Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

Abstract

Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments require 2-5 h.

Figure 1. Changes in intrinsic and extrinsic circular dichroism bands upon the interaction of proteins with denaturants and ligands

a, The change in mean residue ellipticity when Gn-HCl is added to a fragment of tropomodulin 1, Tmod1_160-359, 0.24 mg/ml (10 μM) in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5 at 10 °C. (o) native protein, (Δ) protein + 3.6 M Gn-HCl. b. The change in mean residue ellipticity when calcium binds to the calcium binding subunit of cardiac troponin, TnC, 0.2 mg/ml, at 2 °C in 50 mM NaC1, 2 mM HEPES, pH 7.0, 0.5 mM DTT, with (Δ) 2mM EDTA, or (•) 3 mM CaCl₂. c, Induced molar ellipticity when saturating sodium folate binds to dihydrofolate reductase from e coli.. The enzyme is 18 μM in 100 mM NaCl, pH 7.2 at 27 °C.

Figure 2. Determining the free energy of folding from changes in CD as a function of denaturant

a, The mean residue ellipticity of three fragments of tropomodulin 1 as a function of Gn-HCl in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5 at 10 °C. a, (black circles), Tmod1_160-359, 0.24 mg/ml; (magenta triangles), residues Tmod1_160-344, 0.17 mg/ml; (cyan squares), Tmod1_1-322, 0.22 mg/ml. The data were fit to equations 9-12 by non-linear least squares analysis. b, The data in panel a were transformed using equations 2, 3 and 5 to give a linear plot of ΔG as a function of denaturant. The free energy of folding equals the value of the y intercept.

Figure 3. Determination of affinity constants from changes in the intrinsic CD of the polypeptide backbone as a function of increasing ligand concentration

0.4 ml of chicken muscle Troponin C (TnC), 0.1 mg/ml, in 100 mM KCl, 50 mM Tris-HCl, pH 7.5, 0.9 mM EGTA, 0.9 mM nitriloacetic acid, pH 7.5 at 25 °C, in a 0.2 ml cuvette was titrated with Ca²⁺.

Figure 4. Determination of affinity constants and number of binding sites from the induction of extrinsic CD bands upon enzyme-ligand interactions

When folate binds to Dihydrofolate reductase from E. coli its absorption bands become optically active. (•) The change of the difference in ellipticity between 292.5and 270.0 nm of dihydrofolate reductase from E. coli as a function of increasing folate concentration. (line) The data fit by equation (23) to determine the number of binding sites/mole (0.95), the maximal ellipticity change, 28 millidegrees, and the dissociation constant, 3.1 μM. The enzyme was 9.2 μM in 0.1 N NaCl, pH 7.2, at 27 °C.