Fluorescence quenching studies of γ-butyrolactone binding protein (CprB) from streptomyces coelicolor A3(2)

Anwesha Biswas, Ravi K. Swarnkar, Hussain Bhukya, Suraj K. Sahoo, P. I. Pradeepkumar, G. Naresh Patwari, Ruchi Anand

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Quorum sensing is a cell density dependent phenomenon that utilizes small molecule inducers like γ-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 γ-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. 

Original languageEnglish
Pages (from-to)10035-10042
Number of pages8
JournalJournal of Physical Chemistry B
Issue number34
Publication statusPublished - 28 Aug 2014
Externally publishedYes

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