Synthesis of the Protein Cutting Reagent Iron (S)-1-(p-Bromoacetamidobenzyl)ethylenediaminetetraacetate and Conjugation to Cysteine Side Chains

Abstract

Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced. The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit. The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

“…The efficiencies of Fe⅐BABE conjugation were 83.6 and 81.6% for [269C]␣ and [45A269C]␣, respectively, as determined by measuring free cysteine side chains (19). When the Fe⅐BABE-conjugated RNA polymerases were tested for their activities in CRP-directed transcription using the test promoters, the modified RNA polymerases were as active as the unmodified enzymes (data not shown), indicating that the RNA polymerases retain the CRP contact activity even after Fe⅐BABE conjugation.…”

“…The conjugation of Fe⅐BABE onto the ␣ derivatives, [269C]␣ and [45A269C]␣, and the reconstitution of RNA polymerase carrying Fe⅐BABE on both of two ␣ subunits, ␣(Fe)␤⅐␣(Fe)␤Ј, or only the ␤Ј-associated ␣, ␣␤⅐␣(Fe)␤Ј, were carried out as described by Murakami et al (20). The efficiency of Fe⅐BABE conjugation was determined by measuring free cysteine side chains with the fluorescent reagent N- [4-[7- (19). The hybrid RNA polymerase carrying Fe⅐BABE on the ␤-associated ␣, ␣(Fe)␤⅐␣␤Ј, was reconstituted by mixing Fe⅐BABE-labeled [269C], [45A]␣-His 6 (C), ␤ and ␤Ј in a molar ratio of 1:10:1:1.…”

“…Experiments described herein suggest that the CTDs of the two ␣ subunits can interact with different subunits of both bound CRP dimers, and this results in complexes of different architectures depending on the positions of the upstream CRP dimer. To investigate the effects of CRP in positioning of the two ␣ subunits, we have applied an affinity DNA cleavage method by protein-conjugated EDTA⅐Fe (17)(18)(19)(20), which provides information on the location of each of the two ␣ subunits. (20).…”

Functional Architecture of T7 RNA Polymerase Transcription Complexes

“…An enzyme mutated in the downstream positive patch behaved like the WT RNAP in the 100 and 800 mM NaCl conditions (lanes 9-12), but reproducibly exhibited less transcript release in 0 mM NaCl (lanes 7-8). Enzymes mutated in the upstream positively charged region showed increased transcript release, with a quadruply substituted enzyme releasing most transcripts under all 3 NaCl conditions (lanes 13-18) and a doubly substituted enzyme releasing most of the transcripts in the 100 and 800 mM NaCl reactions (lanes [19][20][21][22][23][24] …”

“…Effects of mutations in up-and downstream positively charged regions on EC stability. A: Transcripts (13mers) retained ("R") or freed ("F") from halted ECs formed with WT (lanes 1-6), K711C/K713E/K714E (lanes 7-12), K407E/K494E/K332E/K412C (lanes 13-18), or K407E/K412E (lanes 19-24) mutant T7RNAPs in the presence of 0 mM (lanes 1,2,7,8,13,14,19,20), 100 mM (lanes 3,4,9,10,15,16,21,22), or 800 mM (lanes 5,6,11,12,17,18,23,24) NaCl. B: Ratio of Free/Retained transcripts from the experiment in A plotted vs. enzyme and NaCl concentration (error bars give ranges from n=2).…”