Analysis of Conformational Changes at the Unique Loop Adjacent U the ATP Binding Site of Smooth Muscle Myosin Using a Fluorescent Probe

2000 ◽  
Vol 127 (2) ◽  
pp. 199-204 ◽  
Author(s):  
S. Maruta ◽  
J. Saitoh ◽  
T. Asakura
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Fluorescent Probe ◽  
Conformational Changes ◽  
Smooth Muscle Myosin ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Muscle Myosin

scholarly journals Affinity labelling of smooth-muscle myosin light-chain kinase with 5′-[p-(fluorosulphonyl)benzoyl]adenosine

1993 ◽  
Vol 296 (1) ◽  
pp. 53-58 ◽  
Author(s):  
H Komatsu ◽  
M Ikebe
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Calmodulin Binding ◽  
Muscle Myosin

5′-(p-(Fluorosulphonyl)[14C]benzoyl)adenosine (FSBA) was synthesized and used as a probe to study the ATP-binding site of smooth-muscle myosin light-chain kinase (MLCK). FSBA modified both free MLCK and calmodulin/MLCK complex, resulting in inactivation of the kinase activity. Nearly complete protection of the calmodulin/MLCK complex against FSBA modification was obtained by addition of excess ATP whereas MLCK activity alone was lost in a dose-dependent manner even in the presence of excess ATP. These results suggest that FSBA modified ATP-binding sites and ATP-independent sites, and the latter sites are protected by calmodulin binding. The results also suggest that the ATP-binding site is accessible to the nucleotide substrate regardless of calmodulin binding. The FSBA-labelled MLCK was completely proteolysed by alpha-chymotrypsin, and the 14C-labelled peptides were isolated and sequenced. The sequence of the labelled peptide was Ala-Gly-X-Phe, where X is the labelled residue. The sequence was compared with the known MLCK sequence, and the labelled residue was identified as lysine-548, which is located downstream of the GXGXXG motif conserved among ATP-utilizing enzymes.

scholarly journals Photoaffinity labelling of smooth-muscle myosin by methylanthraniloyl-8-azido-ATP

1993 ◽  
Vol 292 (2) ◽  
pp. 439-444 ◽  
Author(s):  
S Maruta ◽  
M Ikebe
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Smooth Muscle Myosin ◽  
Stable Complex ◽  
Atp Binding ◽  
Skeletal Muscle Myosin ◽  
Atp Binding Site ◽  
Tryptic Fragment ◽  
Muscle Myosin

Methylanthraniloyl-8-azido-ATP (Mant-8-N3-ATP), which binds to the 20 kDa C-terminal tryptic fragment of skeletal-muscle myosin subfragment-1 [Maruta, Miyanishi and Matsuda (1989) Eur. J. Biochem. 184, 213-221], was synthesized and used as a probe of the conformational change of smooth-muscle myosin. Mant-8-N3-ATP, like ATP, induced the formation of the 10 S conformation at low ionic strength. In the presence of vanadate, smooth-muscle myosin formed a stable complex with Mant-8-N3-ADP, and this complex showed the 10 S→6 S transition of myosin. ATP-binding sites for 6 S (extended state) and 10 S (folded state) myosin were studied by photolabelling of myosin with Mant-8-N3-ADP. For both 6 S and 10 S myosin, Mant-8-N3-ATP was incorporated into the 29 kDa N-terminal tryptic fragment of myosin heavy chain. This is unlike the labelling of skeletal-muscle myosin, in which the 20 kDa C-terminal fragment is labelled. The labelling of 29 kDa fragment was diminished significantly by addition of ATP. These results suggest that the conformation of the ATP-binding site of smooth-muscle myosin is different from that of skeletal-muscle myosin. To examine further the possible differences in the labelling site between 6 S and 10 S myosin, the affinity-labelled 29 kDa fragment was subjected to complete proteolysis by lysylendo-peptidase. The fluorescent-labelled-peptide map suggested that the Mant-8-N3-ADP-binding sites for 6 S and 10 S myosin were identical.

scholarly journals A Variable Domain near the ATP-Binding Site in Drosophila Muscle Myosin Is Part of the Communication Pathway between the Nucleotide and Actin-binding Sites

2007 ◽  
Vol 368 (4) ◽  
pp. 1051-1066 ◽  
Author(s):  
Becky M. Miller ◽  
Marieke J. Bloemink ◽  
Miklós Nyitrai ◽  
Sanford I. Bernstein ◽  
Michael A. Geeves
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Variable Domain ◽  
Actin Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Communication Pathway ◽  
Muscle Myosin

scholarly journals Tryptophan 512 Is Sensitive to Conformational Changes in the Rigid Relay Loop of Smooth Muscle Myosin during the MgATPase Cycle

2000 ◽  
Vol 275 (33) ◽  
pp. 25481-25487 ◽  
Author(s):  
Christopher M. Yengo ◽  
Lyun R. Chrin ◽  
Arthur S. Rovner ◽  
Christopher L. Berger
Keyword(s):  
Smooth Muscle ◽  
Conformational Changes ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin

scholarly journals Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding.

1987 ◽  
Vol 105 (6) ◽  
pp. 3007-3019 ◽  
Author(s):  
K M Trybus ◽  
S Lowey
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Conformational Changes ◽  
Myosin Head ◽  
Smooth Muscle Myosin ◽  
Detectable Effect ◽  
Skeletal Muscle Myosin ◽  
Polar Type ◽  
Low Ionic Strength ◽  
Muscle Myosin

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.

scholarly journals Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recognizes the calmodulin-binding region

1991 ◽  
Vol 275 (3) ◽  
pp. 679-684 ◽  
Author(s):  
Y Araki ◽  
M Ikebe
Keyword(s):  
Monoclonal Antibody ◽  
Smooth Muscle ◽  
Monoclonal Antibodies ◽  
Binding Site ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Calmodulin Binding ◽  
Muscle Myosin ◽  
Active Fragment

The regulatory domain of smooth muscle myosin light chain kinase (MLCK) was studied using monoclonal antibodies. Of the 22 monoclonal antibodies tested, a monoclonal antibody designated LKH-18 was found to activate MLCK in the absence of Ca2+/calmodulin. This activation was even greater when an Fab fragment of LKH-18 was used. Consequently, the actin-dependent smooth muscle myosin ATPase activity and the superprecipitation of actomyosin were significantly activated by MLCK plus LKH-18, even in the absence of Ca2+/calmodulin. The antibody-binding site was studied using proteolytic fragments and synthetic peptide analogues of MLCK. Immunoblot analysis revealed that LKH-18 reacted with the 66 kDa calmodulin-dependent active fragment but not with the 64 kDa inactive fragment or with the 61 kDa calmodulin-independent active fragment. Furthermore, LKH-18 reacted with MLCK-(796-815)-peptide but not with MLCK-(786-801)-peptide or with MLCK-(796-807)-peptide. Therefore the LKH-18-binding site was assigned to amino acid residues 808-815 of MLCK, which are thought to be a part of the calmodulin-binding site. The present results suggest that the binding of ligand to this region induces a conformation change in MLCK and that this abolishes the action of the inhibitory region which exists next to the N-terminal side of the calmodulin-binding site.

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

1995 ◽  
Vol 15 (5) ◽  
pp. 317-326 ◽  
Author(s):  
Tohru Kanazawa ◽  
Hiroshi Suzuki ◽  
Takashi Daiho ◽  
Kazuo Yamasaki
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Tryptophan Fluorescence ◽  
Cytoplasmic Domain ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

scholarly journals Studies of functional amino acid residues in smooth muscle myosin : mutagenesis of actin binding site

2000 ◽  
Vol 40 (supplement) ◽  
pp. S60
Author(s):  
H. Onishi ◽  
S. Kojima ◽  
K. Konishi ◽  
K. Katoh ◽  
K. Fujiwara ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Binding Site ◽  
Smooth Muscle Myosin ◽  
Actin Binding ◽  
Amino Acid Residues ◽  
Muscle Myosin ◽  
Actin Binding Site
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