Isothermal urea denaturation monitored by CD spectroscopy

Urea was prepared at ~ 6 M in 20 mM HEPES–NaOH, 200 mM NaCl, 1 mM 2-mercaptoethanol (β-ME), pH 7.4 and its actual concentration was determined by refractive index measurements. Urea stock solutions were mixed with hPGK1 solutions in 20 mM HEPES–NaOH, 200 mM NaCl, 1 mM β-ME, pH 7.4 to a final concentration of 5 μM (in protein) and 0–5 M (in urea). Samples were incubated for at least 2 h and denaturation experiments were monitored by Far-UV CD spectroscopy similarly to those described in Section "Circular dichroism (CD) spectroscopy". but restricting the acquisition range to 215–260 nm.

Data at 222 nm were used for fittings assuming a two-state model (apparently valid for WT hPGK1 using different conformational probes) ^3,28–30 to provide the values of experimental equilibrium m (m_urea) and C_m (concentration of urea at which half-denaturation occurs) using Eq. (⁶):

where S is the experimental CD signal as a function of the [urea], S_N and S_U are the fitted CD signals for the Native and Unfolded state baselines at 0 M urea, respectively, m_N and m_U are the slopes of the native and unfolded state baselines, m_urea describes the unfolding cooperativity, R is the ideal gas constant and T is the experimental temperature (298.15 K). Although this model is likely an oversimplification, it provides a proxy to compare the cooperativity of reversible chemical unfolding of hPGK1 variants.