Intrinsic Tryptophan Fluorescence Identifies Specific Conformational Changes at the Actomyosin Interface upon Actin Binding and ADP Release†

Biochemistry ◽  
1999 ◽  
Vol 38 (44) ◽  
pp. 14515-14523 ◽  
Author(s):  
C. M. Yengo ◽  
L. Chrin ◽  
A. S. Rovner ◽  
C. L. Berger
Keyword(s):  
Conformational Changes ◽  
Tryptophan Fluorescence ◽  
Actin Binding ◽  
Intrinsic Tryptophan Fluorescence ◽  
Adp Release

scholarly journals Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin

1988 ◽  
Vol 251 (2) ◽  
pp. 515-526 ◽  
Author(s):  
A P Jackson ◽  
C R Bagshaw
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
Adductor Muscle ◽  
Reverse Direction ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.

Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes

Biochemistry ◽  
1993 ◽  
Vol 32 (8) ◽  
pp. 1976-1980 ◽  
Author(s):  
Bruno Sontag ◽  
Anne Marie Reboud ◽  
Gilles Divita ◽  
Attilio Di Pietro ◽  
Dominique Guillot ◽  
...  
Keyword(s):  
Rat Liver ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Tryptophan Fluorescence ◽  
Intrinsic Tryptophan Fluorescence

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Joseph Atherton ◽  
Irene Farabella ◽  
I-Mei Yu ◽  
Steven S Rosenfeld ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Catalytic Site ◽  
Fundamental Question ◽  
Force Generation ◽  
Atp Binding ◽  
Cellular Functions ◽  
Microtubule Binding ◽  
Cryo Electron Microscopy ◽  
Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

scholarly journals Phosphorylation-dependent Conformational Changes Induce a Switch in the Actin-binding Function of MARCKS

1999 ◽  
Vol 274 (51) ◽  
pp. 36472-36478 ◽  
Author(s):  
Michael R. Bubb ◽  
Robert H. Lenox ◽  
Arthur S. Edison
Keyword(s):  
Conformational Changes ◽  
Actin Binding ◽  
Binding Function

scholarly journals Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ksenia Terekhova ◽  
Sabine Pokutta ◽  
Yee S. Kee ◽  
Jing Li ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Protein ◽  
Binding Domain ◽  
Actin Binding ◽  
Titration Calorimetry ◽  
Allosteric Coupling ◽  
Actin Binding Protein ◽  
Binding Partners ◽  
Actin Binding Domain ◽  
Cysteine Labeling

Abstract Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.

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