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 H NMR spectroscopy and amide hydrogen exchange kinetics and compared to those of rabbit cardiac TM and its nonpolymerizable derivative. Analysis of the 300-MHz H NMR spectra of native chicken gizzard and rabbit cardiac TMs and their nonpolymerizable derivatives showed that the line widths of the aromatic and histidine residues were within 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 25 degrees C by H 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, whose rates were retarded by a factor of 10, 10(3), and 10(5), respectively, relative to the random-coil peptide poly(DL-alanine). Measurement of the direct exchange kinetics of both rabbit cardiac and nonpolymerizable gizzard TMs showed that their rate constants and population sizes were within experimental error of those for the gizzard protein, except that the fast exchanging class for cardiac TM was increased in size while that of the nonpolymerizable gizzard TM was reduced, relative to that for gizzard TM. Comparison of the exchange-out kinetics for the cardiac and gizzard proteins at pH 2.0 and 55 degrees C, where only the two slowly exchanging amide hydrogen sets are measured, again demonstrated the similarity of their kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)
Sanders
C
C
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Sykes
B D
BD
Smillie
L B
LB
D000577:Amides; D000818:Animals; D002645:Chickens; D005895:Gizzard, Avian / Q000032:analysis; D006859:Hydrogen; D007474:Ion Exchange; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009206:Myocardium / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D013816:Thermodynamics; D014335:Tropomyosin
D000577:Amides; D014335:Tropomyosin; D006859:Hydrogen