Frequency-domain fluorescence of mutant cytochrome b5

Alexey S. Ladokhin; Henryk M. Malak; Michael L. Johnson; Joseph R. Lakowicz; L. Wang; A. W. Steggles; Peter W. Holloway

doi:10.1117/12.58250

1 April 1992 Frequency-domain fluorescence of mutant cytochrome b₅

Alexey S. Ladokhin, Henryk M. Malak, Michael L. Johnson, Joseph R. Lakowicz, L. Wang, A. W. Steggles, Peter W. Holloway

Proceedings Volume 1640, Time-Resolved Laser Spectroscopy in Biochemistry III; (1992) https://doi.org/10.1117/12.58250
Event: OE/LASE '92, 1992, Los Angeles, CA, United States

Abstract

Cytochrome b₅, isolated by detergent extraction from rabbit liver, has been extensively studied by fluorescence techniques, however, its fluorescence properties are complicated by the presence of three tryptophans in the membrane-binding domain. This protein has now been expressed in E. coli and a mutant form has been isolated which contains only one tryptophan (Trp-109) in the membrane-binding domain. The mutant protein does differ from the native protein in several of its spectroscopic properties, but it interacts with lipids in a similar way and shows the same functional activity. The availability of a mutant cytochrome b₅ with a single Trp in the nonpolar domain enables several frequency-domain experiments: to estimate the distribution of distances between Trp-109 and the heme; to study the incorporation of the protein into the membrane; to study the quenching of the tryptophan fluorescence by lipids with a brominated acyl chain; and to analyze the shape of the lifetime distribution.

Citation Download Citation

Alexey S. Ladokhin, Henryk M. Malak, Michael L. Johnson, Joseph R. Lakowicz, L. Wang, A. W. Steggles, and Peter W. Holloway "Frequency-domain fluorescence of mutant cytochrome b₅", Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); https://doi.org/10.1117/12.58250

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