Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Abstract

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long‐distance signal transmission from the N‐terminal cAMP‐binding domain to the C‐terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N‐terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long‐distance conformational signal transduction within the protein from the C‐terminal DNA‐binding domain to the N‐terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped‐flow and Förster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA–CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.

Fluorescence Imaging of Bacterial Killing by Antimicrobial Peptide Dendrimer G3KL

“…Remarkably, the movie indicated that the fluorescence of G3KL-Fluo slightly decreased in the center of the bacteria at 15 min, which might indicate fluorescence quenching due to binding of the dendrimer to DNA (Figure A,B). , Consistent with this hypothesis, incubating G3KL-Fluo with plasmid DNA (pEt25 rsl ) induced significant quenching of fluorescence at N/P = 0.5 and below (excess plasmid, Figure C). Quenching reflects a direct binding interaction between the dendrimer and DNA, as evidenced by the decrease in unbound DNA upon addition of unlabeled G3KL above N/P = 2.5 as measured by the intercalating dye PicoGreen (Figures D and S4).…”

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…70,2006 PHOSPHOTRANSFERASE SYSTEM-RELATED PHOSPHORYLATION 947 which is synthesized from ATP by adenylate cyclase (gene cyaA) (for recent papers on the structure of the Crp/cAMP regulator, see references 316, 471, 671, and 888). Different genes and operons require different levels of Crp/cAMP for full expression because the Crp/cAMP complex can adopt several conformationally active states and because the affinity for the recognition sites on the DNA varies (149,316,417,484,853,888). The concentration of Crp/cAMP is tightly regulated by the PTS.…”

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

“…The evidence that both mutants, Δ crp (Q-type) and Δ crp (R-type), shared some common target genes (13.3% in the up-regulated genes, 16% in the down-regulated genes, Figure 4 ) during the exponential phase was somewhat unexpected. A first, simple interpretation of this phenomenon could suggest that transcription of these sets of genes does not depend on the acetylation stage of CRP, but rather on the presence of several conformer complexes between c-AMP and CRP (Heyduk and Lee, 1989 ; Mukhopadhyay et al, 1999 ; Tworzydło et al, 2005 ; Tutar, 2008 ; Saha et al, 2015 ).…”