In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET

Seidel, Thorsten; Seefeldt, Britta; Sauer, Markus; Dietz, Karl‐Josef

doi:10.1016/j.jbiotec.2010.06.016

Cited by 23 publications

(20 citation statements)

References 39 publications

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“…Simultaneous cotransfection of protoplasts with 2-CysPrx fused to CFP, YFP, and mCherry, respectively, enables radiationless energy transfer from CFP via YFP to mCherry (two-step FRET). Occurrence of two-step FRET depends on close proximity of three flagged subunits and in the case of 2-CysPrx indicates existence of oligomeric 2-CysPrx in the chloroplast (159). These data sets prove that Prxs are parts of functional modules that may form stable but also dynamic complexes in dependence on redox environment and interacting partners.…”

Section: Conformational Dynamics and Interacting Partners Of Prx mentioning

confidence: 68%

Peroxiredoxins in Plants and Cyanobacteria

Dietz

2011

Antioxidants & Redox Signaling

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Peroxiredoxins (Prx) are central elements of the antioxidant defense system and the dithiol-disulfide redox regulatory network of the plant and cyanobacterial cell. They employ a thiol-based catalytic mechanism to reduce H 2 O 2 , alkylhydroperoxide, and peroxinitrite. In plants and cyanobacteria, there exist 2-CysPrx, 1-CysPrx, PrxQ, and type II Prx. Higher plants typically contain at least one plastid 2-CysPrx, one nucleo-cytoplasmic 1-CysPrx, one chloroplast PrxQ, and one each of cytosolic, mitochondrial, and plastidic type II Prx. Cyanobacteria express variable sets of three or more Prxs. The catalytic cycle consists of three steps: (i) peroxidative reduction, (ii) resolving step, and (iii) regeneration using diverse electron donors such as thioredoxins, glutaredoxins, cyclophilins, glutathione, and ascorbic acid. Prx proteins undergo major conformational changes in dependence of their redox state. Thus, they not only modulate cellular reactive oxygen species-and reactive nitrogen speciesdependent signaling, but depending on the Prx type they sense the redox state, transmit redox information to binding partners, and function as chaperone. They serve in context of photosynthesis and respiration, but also in metabolism and development of all tissues, for example, in nodules as well as during seed and fruit development. The article surveys the current literature and attempts a mostly comprehensive coverage of present day knowledge and concepts on Prx mechanism, regulation, and function and thus on the whole Prx systems in plants. Antioxid. Redox Signal. 15, 1129-1159

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Section: Conformational Dynamics and Interacting Partners Of Prx mentioning

confidence: 68%

Peroxiredoxins in Plants and Cyanobacteria

Dietz

2011

Antioxidants & Redox Signaling

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“…Evidence from PhoA fusion experiments confirmed that the topological structure of NhaD2 is N in -C out , in which sites 39 and 486 are located in the periplasmic space. The principle use of FRET is to monitor the interactions between molecules and to estimate the distance between those molecules, even in living cells (24). In this study, cyan fluorescent protein (CFP) and YFP fusion strains at site 39 and the N terminus of NhaD2 were constructed for FRET analysis.…”

Section: Discussionmentioning

confidence: 99%

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

et al. 2017

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Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na ϩ and Li ϩ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na ϩ and Li ϩ expulsion, as well as complementary growth following exposure to 300 mM Na ϩ and 100 mM Li ϩ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu 38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile.IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu 38 , in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

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“…FRET between three fluorescent proteins has been described before and was denominated 2-step FRET or 3-chromophore FRET (Watrob et al, 2003; Galperin et al, 2004; He et al, 2004, 2005; Seidel et al, 2010; Sun et al, 2010). For such 2-step FRET-measurements in living cells the commonly used FRET-pair CFP-YFP was supplemented by the red fluorescent proteins HcRed (He et al, 2004), mRFP1 (Galperin et al, 2004; He et al, 2005) or mCherry (Seidel et al, 2010). So the energy is ideally transferred from the donor (CFP) via the mediator (YFP) to the acceptor (red fluorescent protein), both steps were analyzed by sensitized emission.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…So the energy is ideally transferred from the donor (CFP) via the mediator (YFP) to the acceptor (red fluorescent protein), both steps were analyzed by sensitized emission. However, the situation becomes complex since three distinct FRET-pairs have to be considered, resulting e.g., in direct energy transfer between donor and acceptor bypassing the mediator (Seidel et al, 2010). This can be overcome by control measurements e.g., with REACh as a “mediator” that is no longer capable to transfer energy to the acceptor (Seidel et al, 2010) or by photoswitchable mediators that allow the direct estimation of direct energy transfer between donor and acceptor, although the absence of the mediator's absorption is expected to enhance the probability of FRET between donor and acceptor.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…However, the situation becomes complex since three distinct FRET-pairs have to be considered, resulting e.g., in direct energy transfer between donor and acceptor bypassing the mediator (Seidel et al, 2010). This can be overcome by control measurements e.g., with REACh as a “mediator” that is no longer capable to transfer energy to the acceptor (Seidel et al, 2010) or by photoswitchable mediators that allow the direct estimation of direct energy transfer between donor and acceptor, although the absence of the mediator's absorption is expected to enhance the probability of FRET between donor and acceptor. Theoretically, FRET measurements by sensitized emission have the potential to be supplied by further fluorescent proteins like UV-fluorescent proteins such as Sirius (Tomosugi et al, 2009) as donors and infrared-fluorescent proteins as acceptors resulting in a cascade of 3–4 steps of FRET.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

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Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

et al. 2013

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Förster resonance energy transfer (FRET) describes excitation energy exchange between two adjacent molecules typically in distances ranging from 2 to 10 nm. The process depends on dipole-dipole coupling of the molecules and its probability of occurrence cannot be proven directly. Mostly, fluorescence is employed for quantification as it represents a concurring process of relaxation of the excited singlet state S1 so that the probability of fluorescence decreases as the probability of FRET increases. This reflects closer proximity of the molecules or an orientation of donor and acceptor transition dipoles that facilitates FRET. Monitoring sensitized emission by 3-Filter-FRET allows for fast image acquisition and is suitable for quantifying FRET in dynamic systems such as living cells. In recent years, several calibration protocols were established to overcome to previous difficulties in measuring FRET-efficiencies. Thus, we can now obtain by 3-filter FRET FRET-efficiencies that are comparable to results from sophisticated fluorescence lifetime measurements. With the discovery of fluorescent proteins and their improvement toward spectral variants and usability in plant cells, the tool box for in vivo FRET-analyses in plant cells was provided and FRET became applicable for the in vivo detection of protein-protein interactions and for monitoring conformational dynamics. The latter opened the door toward a multitude of FRET-sensors such as the widely applied Ca2+-sensor Cameleon. Recently, FRET-couples of two fluorescent proteins were supplemented by additional fluorescent proteins toward FRET-cascades in order to monitor more complex arrangements. Novel FRET-couples involving switchable fluorescent proteins promise to increase the utility of FRET through combination with photoactivation-based super-resolution microscopy.

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In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET

Cited by 23 publications

References 39 publications

Peroxiredoxins in Plants and Cyanobacteria

Peroxiredoxins in Plants and Cyanobacteria

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

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