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1.
Anal Biochem ; 581: 113338, 2019 09 15.
Article in English | MEDLINE | ID: mdl-31201789

ABSTRACT

Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical reactions. The technique however is often limited by the volumes of reactants needed to load the system. Here we present a simple adaptation of commercial stopped-flow system that reduces the volume needed by a factor of 4 to ≈120 µl. After evaluation the volume requirements of the system we show that many standard myosin based assays can be performed using <100 µg of myosin. This adaptation both reduces the volume and therefore mass of protein required and also produces data of similar quality to that produced using the standard set up. The 100 µg of myosin required for these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used to assist diagnoses, to examine the effects of post translational modifications on muscle proteins and to test potential therapeutic drugs using patient derived samples.


Subject(s)
Myosins/analysis , Spectrum Analysis , Animals , Humans , Rabbits
2.
FEBS Lett ; 593(3): 296-307, 2019 02.
Article in English | MEDLINE | ID: mdl-30575960

ABSTRACT

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 µm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.


Subject(s)
Adenosine Triphosphate/chemistry , Cytoplasm/chemistry , Dyneins/chemistry , Optical Tweezers , Adenosine Triphosphate/metabolism , Animals , Cytoplasm/metabolism , Dyneins/metabolism , Swine
3.
Sci Rep ; 7(1): 7650, 2017 08 09.
Article in English | MEDLINE | ID: mdl-28794442

ABSTRACT

Myosin motor proteins convert chemical energy into force and movement through their interactions with nucleotide and filamentous actin (F-actin). The evolutionarily conserved lysine-265 (K265) of the myosin-2 motor from Dictyostelium discoideum (Dd) is proposed to be a key residue in an allosteric communication pathway that mediates actin-nucleotide coupling. To better understand the role of K265, point mutations were introduced within the Dd myosin-2 M765-2R framework, replacing this lysine with alanine (K265A), glutamic acid (K265E) or glutamine (K265Q), and the functional and kinetic properties of the resulting myosin motors were assessed. The alanine and glutamic acid substitutions reduced actin-activated ATPase activity, slowed the in vitro sliding velocity and attenuated the inhibitory potential of the allosteric myosin inhibitor pentabromopseudilin (PBP). However, glutamine substitution did not substantially change these parameters. Structural modelling suggests that K265 interacts with D590 and Q633 to establish a pivotal allosteric branching point. Based on our results, we propose: (1) that the K265-D590 interaction functions to reduce myosins basal ATPase activity in the absence of F-actin, and (2) that the dynamic formation of the K265-Q633 salt bridge upon actin cleft closure regulates the activation of product release by actin filaments.


Subject(s)
Actins/metabolism , Binding Sites , Lysine/metabolism , Myosin Type II/chemistry , Myosin Type II/metabolism , Nucleotides/metabolism , Actins/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Alanine/metabolism , Allosteric Regulation , Enzyme Activation , Gene Expression , Glutamic Acid , Kinetics , Models, Molecular , Mutation , Myosin Type II/genetics , Nucleotides/chemistry , Protein Binding , Structure-Activity Relationship
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