Thermodynamic analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol

• 2,2,2-Trifluoroethanol induced molten globule state of myoglobin characterised.
• Thermodynamics of unfolding addressed via formation of molten globule state.
• ANS binds to molten globule state of myoglobin at two types of binding sites.
• Common structural properties of molten globule addressed by energetics of ANS binding.

The thermal denaturation of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using differential scanning calorimetry and UV–visible spectroscopy. The most obvious effect of TFE was lowering the transition temperature up to 1.5 mol · kg−1, beyond which no thermal transitions were observed. The protein conformation was analysed by fluorescence and circular dichroism measurements. Quantitative binding of ANS to the TFE induced molten globule state of myoglobin was studied by using isothermal titration calorimetry. The results enable quantitative estimation of the binding strength of ANS with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. The results suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ANS molecules which are a widely used fluorescence probe to characterise partially folded states of proteins.

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