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Query: UMLS:C0038454 (stroke)
 147,016 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin-myosin-ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1-SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1-SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.
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PMID:Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II. 978 2

In muscle, the myosin head ('crossbridge') performs the 'working stroke', in which ATP is hydrolysed to generate the sliding of actin and myosin filaments. The myosin head consists of a globular motor domain and a long lever-arm domain. The 'lever-arm hypothesis' predicts that during the working stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working stroke in operation, we constructed fusion proteins by connecting Aequorea victoria green fluorescent protein and blue fluorescent protein to the amino and carboxyl termini of the motor domain of myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the ATP hydrolysis cycle, and then swings back at the subsequent step in which inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the phosphate-release step may correspond to the working stroke, and the swing at the isomerization step to the recovery stroke.
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PMID:Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps. 984 66

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.
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PMID:Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins. 1113 41

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is approximately 30 degrees further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain.
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PMID:Crystal structure of the motor domain of a class-I myosin. 1203 65

Myosin is a molecular motor and a member of a protein family comprising at least 18 classes. There is an about 1,000-fold difference in the in vitro sliding velocity between the fastest myosin and the slowest one. Previous studies revealed that the hydrophobic triplet in the motor domain (Val534, Phe535, and Pro536 in Dictyostelium myosin) is important for the strong binding of myosin to actin. We studied the role of the triplet in the sliding motion of myosin by means of site directed mutagenesis because the sliding velocity is determined by the time that myosin interacts with actin strongly. We produced mutant Dictyostelium myosins and subfragment-1s that have the triplet sequences of various classes of myosin with different sliding velocities. The V(max) and K(actin) values of the actin-activated ATPase for all these mutant subfragment-1s were lower than those of the wild-type Dictyostelium myosin. The mutant myosins exhibited much lower sliding velocities than the wild type. The time that the mutant subfragment-1s are in the strongly bound state did not correlate well with the sliding velocity. Our results suggested that (i) the hydrophobic triplet alone does not determine the sliding velocity of myosin, (ii) the size of the amino acid side chain in the triplet is crucial for the ATPase activity and the motility of myosin, and (iii) the hydrophobic triplet is important not only for strong binding to actin but also for the structural change of the myosin motor domain during the power stroke.
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PMID:Roles of the hydrophobic triplet in the motor domain of myosin in the interaction between myosin and actin. 1294 84

A cytoplasmic dynein is a microtubule-based motor protein involved in diverse cellular functions, such as organelle transport and chromosome segregation. The dynein has two ring-shaped heads that contain six repeats of the AAA domain responsible for ATP hydrolysis. It has been proposed that the ATPase-dependent swing of a stalk and a stem emerging from each of the heads generates the power stroke (Burgess, S.A. (2003) Nature 421, 715-718). To understand the molecular mechanism of the dynein power stroke, it is essential to establish an easy and reproducible method to express and purify the recombinant dynein with full motor activities. Here we report the expression and purification of the C-terminal 380-kDa fragment of the Dictyostelium cytoplasmic dynein heavy-chain fused with an affinity tag and green fluorescent protein. The purified single-headed recombinant protein drove the robust minus-end-directed sliding of microtubules at a velocity of 1.2 microm/s. This recombinant protein had a high basal ATPase activity (approximately 4s(-1)), which was further activated by >15-fold on the addition of 40 microM microtubules. These results show that the 380-kDa recombinant fragment retains all the structures required for motor functions, i.e. the ATPase activity highly stimulated by microtubules and the robust motility.
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PMID:A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules. 1505 17

Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles the previously published DFNA11 family. The affected family members show a flat audiogram at young ages and only modest progression, most clearly at the high frequencies. In addition, they suffer from minor vestibular symptoms. Linkage analysis yielded a maximum two-point lodscore of 3.43 for marker D11S937 located within 1 cM of the myosin VIIA gene. The myosin VIIA gene was sequenced and 11 nucleotide variations were found. Ten nucleotide changes represent benign intronic variants, silent exon mutations or non-pathologic amino acid substitutions. One variant, a c.1373A-->T transversion that is heterozygously present in all affected family members and absent in 300 healthy individuals, is predicted to result in an Asn458Ile amino acid substitution. Asn458 is located in a region of the myosin VIIA motor domain that is highly conserved in different classes of myosins and in myosins of different species. To evaluate whether the Asn458Ile mutation was indeed responsible for the hearing impairment, a molecular model of myosin VIIA was built based on the known structure of the myosin II heavy chain from Dictyostelium discoideum. In this model, conformational changes in the protein caused by the amino acid substitution Asn458Ile are predicted to disrupt ATP/ADP binding and impair the myosin power-stroke, which would have a severe effect on the function of the myosin VIIA protein.
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PMID:Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11). 1522 49

The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.
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PMID:The effect of F-actin on the relay helix position of myosin II, as revealed by tryptophan fluorescence, and its implications for mechanochemical coupling. 1558 52

During the recovery stroke, the myosin motor is primed for the next power stroke by a 60 degree rotation of its lever arm. This reversible motion is coupled to the activation of the ATPase function of myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456 peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and gamma-phosphate that is essential for ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum myosin II in the two end conformations of the recovery stroke with different nucleotide states (ATP, ADP x Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the nucleotide state is achieved by a small displacement of the cleaved gamma-phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery stroke while the nucleotide is in the ADP x Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when ATP is added to the pre-recovery stroke conformer and remains stable after the recovery stroke, indicating that it has a role in shaping the ATP binding site by induced fit.
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PMID:Simulations of the myosin II motor reveal a nucleotide-state sensing element that controls the recovery stroke. 1685 3

The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.
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PMID:Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin. 1741 46


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