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Article

Unfolding Thermodynamics of Trp-Cage, a 20 Residue Miniprotein, Studied by Differential Scanning Calorimetry and Circular Dichroism Spectroscopy^†

Werner W. Streicher^‡ and George I. Makhatadze*^‡^§

Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, and Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

Biochemistry, 2007, 46 (10), pp 2876–2880

DOI: 10.1021/bi602424x

Publication Date (Web): February 13, 2007

†

This work was supported by a grant (GM54537) from the National Institutes of Health.

‡

The Pennsylvania State University College of Medicine.

Corresponding author. Tel: (717) 531-0712. Fax: (717) 531-7072. E-mail: makhatadze@psu.edu.

The Pennsylvania State University.

Abstract

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only 90−95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, ΔC_p. This can be circumvented by obtaining ΔC_p directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: T_m = 43.9 ± 0.8 °C, ΔH(T_m) = 56 ± 2 kJ/mol, ΔC_p = 0.3 ± 0.1 kJ/(mol·K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 °C, ΔG(25 °C) = 3.2 ± 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.