea-proteins, phospholipids
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-136033ISBN: 978-91-7649-065-5 (print)ISBN: 978-91-7649-599-5 (print)OAI: oai:DiVA.org:su-136033DiVA, id: diva2:1050509
Public defence
2017-01-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2016-12-20 Created: 2016-11-29 Last updated: 2016-12-21Bibliographically approved
List of papers
1. Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse
Open this publication in new window or tab >>Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse
2008 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 4, p. 1925-1937Article in journal (Refereed) Published
Abstract [en]

The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-dimensional structure. Their specific molecular action, as well as the reason for their disordered character, is as yet poorly understood. It has been speculated, however, that the dehydrins are tuned to acquire a biologically active structure only under the conditions at which they normally function, i.e. upon dehydration. To test this idea, we here investigate the effect of reduced water content and macromolecular crowding on three dehydrins from Arabidopsis thaliana. As a simplistic model for mimicking cellular dehydration we used polyethylene glycol (PEG), glycerol, and sugars which plants naturally employ as compatible solutes, i.e. sucrose and glucose. Macromolecular crowding was induced by the large polysaccharides ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by the solvent alterations. A notable exception is the dehydrin Cor47 which shows a small, intrinsic increase in helical structure at high concentrations of osmolytes. We also examined the effect of phosphorylation but found no evidence that such post-translational modifications of the dehydrin sequences modulate their structural response to osmolytes and crowding agents. The results suggest that the dehydrins are highly specialised proteins that have evolved to maintain their disordered character under conditions where unfolded states of several globular proteins would tend to collapse.

Keywords
dehydrin, intrinsically disordered protein, macromolecular crowding, circular dichroism (CD)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-17653 (URN)10.1104/pp.108.124099 (DOI)000261501500016 ()
Available from: 2009-01-19 Created: 2009-01-19 Last updated: 2017-12-13Bibliographically approved
2. Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein
Open this publication in new window or tab >>Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein
Show others...
2011 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 23, no 6, p. 2391-2404Article in journal (Refereed) Published
Abstract [en]

Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30's proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-67573 (URN)10.1105/tpc.111.085183 (DOI)000293224200028 ()
Note

authorCount :5

Available from: 2011-12-29 Created: 2011-12-29 Last updated: 2017-12-08Bibliographically approved
3. Membrane-Induced Folding of the Plant Stress Dehydrin Lti30
Open this publication in new window or tab >>Membrane-Induced Folding of the Plant Stress Dehydrin Lti30
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2016 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 171, no 2, p. 932-943Article in journal (Refereed) Published
Abstract [en]

Dehydrins are disordered proteins that are expressed in plants as a response to embryogenesis and water-related stress. The molecular function and structural action of the dehydrins are yet elusive, but increasing evidence points to a role in protecting the structure and functional dynamics of cell membranes. An intriguing example is the cold-induced dehydrin Lti30 that binds to membranes by its conserved K segments. Moreover, this binding can be regulated by pH and phosphorylation and shifts the membrane phase transition to lower temperatures, consistent with the protein's postulated function in cold stress. In this study, we reveal how the Lti30-membrane interplay works structurally at atomic level resolution in Arabidopsis (Arabidopsis thaliana). Nuclear magnetic resonance analysis suggests that negatively charged lipid head groups electrostatically capture the protein's disordered K segments, which locally fold up into a-helical segments on the membrane surface. Thus, Lti30 conforms to the general theme of structure-function relationships by folding upon binding, in spite of its disordered, atypically hydrophilic and repetitive sequence signatures. Moreover, the fixed and well-defined structure of the membrane-bound K segments suggests that dehydrins have the molecular prerequisites for higher level binding specificity and regulation, raising new questions about the complexity of their biological function.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-133244 (URN)10.1104/pp.15.01531 (DOI)000380699200015 ()27208263 (PubMedID)
Available from: 2016-09-07 Created: 2016-09-05 Last updated: 2017-11-21Bibliographically approved
4. Membrane binding of disordered plant dehydrins is tuned by phosphorylation and coordination of Ca2+ and Zn2+ ions.
Open this publication in new window or tab >>Membrane binding of disordered plant dehydrins is tuned by phosphorylation and coordination of Ca2+ and Zn2+ ions.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Dehydrins are intrinsically disordered proteins expressed under water- related stress in plants. As a clue to their function, some dehydrins are found to interact in an orderly manner with negatively-charged lipids, supporting the idea of a key role in safeguarding membrane integrity. We have earlier reported that this lipid interaction is modulated electrostatically. Of particular interest is the pronounced effect of local charge that shed light on how dehydrin function is regulated in vivo. In this study we test the generality of this proposition on four dehydrins from Arabidopsis thaliana representing different dehydrin subgroups. The results show that membrane interaction of dehydrins in their apo state is correlated to their protein net charge. Also, we explore further putative regulation mechanism by investigating the additive role of ion coordination and phosphorylation on membrane binding. The results show that coordination of Ca2+ and Zn2+ have markedly different effects. Coordination of Ca2+ augments mainly the membrane affinity of dehydrins that already bind lipids in their apo states (Lti30 and Rab18). Coordination of Zn2+, on the other hand, induces membrane binding and vesicle assembly of all tested proteins, also those that fail to bind membranes in the absence of metal ions (Cor47 or Lti29). Finally, we observe that the effect of Ca2+ is effectively enhanced by phosphorylation. The observations corroborate the idea of a sensitive and multifaceted regulatory mechanism of the dehydrin function in stressed plant cells but point also at a functional diversity. 

Keywords
Dehydrin, intrinsically disordered proteins, Lea-proteins, membrane binding, calcium, zinc, phosphorylation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136023 (URN)
Available from: 2016-11-29 Created: 2016-11-29 Last updated: 2017-01-10Bibliographically approved
5. The plant Rab18 dehydrin - a disordered stressed induced conditional peripheral membrane protein. Specific interaction with PI(4,5)P2
Open this publication in new window or tab >>The plant Rab18 dehydrin - a disordered stressed induced conditional peripheral membrane protein. Specific interaction with PI(4,5)P2
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Arabidosis dehydrin Rab18 is expressed in response to drought and the phytohormone ABA. As a clue to a general dehydrin function, some dehydrins are found to interact in an orderly manner with negatively-charged lipids, supporting the idea of a key role in safeguarding membrane integrity. Interaction between dehydrins and membranes studied so far has been driven by general electrostatic attractions. Here, we report on specific binding of Rab18 with inositol lipids, especially PI(4,5)P2. This binding is not purely under the control of global protein electrostatics since Rab18 fails to bind to lipid vesicles with a high negative net charge (DOPC:DOPG, 3:1). Instead, Rab18 binds strongly to inositol lipids even at low negative vesicle net charge (i.e. DOPC:DOPI(4,5)P2, 98:2). Further, Rab18 show a high specificity to inositol lipids with a phosphate in the 5th position on the inositol ring i.e. PI(5)P and PI(4,5)P2 whereas a phosphate at 3rd position restrains Rab18 binding (i.e. PI(4,5)P2>PI(3,5)P2>PI(3,4)P2 and PI(5)P>> PI(3)P). Moreover, Rab18 specificity to inositol lipids is mainly augmented by the Arg in the protein sequence and when all six Arg are replaced by Lys is the binding of Rab18 to PI(4,5)P2 almost abolished. The two Arg preceding the first K-seg (Rab18Arg125-126) are key residues in binding PI(4,5)P2 and the mechanistic implication of these Arg is elucidated. We put forward the idea that Rab18 is a conditional peripheral membrane protein that sense and respond to lipid alterations at membrane surfaces during stress. Moreover, by binding to PI(4,5)P2 Rab18 could take part in the regulation of different cellular processes, showing a new role of dehydrins as regulators of plant stress. A possible role of Rab18 in the regulation of ABA induced stomata movements are presented. 

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136030 (URN)
Available from: 2016-11-29 Created: 2016-11-29 Last updated: 2017-01-10Bibliographically approved

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