Optical Spectroscopic Characterization of Single Tryptophan Mutants of Chicken Skeletal Troponin C: Evidence for Interdomain Interaction

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• 作者：Martin C. Moncrieffe ; Sergei Yu. Venyaminov ; Todd E. Miller ; Georgiana Guzman ; James D. Potter ; and Franklyn G. Prendergast
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 14, 1999
• 年：1999
• 卷：38
• 期：37
• 页码：11973 - 11983
• 全文大小：114K
• 年卷期：v.38,no.37(September 14, 1999)
• ISSN：1520-4995

文摘

The effects of metal ion binding on the optical spectroscopic properties and temperature stabilityof two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. Theabsence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectralsignal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectraof the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that theintroduction of the Trp residue had no effect on the total secondary structure content. The fluorescenceand near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed tosolvent. Additionally, the highly resolved ¹L_b band of Trp-78 seen in the near-UV absorbance and CDspectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. Inthe 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 calciumbinding at the C-terminal domain sites. Measurements of the temperature stability reveal that eventsoccurring 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-terminaldomains of TnC.