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|Title:||Fluorescence and transient kinetic analysis of the dictyostelium myosin-II motor domain using green fluorescent protein (GFP) and its variants as probes|
|Presented at:||University of Leicester|
|Abstract:||The determination of the structure of the myosin motor domain has made it possible to introduce fluorescent probes at defined sites, thereby allowing the resolution of the mechanochemical steps in greater detail. Here, modern genetic cloning techniques were utilised to create and express within Dictyostelium discoideum novel myosin-II motor fusion proteins containing various fluorescent probes, in an attempt to investigate conformational changes within the motor domain during the actin-bound stages of the crossbridge cycle. Stopped-flow analysis showed that the myosin ATPase of the single tryptophan myosin-II motor W501 was unaffected by N- and C-terminal YFP and CFP probes, whereas ATP-induced actomyosin dissociation was disrupted (potentially by the probes' propensity to dimerise in close proximity), thus rendering the system unsuitable for investigation of the actin-bound stages of the actomyosin ATPase. By combining total internal reflection microscopy with flash photolysis of an inert caged-ATP precursor, it was shown that kinetic information for the ATP-induced dissociation of fluorescently-labelled myosin motor domains may be achieved using only nanogram quantities, while simultaneously avoiding bundling artefacts common to solution kinetics. A slight adaptation of this process could yield a highly sensitive assay for the processivity of non-classical myosin types, yielding information on their rotational and lateral movement concurrently. Lever arm movement was assessed via the analysis of fluorescence resonance energy transfer (FRET) changes between the YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) probes. A FRET efficiency increase was observed upon nucleotide occupancy of the active site, in direct contrast to previous FRET studies. Anisotropy studies showed no change upon nucleotide binding, suggesting the FRET increase was due to a distance change, rather than a variation of relative dipole orientations. However, due to the high anisotropy (i.e. slow rotation in solution) of the protein, it was also shown that results from this type of FRET system are qualitative rather than quantitative.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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