Methodology

Many techniques can be used to determine complex protein structures and perform functional assays. Here we discuss some of the most important methods mentioned in the paper.

• QscR Purification: The receptor-ligand complex was expressed using a plasmid in E.coli host. Heterologous expression was used since the native source, P.aeruginosa, is a pathogen and thus more complex to work with. Cell extract was subjected to purification in an anion exchange chromatography column where QscR (with net positive charge) was retained by the negatively charged beads and then subsequently eluted using NaCl gradient (increasing concentrations from 0.1M to 0.4M). Eluant was then run in SDS-PAGE to identify QscR containing fractions which were stored at -80°C.

Fig.1: Anion Exchange Column. a) Protein mixture added in the column which is covered with  cationic resin. b) Positively charged proteins are eluted first, since they do not bind the resin. c), d) Increasing concentration of the ion is used to elute the bound protein in later fractions.[Source: OnlineWaterStore.com ]

• Structure Determination and Analysis (Crystallization): Crystals of ligand bound receptor were obtained in the presence and absence of DNA using the hanging drop vapor diffusion method (Fig.2). Molecular Replacement was primarily used to solve the structure at a resolution of 2.5Å. Domains of homologous LuxR proteins such as TraR and LasR from PDB were used as search models. These structures were used to suggest possible orientations of QscR which could not be derived by crystallization. For example, TraR DNA-binding domain was exploited to produce a model for QscR-DNA complex through superposition of QscR and TraR DBDs. (See more in Structural Insights) Analysis of the structure's stereo-chemical and geometrical properties followed using various Bioinformatics programs.

Fig.2: Simplified Diagram of Hanging Drop Crystallization Method. [Source: Protein Crystallization.]

•  Activity Assay (in vivo): lacZ gene was incorporated in the host DNA and was used to determine whether the protein was successfully expressed. β-galactosidase activity was measured indicating the relative presence of the protein of interest in the bacteria.

• Circular Dichroism: Used to assess the effect of DNA-binding on the ligand-receptor complex by determining α-helical content. CD spectra can determine changes in secondary structure because amino acid residues are optically active, therefore able to rotate the plane of polarisation of light which is measured by CD apparatus. This allows identification of an increase in thermal stability of QscR in the presence of DNA.

• Limited Proteolysis: Trypsin digestion of the receptor molecule bound or unbound to DNA. Reaction was quenched by addition of buffer followed by boiling.

Fig 3: Resulting blot after SDS-PAGE of protein pre- and post-trypsin digestion. The figure shows that DNA-binding protects the receptor from proteolytic cleavage.
 [Source: SI Appendix.]

• Site Directed Mutagenesis: Point mutations were introduced on QscR expression vector in order to identify key amino acid residues in DBDs and LBDs. DNA sequencing was used to verify the resulting sequence before transformation into the E.coli host.