Figure 2.

Urea-induced equilibrium denaturation of MSG. A, Trp fluorescence emission scans (excitation at 295 nm) recorded for 0–9 m denaturant are represented as color ramped from blue to red. B and C, solid line represents the global two-state fit of relative fluorescence intensity at 340 nm and relative ellipticity at 222 nm against urea concentration. The transition regions in 1.5–2.5 m urea and 3.25–6.5 m urea in relative fluorescence plots have been excluded from fitting due to the absence of corresponding baselines for the intermediate states. Insets display reversibility of MSG unfolding to various urea concentrations on the denaturation curve. Error bars represent standard deviation from three individual samples. D, significant basis spectra obtained by SVD analysis of the fluorescence scans in A. Only two distinguishable species corresponding to buried and exposed Trp residues (peak maximums near 345 and 377 nm, respectively) can be identified. E and F, amplitudes corresponding to major (weight = 84.6%) and minor (weight = 13.6%) basis spectra representing their population variation with urea concentration. G, molar residue ellipticity (MRE) scans of the protein equilibrated from 0 to 8 m of urea are represented as color ramped from blue to red. H, only significant SVD basis spectrum (weight = 77.9%) obtained from CD spectra. I, amplitudes corresponding to the basis spectrum from CD, showing respective population variation with denaturant concentration.