Domain motions in phosphoglycerate kinase: Determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer

G. Haran, E. Haas, B. K. Szpikowska, M. T. Mas

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    86 Scopus citations

    Abstract

    3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 Å or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 ± 1 Å, was 8 Å smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 ± 1 Å and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.

    Original languageEnglish
    Pages (from-to)11764-11768
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume89
    Issue number24
    DOIs
    StatePublished - 15 Dec 1992

    Keywords

    • Domain closure
    • Emission anisotropy
    • Global analysis
    • Hinge bending
    • Site-directed mutagenesis

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