Table 1.
A summary of the LacI variants used in this study.
In all LacI variants the native 22 C-terminal amino acids have been replaced with a 6xHis purification tag. LacIΔCterm is wild-type with respect to the protein sequence outside of the 22 C-terminal amino acids and served as the basis for the construction of the remaining LacI variants.
Fig 1.
Location of the native cysteine residues of LacI.
Cartoon representation of the LacI dimer-operator complex structure (PDB ID: 1EFA) [3]. The two LacI monomers are shown in orange and blue with their native cysteines highlighted as green and red spheres, respectively. The operator DNA is shown as a grey surface. The structure of the LacI-operator complex was rendered in Pymol (PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Fig 2.
Analysis of the dimerization and DNA binding activities of the LacICys-Lite and LacIDim variants.
(A) The C-terminally 6xHis-tagged LacI variants were purified by Ni-agarose affinity chromatography followed by a size exclusion chromatography on Superdex 200. The homogeneity of the purified LacI variants was analyzed on a 4–20% denaturing polyacrylamide gel. The protein was visualized by staining the gel with Coomassie Brilliant Blue. (B) The molecular weights of the LacI variants were estimated by analytical gel filtration on a HiLoad 16/600 Superdex 200 size exclusion column (see S1 Fig for the calibration curve). (C) The mean hydrodynamic diameters of the LacI variants were estimated by dynamic light scattering using a W130i DLS system at 4°C. Error bars represent SEM (N = 4 independent experiments with 10 technical replicates each). (D) The operator binding activities of the LacI variants were analyzed in an electrophoretic mobility shift assay. A constant amount of a 5’-Cy5-labeled dsDNA harboring the O1 operator sequence was incubated with increasing concentrations of one of each of the LacI variants at room temperature for 1 h. The LacI-operator complexes were resolved on an 8% native polyacrylamide gel and the free and LacI-bound DNA in the gels was visualized by Cy5 fluorescence.
Fig 3.
Introduction of additional cysteines into LacIDimer for fluorescence labeling.
(A). The positions where additional cysteines were introduced for labeling are shown as spheres in the structure of the wild-type LacI dimer-operator complex (PDB ID: 1EFA) [3] color-coded for residue conservation. The residue conservation score from a multiple sequence alignment was mapped onto the structure of LacI using the Consurf web server (at http://consurf.tau.ac.il) [74,75]. Insert: a close-up view of the positions for cysteine introduction. (B) Affinities of the LacI variants LacIDimer-Y12C, -S28C, and -L62C for the O1 operator sequence were analyzed in an electrophoretic mobility shift assay performed as in Fig 2C. The apparent Kd values of the LacI-operator O1 complexes were estimated by a nonlinear regression of the LacI-DNA band intensity versus LacI concentration curves (S2 Fig) as described in the Methods section. Error bars represent SEM (N = 3 independent experiments).
Fig 4.
Analysis of the functionality of the LacI variants LacIDimer- Y12C, -S28C, and -L62C.
(A) A β-galactosidase assay of the repressor activities of the LacI variants in a derivative of E. coli strain BW25993 lacking endogenous LacI. The LacI variants were expressed from pBAD24 in the presence of 0.02% L-arabinose at 37° C. β-galactosidase expression was induced with 1 mM IPTG for 2.5 h at 37° C. Y-axis: log10 of Miller units. Miller units were calculated as: 1000*ΔA420/[ΔOD600*t*V] where ΔA420 is the sample optical density measured at 420 nm, ΔOD600 is the culture density at the beginning of the assay (ΔOD600 = 0.3), t is the time of sample incubation with 2-nitrophenyl-β-D-galactopyranoside (1.5 min) and V is the culture volume (0.4 mL). Each sample was measured in triplicate. (B) Schematic for the single-molecule detection of individual Cy3-labeled LacIDimer-S28C (LacI-Cy3) binding to surface-immobilized (biotin-streptavidin linkage) dsDNA harboring the O1 operator (O1 DNA) sequence. (C) Left: Distinct fluorescence peaks indicating binding could only be detected (areas of 150 x 150 pixels are shown) in the presence of both LacI-Cy3 as well as a specific operator sequence. Right: Quantification of the number of fluorescent peaks above background recorded in an area of 300 x 300 pixels, determined in triplicates for each condition.