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

Abstract

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.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

“…The dependency of Trp fluorescence properties (e.g., absorption and emission maxima, fluorescence intensity, anisotropy and quantum yield) on its micro environment has been exploited in numerous studies to derive inference with respect to protein conformational changes and/or protein denaturation [ 14 , 56 , 57 , 58 , 59 , 60 , 61 ]. Gorinstein et al utilized the change in intrinsic Trp fluorescence and surface hydrophobicity of human serum proteins to evaluate the effect of beer consumption on protein integrity in patients suffering from coronary artery disease [ 62 ].…”

Estimation of a stronger heparin binding locus in fibronectin domain III14using thermodynamics and molecular dynamics

“…5a that the free heparin showed a broad minimum between 208 nm and 214 nm, which is a clear sign of the presence of helical structures. 40 The helical content of heparin was found to be reduced at pH 7.4 compared to pH 4.0, which would suggest an alteration in heparin structure. Heparin is a negatively charged structure due to the presence of sulfate and carboxylate moieties, which gets protonated at lower pH, resulting in more structured helical arrangement.…”

“…The complex has a broad positive peak between 220 nm and 240 nm resulting from the involvement of intermolecular disulde bonds present in the domain I and II as well as the two intramolecular disulde bridges that connect the two bronectin units. 40,42,43 CD of Free a Errors were about 15 to 20% in K A and DG. FN (Fig.…”

Volatile Organic Compound Gamma-Butyrolactone Released upon Herpes Simplex Virus Type -1 Acute Infection Modulated Membrane Potential and Repressed Viral Infection in Human Neuron-Like Cells

“…It is not known how the virus triggered the production of GBL, but it is quite likely that the infectious process stimulates the production of Gamma-Hydroxybutyric acid or 4-hydroxybutanoic acid (GHB), which is then converted to GBL, since GHB and GBL have spontaneous inter-conversion [ 21 , 22 ]. In Streptomyces , GBL serves as a quorum sensing molecule [ 9 , 23 – 25 ]. Production of GBL is regulated and the intracellular concentration is positively co-related with the production of secondary metabolites such as antibiotics.…”