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November 12, 2008

Paper published in J. Biol. Chem. about engineering the processivity of the myosin 5 vesicle transporter by a designed point mutation

Switch 1 Mutation S217A Converts Myosin V into a Low Duty Ratio Motor.

Forgacs E, Sakamoto T, Cartwright S, Belknap B, Kovács M, Tóth J, Webb MR, Sellers JR, White HD

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-P(i) (inorganic phosphate) and actomyosin V-ADP-P(i) and the rate of the hydrolysis step (myosin V-ATP --> myosin V-ADP-P(i)) were all approximately 10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from approximately 0.85 in WT myosin V to approximately 0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.

PubMed Abstract



July 5, 2008

Nikolett Nagy and Kata Sarlós admitted to Graduate Program in Structural Biochemistry, Eötvös University



June 2, 2008

Balázs Jelinek’s Ph.D. thesis defense



May 19, 2008

Mihály Kovács receives 1st Prize of Sigma-Aldrich Award



May 9-10, 2008

Retreat Conference held at Lake Balaton



May 8, 2008

Kata Sarlós receives Student Excellence Award of Eötvös Loránd University



May 8, 2008

Mihály Kovács’ habilitation at Eötvös Loránd University



May 7, 2008

Máté Gyimesi’s Ph.D. thesis defense



January 30, 2008

Group receives research grant of Hungarian Scientific Research Fund



January 16, 2008

Paper published in J. Biol. Chem. about the mechanism of action of myosin 5c, which strikingly differs from that of other myosin 5 motors in vertebrates

Human myosin Vc is a low duty ratio, non-processive molecular motor.

Takagi Y, Yang Y, Fujiwara I, Jacobs D, Cheney RE, Sellers JR, Kovacs M.

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin activated ATPase measurements revealed a Vmax of 1.8+/-0.3 s-1 and a KATPase of 43+/-11 M. Unlike previously studied vertebrate myosin-Vs, the rate-limiting step in the acto-myosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s-1), rather than the ADP release step (~12.0 - 16.0 s-1). Nevertheless, the ADP affinity of acto-myosin Vc (Kd = 0.25+/-0.02 M) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was ~10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this a fluorescently-labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but did move actin filaments at a velocity of ~24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of acto-myosin Vc may lead to elevations of the duty ratio of MVc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

PubMed Abstract


 
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