GTPase Assays and Tryptophan Emission in FtsZ

High speed pelleting and co-pelleting, and GTPase assays were performed in HEK50 buffer (50 mM HEPES-KOH pH 7.2, 50 mM KCl, 0.1 mM EDTA) as described previously (Goley et al., 2010 (link); Sundararajan et al., 2015 (link)). When indicated, MgCl₂ was added to 1, 2.5 or 10 mM, GTP was added to 2 mM, or GMP-CPP (Jena Bioscience) was added to 0.2 mM. Tryptophan assay was performed by measuring tryptophan emission at 344 nm of FtsZ L72W ± FtsZ in HEK50 buffer with ZapA or ZauP at after exciting at 295 nm using Fluoromax-3 as described (Milam and Erickson, 2013 (link)).

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Woldemeskel S., McQuillen R., Hessel A.M., Xiao J, & Goley E.D. (2017). A Conserved Coiled-Coil Protein Pair Focuses the Cytokinetic Z-Ring in Caulobacter crescentus. Molecular microbiology, 105(5), 721-740.

Publication 2017

GMP-CPP

Assay Buffer Edta Gtpase Hepes Mgcl2 Nm 295 Tryptophan Zapa

Corresponding Organization : Johns Hopkins University

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Variable analysis

independent variables

MgCl2 concentration (1 mM, 2.5 mM, or 10 mM)
Nucleotide addition (GTP at 2 mM or GMP-CPP at 0.2 mM)

dependent variables

High speed pelleting and co-pelleting
GTPase activity
Tryptophan emission at 344 nm of FtsZ L72W ± FtsZ in the presence of ZapA or ZauP

control variables

HEK50 buffer (50 mM HEPES-KOH pH 7.2, 50 mM KCl, 0.1 mM EDTA)
Experimental methods described in previous publications (Goley et al., 2010; Sundararajan et al., 2015; Milam and Erickson, 2013)

controls

Positive control: FtsZ L72W ± FtsZ in the presence of ZapA or ZauP (for tryptophan assay)
Negative control: Not explicitly mentioned

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