Stability and folding studies of the N-domain of troponin C. Evidence for the formation of an intermediate Carlos H.I. Ramos, a,b, * Milton V. Lima Jr., a Silvia L.F. Silva, a Paula F.L. Borin, a Wiliam C.B. R egis, a,b and Marcelo M. Santoro c a Centro de Biologia Molecular Estrutural, Laborat orio Nacional de Luz S  ıncrotron, CP 6192, Campinas SP, 13084-971, Brazil b Departamento de Bioqu  ımica, Instituto de Biologia, Universidade Estadual de Campinas, Brazil c Departamento de Bioqu  ımica e Imunologia, Instituto de Ci^ encias Biol ogicas, Universidade Federal de Minas Gerais, Belo Horizonte MG, Brazil Received 11 February 2004, and in revised form 15 April 2004 Available online 18 May 2004 Abstract We report here on the stability and folding of the 91 residue a-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 cal- orimetry, 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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Protein folding; Folding intermediate; Troponin C; Spectroscopy; Thermodynamic; Muscle contraction; Calcium binding The identification of intermediates is important for our understanding of the process of protein folding, a basic problem in biochemistry. Molten globule-like in- termediates are characterized by a native-like secondary structure and a disordered tertiary structure, with the native-like intermediates showing characteristics of the fully folded protein and evidence of two-state unfolding [1–5]. Numerous proteins have one or more intermedi- ates in their folding pathways (for reviews, see [6–12]). On the other hand, the two-state approximation, which applies to very cooperative transitions, is frequently valid for small proteins and isolated domains [13], and most small proteins are considered to fold via an ap- parent two-state process in which only the native and unfolded states are detected [14–17]. However, all pro- teins may fold via intermediates. The lack of evidence for the presence of intermediates in the folding pathway of some proteins may reflect insufficient sensitivity of the detection methods available, particularly since interme- diates are unstable and poorly populated under equi- librium conditions [7,18]. Thus, it is important to study the folding of small proteins and isolated domains to test whether or not they obey the two-state model of folding and to search for the presence of intermediates. Troponin C (TnC) 1 is a dumbbell-shaped protein with 163 residues formed primarily by a-helices, the N-terminus of which binds calcium and initiates muscle contraction [19–22]. The N-terminus of TnC is an a-helical domain with two EF-hands (Fig. 1). Func- tional studies of the isolated N-domain of TnC (N-TnC) have shown that this region has a regulatory role [21,23]. * Corresponding author. Fax: +55-19-3287-7110. E-mail address: cramos@lnls.br (C.H.I. Ramos). 1 Abbreviations used: N-TnC, N-domain (residues 1–91) Troponin C; C m , urea concentration at the midpoint of the unfolding transition; T m , temperature of the midpoint of the thermal unfolding transition; CD, circular dichroism; [H], mean residue molar ellipticity; DSC, differential scanning calorimetry; DH cal , enthalpy of unfolding, DH vH , vanÕt Hoff enthalpy; Gdm-Cl, guanidinium chloride. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.05.002 Archives of Biochemistry and Biophysics 427 (2004) 135–142 ABB www.elsevier.com/locate/yabbi