Low-Temperature-Induced Structural Changes in the Apo Regulatory Domain of Skeletal Muscle Troponin C†,‡
journal contributionposted on 16.04.1999 by Sakae Tsuda, Ai Miura, Stéphane M. Gagné, Leo Spyracopoulos, Brian D. Sykes
Any type of content formally published in an academic journal, usually following a peer-review process.
Contractile activity of skeletal muscle is triggered by a Ca2+-induced “opening” of the regulatory N-domain of troponin C (apo-NTnC residues 1−90). This structural transition has become a paradigm for large-scale conformational changes that affect the interaction between proteins. The regulatory domain 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 unit). The Ca2+-induced opening is characterized by a movement of the BC unit away from the NAD unit with a concomitant change in conformation at two hinges (Glu41 and Val65) of the BC unit. To examine the effect of low temperatures on this Ca2+-induced structural change and the implications for contractile regulation, we have examined nuclear magnetic resonance (NMR) spectral changes of apo-NTnC upon decreasing the temperature from 30 to 4 °C. In addition, we have determined the solution structure of apo-NTnC at 4 °C using multinuclear multidimensional NMR spectroscopy. Decreasing temperatures induce a decrease in the rates and amplitudes of pico to nanosecond time scale backbone dynamics and an increase in α-helical content for the terminal helices of apo-NTnC. In addition, chemical shift changes for the Hα resonances of Val65 and Asp66, the hinge residues of the BC, unit were observed. Compared to the solution structure of apo-NTnC determined at 30 °C, the BC unit packs more tightly against the NAD unit in the solution structure determined at 4 °C. Concomitant with the tighter packing of the BC and NAD structural units, a decrease in the total exposed hydrophobic surface area is observed. The results have broad implications 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, reflecting forces ranging from crystal packing to solution dynamics.