Spectroscopic analysis of a methionine-48 to tyrosine mutant of chicken troponin C.
A mutant (M48Y) of chicken skeletal muscle troponin C was prepared in which Tyr replaced Met-48 of the recombinant protein (rTnC). Since Tyr and Trp are normally absent, spectral properties could be unambiguously assigned to the site of substitution. In the crystal structure, this residue lies at the COOH-terminal end of the B-helix of the N domain in a region postulated to undergo a significant conformational change to a more polar environment upon Ca2+ binding [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644]. Comparison of the far-UV CD spectra of M48Y and rTnC in the absence and presence of Ca2+ indicated no overall structural alteration due to the mutation. However, Ca2+ titration of the ellipticity change showed a reduction in Ca2+ affinity and cooperativity of sites I and II. A Ca(2+)-induced increase in the near-UV ellipticity of M48Y at pH 7.12 and a red shift in its UV absorbance spectrum occurred over a range of free [Ca2+] attributable to the N-domain transition only. This was largely abolished at pH 5.3 where Ca2+ no longer binds to sites I and II. That region of the 1H NMR spectrum attributable to Tyr was broadened upon Ca2+ binding. These Ca(2+)-induced changes are consistent with the environment of the Tyr side chain becoming chiral, less polar, and more immobile, all in a direction opposite to that predicted. These observations indicate that while the general features of the postulated model are valid, it is unlikely to be correct in detail.
Pearlstone
J R
JR
Department of Biochemistry, University of Alberta, Edmonton, Canada.
McCubbin
W D
WD
Kay
C M
CM
Sykes
B D
BD
Smillie
L B
LB
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002645:Chickens; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D013056:Spectrophotometry, Ultraviolet; D014336:Troponin / Q000737:chemistry / Q000235:genetics*; D019209:Troponin C; D014443:Tyrosine / Q000235:genetics*
D002413:Cations, Divalent; D009841:Oligonucleotides; D014336:Troponin; D019209:Troponin C; D014443:Tyrosine; D008715:Methionine; D002118:Calcium