Effects of high pressure and temperature on the wild-type and F29W mutant forms of the N-domain of avian troponin C

The N-domain of troponin C (residues 1-90) regulates muscle contraction thr ough conformational changes induced by Ca2+ binding. A mutant form of the i solated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressu re and temperature changes. Here we set out to determine whether the point mutation itself has any effects on the protein structure and its stability to pressure and temperature in the absence of Ca2+. Molecular dynamics simu lations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region nea r the mutation site. Also, the simulations proposed that an additional cavi ty had been created in the core of the mutant protein. To determine whether such a cavity would affect the behavior of the protein when subjected to h igh pressures and temperatures, we performed H-1-NMR experiments at 300, 40 0, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-doma in troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized beta-sheet between the Ca2+-binding loop s, an altered environment near Phe-26, and reduced local motions of Phe-26 and Phe-75 in the core of the protein, probably due to a higher compressibi lity of the mutant, Under the same pressure conditions, the wild-type domai n exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type w as resistant to denaturation up to 56 degrees C, This suggests that the par tially exposed surface mutation (F29W) significantly destabilizes the N-dom ain of troponin C by altering the packing and dynamics of the hydrophobic c ore. (C) 1999 Elsevier Science B.V, All rights reserved.