Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.
The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.
Moncrieffe
M C
MC
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Venyaminov
S Y
SY
Miller
T E
TE
Guzman
G
G
Potter
J D
JD
Prendergast
F G
FG
D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002645:Chickens; D002942:Circular Dichroism; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011193:Potassium Iodide / Q000737:chemistry; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D017186:Titrimetry; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics*
D010446:Peptide Fragments; D019209:Troponin C; D011193:Potassium Iodide; D020106:Acrylamide; D014364:Tryptophan; D002118:Calcium