Comparison of the structure and dynamics of chicken gizzard and rabbit cardiac tropomyosins: 1H NMR spectroscopy and measurement of amide hydrogen exchange rates

The side chain and backbone mobilities of chicken gizzard tropomyosin (TM) and its nonpolymerizable derivative have been investigated by 1H NMR spectroscopy and amide hydrogen exchange kinetics and compared to those of rabbit cardiac TM and its nonpolymerizable derivative. Analysis of the 300-MHz 1H NMR spectra of native chicken gizzard and rabbit cardiac TMs and their nonpolymerizable derivative showed that the line widths of the aromatic and histidine residues were within in a factor of 2 for all four proteins, demonstrating that the side chain mobility of these residues is similar in the different TMs. Direct proton exchange-out kinetics were determined in D2O in the pD range 1.5-3.0 at 250C by 1H NMR spectroscopy. Multiple exponential fitting of the exchange data indicated the presence in gizzard TM of at least three kinetically distinct classes of amide hydrogens at pD 1.7 with average population sizes of 147, 74, and 61. Comparison of the exchange-out kinetics for the cardiac and gizzard proteins at pH 2.0 and 550C, where only the two slowly exchanging amide hydrogen sets are measured, again demonstrated the similarity of their kinetic parameters. Amide hydrogen exchange in the pH range 6-11 was studied by NMR measurement of the transfer of saturation from water to the amide hydrogens. Differential scanning calorimetry of gizzard TM and its nonpolymerizable derivative indicated the presence of domains of differing thermal stability in both proteins at acidic and alkaline pH. Circular dichroism measurement on the variation of the α-helical content with temperature of gizzard TM and its nonpolymerizable derivative at pH 2.0 and 7.0 also showed that the two proteins' structures were of comparable stability