Anisotropy, Fluorescence

Fluorescence polarization data were measured on a Spectramax M5 microplate reader (Molecular Devices) at 24°C. For K_d measurements with a given fluorescently labeled peptide, a stock solution of protein was incubated for 10–30 minutes at room temperature in FP buffer (storage buffer, supplemented to a final concentration of 0.1 mg/mL bovine IgG (Sigma) and 0.5 mM Thesit (Fluka)) containing 30 nM fluorescent peptide. The pre-equilibrated protein:reporter peptide mixture was then serially diluted into FP buffer containing 30 nM fluorescent peptide and allowed to incubate for 10 minutes. Following centrifugation for 4 minutes at 1200 × g to remove air bubbles, 40 μL aliquots were transferred to HE low-volume, black 96-well plates (Molecular Devices). The plates were again centrifuged for 4 minutes at 1200 × g, placed in the microplate reader, and allowed to equilibrate at 24 °C for an additional 15 minutes prior to measurement.
Competition binding experiments were performed similarly. A single stock solution was prepared in FP buffer containing fixed concentrations of both fluorescently labeled reporter peptide and protein. This mixture was allowed to equilibrate for 20–60 minutes at RT. Unlabeled competitor peptide was dissolved and serially diluted in DMSO (Fluka). Each serial dilution was aliquoted at 1/20^th final volume, to which was added 19/20^th volume of the protein:reporter mixture. The final reporter peptide concentration was 30 nM, and the final protein concentration was 0.25 – 3.0 × K_d, depending on the measurement. Plates were mixed by vibration, centrifuged, and allowed to incubate for an additional 15 minutes at 24 °C in the microplate reader before measurement.
Fluorescence polarization was determined at an excitation wavelength of 485 nm and an emission wavelength of 525 nm as
$$F{P}_{exp}=\frac{I_{vv}-g{I}_{vh}}{I_{vv}+g{I}_{vh}},$$ where I_vv and I_vh represent the vertically and horizontally polarized emission signals obtained with vertically polarized excitation, and g represents the assay-specific polarization bias, which was separately determined in each buffer. The time of equilibration and salt concentration, and the requirement for detergent or carrier protein, were determined to minimize nonspecific peptide binding. Fluorescence intensities were monitored to ensure no change in reporter fluorescence quantum yield upon binding, and also to exclude any light-scattering contribution to the measured polarization. For analysis, data were converted to anisotropy values.
Direct binding data were fit to a model for a single-site
$$P+L\stackrel{k_d}{\leftrightarrow }PL$$ binding equilibrium. A non-linear least-squares algorithm (Kaleidagraph) was used to fit the experimental anisotropy (r_exp) to the anisotropy calculated by the following equation:
$$r_{\mathit{calc}}=r_L+(r_{PL}-r_L)[PL]/{[L]}_{\mathit{tot}},$$ where [L]_tot = total reporter peptide concentration and r_L and r_PL = the fluorescence anisotropies of the free and bound ligands, respectively, for the case in which fluorescence lifetime and quantum yield are unaffected by protein binding. The concentration of protein:ligand complex [PL] was determined as:
$$[PL]=\frac{{[L]}_{\mathit{tot}}+{[P]}_{\mathit{tot}}+K_d-\sqrt{{({[L]}_{\mathit{tot}}+{[P]}_{\mathit{tot}}+K_d)}^2-4{[L]}_{\mathit{tot}}{[P]}_{\mathit{tot}}}}{2}$$ where [P]_tot = total protein concentration and K_d = equilibrium dissociation constant of complex formation.
Competition isotherms were fit using a non-linear least-squares fitting algorithm implemented in Excel using the SOLVER function. r_exp was fit to
$$r_{\mathit{calc}}=\frac{r_L+r_{PL}[P]/K_d}{1+[P]/K_d},$$ where the free protein concentration [P] in the presence of both reporter and competitive inhibitor was calculated as a function of the total protein and ligand concentrations and the equilibrium dissociation constants K_d (known) and K_i (fit), respectively, by exact analytical solution of the resulting cubic equation (see eqn. 13, ref. 23 (link)). For illustrative purposes, four-parameter logistic curve fits are shown in figures to illustrate relative IC₅₀ values.

Recombinant full-length RIG-I (1–925), helicase-RD (232–925), helicase (232–794), RD (795–925), and selenomethione derivatized helicase-RD were expressed in Escherichia coli and purified to homogeneity using immobilized metal ion affinity, hydroxyapatite and heparin affinity chromatography. Fluorescence anisotropy titrations were performed at 25°C^{30 (link)} (λ_excitation 494 nm and λ_emission 516 nm) using a fluorescein-labeled 14 base pair dsRNA prepared by annealing 5′GGAGAGAACCGCCU and 3′CCUCUCUUGGCGGA-F RNA, where F is fluorescein. Crystals of the native and selenomethione helicase-RD with pal-dsRNA (5′CGACGCUAGCGUCG) and ADP•BeF₃•Mg²⁺ were obtained in 25% (w/v) PEG 3350, 0.25 M NaSCN, 100 mM MOPS (pH 7.8), 3% (v/v) 2,2,2-Trifluoroethanol at 20°C by hanging drop. The crystals belong to space group P6₅22 with cell parameters a=b=174.9 Å and c=110.9 Å. The structure was determined by SAD to 3.2 Å resolution and refined against a 2.9 Å resolution native data set. The final model has an R_work and R_free of 0.199 and 0.287, respectively. SAXS measurements were preformed on full-length RIG-I and helicase-RD in the absence and presence of dsRNA (5′GCGCGCGCGC). Buffer subtraction and R_g were calculated from Guinier plots. D_max was determined by scanning a range of values and comparing experimental I(s) values to P(r) transforms. Ten ab initio models were averaged and normalized spatial discrepancy (NSD) values were calculated. The helicase-RD•dsRNA and a homologous CARD structure were positioned into the ab initio model and χ² were determined.