Synthesis of [32P]Phosphoramidate for Use as a Low Molecular Weight Phosphodonor Reagent

Buckler, David R.; Stock, Ann

doi:10.1006/abio.2000.4639

Cited by 17 publications

(14 citation statements)

References 27 publications

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“…Non-radioactive phosphoramidate was synthesized [43] and used to phosphorylate the Spo0A. On a Superdex 200 column the resulting Spo0A appeared to be predominantly in the dimer form.…”

Section: Methodsmentioning

confidence: 99%

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

et al. 2012

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ComK transcriptionally controls competence for the uptake of transforming DNA in Bacillus subtilis. Only 10%–20% of the cells in a clonal population are randomly selected for competence. Because ComK activates its own promoter, cells exceeding a threshold amount of ComK trigger a positive feedback loop, transitioning to the competence ON state. The transition rate increases to a maximum during the approach to stationary phase and then decreases, with most cells remaining OFF. The average basal rate of comK transcription increases transiently, defining a window of opportunity for transitions and accounting for the heterogeneity of competent populations. We show that as the concentration of the response regulator Spo0A∼P increases during the entry to stationary phase it first induces comK promoter activity and then represses it by direct binding. Spo0A∼P activates by antagonizing the repressor, Rok. This amplifies an inherent increase in basal level comK promoter activity that takes place during the approach to stationary phase and is a general feature of core promoters, serving to couple the probability of competence transitions to growth rate. Competence transitions are thus regulated by growth rate and temporally controlled by the complex mechanisms that govern the formation of Spo0A∼P. On the level of individual cells, the fate-determining noise for competence is intrinsic to the comK promoter. This overall mechanism has been stochastically simulated and shown to be plausible. Thus, a deterministic mechanism modulates an inherently stochastic process.

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“…Non-radioactive phosphoramidate was synthesized [43] and used to phosphorylate the Spo0A. On a Superdex 200 column the resulting Spo0A appeared to be predominantly in the dimer form.…”

Section: Methodsmentioning

confidence: 99%

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

et al. 2012

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“…Removal of the His 6 tag had no effect on subsequent assays. Although His residues can be phosphorylated by phosphoramidate (PA), the rates are negligible compared with phosphorylation of RRs at the active-site Asp (26). …”

Section: Methodsmentioning

confidence: 99%

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

Barbieri

Mack

Robinson

et al. 2010

Journal of Biological Chemistry

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DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.

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“…Secondary polyclonal swine anti-rabbit antibodies, conjugated with alkaline phosphatase, were from Dako, Denmark and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was from Sigma, USA. Non-radioactive phosphoramidate was synthesized by applying the method described for [ 32 P]phosphoramidate by Buckler and Stock (18). It was lyophilized and stored at -20°C until use, when it was dissolved in deionized water to a concentration of 0.2 M. Solutions of phosphoramidate were then stored at -80°C in aliquots that were thawed only once before use.…”

Section: Methodsmentioning

confidence: 99%

A screening method for phosphohistidine phosphatase 1 activity

Beckman-Sundh

Zetterqvist

et al. 2011

Upsala Journal of Medical Sciences

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IntroductionResearch in the field of protein-bound phosphohistidine phosphorylation has been hampered by the difficulties in analysis and detection of phosphohistidine. Therefore a screening method was developed primarily for the analysis of phosphohistidine phosphatase 1 (PHPT1) activity.MethodsA highly positively charged substrate, Ac-Val-Arg-Leu-Lys-His-Arg-Lys-Leu-Arg-pNA, containing the peptide surrounding the phosphorylated histidine in ion channel KCa3.1 was chemically phosphorylated using phosphoramidate. Excess phosphoramidate was removed by anion exchange chromatography using a micro spin column. After incubation of the eluate with PHPT1, the removed phosphate was bound on a consecutive anion exchange spin column. The eluate was assayed in a micro plate format for remaining phosphate in the substrate Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA. Histone H4, also highly positive in charge, was subjected to the same procedure to explore the possibility to use other substrates to PHPT1 in this assay format.ResultsIt was found that Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA and phosphohistone H4 were dephosphorylated by PHPT1. The apparent Km for Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA was in the order of 10 μM.Using this method, phosphohistidine phosphatase activity was detected in mouse liver cell sap with Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA as substrate.DiscussionThe described method for determination of PHPT1 activity is comparably much easier and faster than presently used methods for detection of phosphohistidine phosphatase activity. It is also sensitive, since the lower activity limit was 5 pmol phosphate released per min. It has the potential to be used both for more rapid screening for inhibitors and activators to phosphohistidine phosphatases and for screening of histidine kinases.

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Synthesis of [32P]Phosphoramidate for Use as a Low Molecular Weight Phosphodonor Reagent

Cited by 17 publications

References 27 publications

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

A screening method for phosphohistidine phosphatase 1 activity

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