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 through conformational changes induced by Ca2+ binding. A mutant form of the isolated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure 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 simulations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region near the mutation site. Also, the simulations proposed that an additional cavity 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 high pressures and temperatures, we performed 1H-NMR experiments at 300, 400, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-domain 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 loops, 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 compressibility of the mutant. Under the same pressure conditions, the wild-type domain exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type was resistant to denaturation up to 56 degrees C. This suggests that the partially exposed surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.
Yu
A
A
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Ballard
L
L
Smillie
L
L
Pearlstone
J
J
Foguel
D
D
Silva
J
J
Jonas
A
A
Jonas
J
J
D000818:Animals; D002645:Chickens; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017354:Point Mutation; D011312:Pressure; D013696:Temperature; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D019209:Troponin C