Fluorescence Characterization of VU-9 Calmodulin, an Engineered Calmodulin with One Tryptophan in Calcium Binding Domain III

M. C. Kilhoffer, D. M. Roberts, A. Adibi, D. M. Watterson, J. Haiech

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Absorption and fluorescence properties of VU-9 calmodulin, an engineered calmodulin in which a tryptophan residue has been introduced in position 99, have been investigated. Tryptophan 99 fluoresces with a maximum around 348 nm and is easily quenched by fluorescence quenchers such as acrylamide, indicating that the chromophore is in a polar environment and well exposed to the solvent, a location which has been reported previously for tyrosine 99 in mammalian calmodulin [Kilhoffer, M. C., Demaille, J. G., & Gérard, D. (1981) Biochemistry 20, 4407-4414]. The quantum yields of tryptophan 99 were found to be 0.19 in the absence of calcium and 0.15 in its presence. These values indicate that the chromophore is in a particular microenvironment where it is protected from the quenching mechanisms normally occurring in proteins. Steady-state fluorescence polarization measurements indicate that the protein exhibits segmental mobility both in the absence and in the presence of calcium. Binding of calcium decreases the mobility of the chromophore, a good indication for a rigidification of the protein structure. A quite rigid structure of at least the carboxy-terminal part of VU-9 calmodulin in the presence of Ca2+ is also suggested by Förster energy-transfer measurements.

Original languageEnglish (US)
Pages (from-to)6086-6092
Number of pages7
JournalBiochemistry
Volume28
Issue number14
DOIs
StatePublished - Jul 1 1989

ASJC Scopus subject areas

  • Biochemistry

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