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Fluorescence Quenching Studies of gamma-Butyrolactone Binding Protein (CprB) from Streptomyces coelicolor A3(2)

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Title Fluorescence Quenching Studies of gamma-Butyrolactone Binding Protein (CprB) from Streptomyces coelicolor A3(2)
 
Creator BISWAS, A
SWARNKAR, RK
HUSSAIN, B
SAHOO, SK
PRADEEPKUMAR, PI
PATWARI, GN
ANAND, R
 
Subject AUTOREGULATOR-RECEPTOR PROTEIN
REGULATING ANTIBIOTIC PRODUCTION
TIME-RESOLVED FLUORESCENCE
TO-CELL COMMUNICATION
A-FACTOR
PSEUDOMONAS-AERUGINOSA
TRYPTOPHAN FLUORESCENCE
SECONDARY METABOLISM
TRANSCRIPTIONAL REGULATOR
SIGNALING MOLECULES
 
Description Quorum sensing is a cell density dependent phenomenon that utilizes small molecule inducers like gamma-butyrolactones (GBLs) and their receptor proteins for adaptation to the environment. The cognate GBLs that bind to several of this GBL receptor family of proteins remain elusive. Here, using CprB protein from Streptomyces coelicolor A3(2) as a model system, we devise a method suited for ligand screening that would be applicable to the entire family of GBL receptors. Docking studies were performed to confirm the identity of the ligand binding pocket, and it was ascertained that the common gamma-butyrolactone moiety interacts with the conserved tryptophan residue (W127) residing in the ligand binding pocket. The presence of W127 in the cavity was exploited to monitor its fluorescence quenching on the addition of two chemically synthesized GBLs. Analysis of the data with both the native and W185L, mutant versions of the protein confirmed that the compounds used as quenchers reside in the ligand binding pocket. Furthermore, fluorescence lifetime and potassium iodide (KI) quenching studies established that the quenching is static in nature and that the tryptophan residue is buried and inaccessible to surface quenchers. Additionally, a combination of concentration dependent fluorescence quenching and dynamic light scattering experiments revealed that the binding properties of the protein are concentration dependent and it was concluded that the most efficient binding of the ligand is evoked by working at the lowest concentration of protein, providing a sufficient signal, where the aggregation effects are negligible.
 
Publisher AMER CHEMICAL SOC
 
Date 2014-12-29T05:52:06Z
2014-12-29T05:52:06Z
2014
 
Type Article
 
Identifier JOURNAL OF PHYSICAL CHEMISTRY B, 118(34)10035-10042
1520-6106
http://dx.doi.org/10.1021/jp503589h
http://dspace.library.iitb.ac.in/jspui/handle/100/17240
 
Language English