Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: Implications for the open-closed transition identified by crystallography

A. Málnási-Csizmadia, R. J. Woolley, C. R. Bagshaw

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99 Citations (Scopus)

Abstract

When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the Converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20° C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.

Original languageEnglish
Pages (from-to)16135-16146
Number of pages12
JournalBiochemistry
Volume39
Issue number51
DOIs
Publication statusPublished - Dec 26 2000

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Myosin Type II
Crystallography
Dictyostelium
Tryptophan
Adenosine Triphosphate
Fluorescence
Hydrolysis
Myosins
Nucleotides
Acrylamide
Flow measurement
Isomerization
Quenching
Assays
Crystal structure
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: Implications for the open-closed transition identified by crystallography",
abstract = "When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the Converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20° C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80{\%} enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.",
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T1 - Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants

T2 - Implications for the open-closed transition identified by crystallography

AU - Málnási-Csizmadia, A.

AU - Woolley, R. J.

AU - Bagshaw, C. R.

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Y1 - 2000/12/26

N2 - When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the Converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20° C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.

AB - When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the Converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20° C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.

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