We report here on the stability and folding of the 91 residue alpha-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC(1-91)F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC(1-91)F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed. (C) 2004 Elsevier Inc. All rights reserved.4272135142