Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Abstract

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires approximately 8 h.

Figure 1. Circular dichroism (CD) spectra of polypeptides and proteins with some representative secondary structures

a, CD spectra of poly-L-lysine in the 1, α-helical (black) and 2 antiparallel β-sheet (red) conformations at pH 11.1, and 3 extended conformation at pH 5.7, 3 (green) and placental collagen in its 4, native triple-helical (blue) and 5, denatured (cyan) forms. Note that the extended conformation of poly-L-lysine was original described as a “random coil” but its spectrum is similar to the conformation of poly-L-proline II,, which forms an extended left-handed helix.

Figure 2. Spectra of unbound tropomyosin and troponin model proteins and protein-protein complexes

a, raw data. b, mean residue ellipticity. (green circles) CTD; red squares (NTD); (blue triangles up); TNT_70-170I79N; pink triangles down (1:1 mixture of CTD and NTD); (cyan diamonds) 1:1:1 mixtures of the NTD, CTD and TnT_70-170I79N model peptides. (open triangles up) add the curves for the unbound CTD and NTD peptides; (open triangles down) add the curves for the CTD, NTD and TnT_70-170I79N model peptides. The peptide concentrations were all 17.5 μM in 10 mM potassium phosphate, pH 6.5. Data were collected in cells with 1 mm path lengths. Abbreviations: NTD, model protein of the N-terminal domain of tropomyosin containing residues 1-14 of rat striated alpha tropomyosin and the last 18 residues of the yeast transcription factor, GCN4. The peptide is N-acetylated,. CTD, peptide containing residues 251-284 of rat striated tropomyosin with GCG at N-terminus. The peptides are cross-linked by a disulfide bond. TNT_70-170I79N, residues 70-170 of human cardiac troponin with the mutation I79N and glycine at the N-terminus.

Figure 3. Typical curves of ellipticity as a function of temperature used to determine the thermodynamics of folding

In all the panels the proteins, symbols and conditions are the same as Figure 2. a, Changes in ellipticity of tropomyosin and troponin model proteins and protein complexes as a function of temperature. The raw data are fit with equations for the unfolding of monomers (CTD and TnT_70-170I79N), a dimer (NTD); a heterotrimer (NTD/CTD complex) and a generic hyperbolic curve (CTD/NTD/ TnT_70-170I79N complex), respectively. b, Van't Hoff plots of the data in panel a. c, the first derivatives of the curves in panel a. The maxima correspond to the midpoints (T_Ms) of the unfolding/folding curves.

Figure 4. Example of the analysis of set of spectra using the convex constraint algorithm.

a and d, spectra of a model protein containing the C-terminal domain of rat striated muscle tropomyosin (CTD) and a fragment containing the mutation of a glutamine to leucine (CTDQ263L) collected as a function of temperature, respectively. b, and e, the data in panels a and d deconvoluted into 3 basis curves using the convex constraint algorithm. c and f, the fraction of each basis curve contributing to each spectrum at each temperature. The fractional weights of the two curves corresponding to the unfolded protein are summed in panel c because they were almost identical. The protein samples were 1.67 μM in 100 mM NaCl, 10 mM sodium phosphate buffer pH 6.5. Data were collected at 5 deg increments with two minutes equilibration at each point in cells with path lengths of 1 cm. Abbreviations: CTD, model protein containing residues 251-284 of rat striated tropomyosin with GCG at N-terminus. CTDQ263L, the same peptide with the mutation Q263L. The peptides are cross-linked by disulfide bonds.