Low-temperature-induced structural changes in the Apo regulatory domain ofskeletal muscle troponin C

Contractile activity of skeletal muscle is triggered by a Ca2+-induced "ope ning" of the regulatory N-domain of troponin C (apo-NTnC residues 1-90). Th is structural transition has become a paradigm for large-scale conformation al changes that affect the interaction between proteins. The regulatory dom ain is comprised of two basic structural elements: one contributed by the N -, A-, and D-helices (NAD unit) and the other by the B- and C-helices (BC u nit). The Ca2+-induced opening is characterized by a movement of the BC uni t away from the NAD unit with a concomitant change in conformation at two h inges (Glu(41) and Val(65)) of the BC unit. To examine the effect of low te mperatures on this Ca2+-induced structural change and the implications for contractile regulation, we have examined nuclear magnetic resonance (NMR) s pectral changes of apo-NTnC upon decreasing the temperature from 30 to 4 de grees C. In addition, we have determined the solution structure of apo-NTnC at 4 degrees C using multinuclear multidimensional NMR spectroscopy. Decre asing temperatures induce a decrease in the rates and amplitudes of pico to nanosecond time scale backbone dynamics and an increase in alpha-helical c ontent for the terminal helices of apo-NTnC. In addition, chemical shift ch anges for the H-alpha resonances of Val(65) and Asp(66), the hinge residues of the BC, unit were observed. Compared to the solution structure of apo-N TnC determined at 30 OC, the BC unit packs more tightly against the NAD uni t in the solution structure determined at 4 degrees C. Concomitant with the tighter packing of the BC and NAD structural units, a decrease in the tota l exposed hydrophobic surface area is observed, The results have broad impl ications relative to structure determination of proteins in the presence of large domain movements, and help to elucidate the relevance of structures determined under different conditions of physical state and temperature, re flecting forces ranging from crystal packing to solution dynamics.