Project R4 (Sweeney): Tuning the lever arms of myosin V and VI Myosin V and VI use different mechanisms to accomplish processive movements of similar step sizes, but ofopposite directionality. Both motors bind calmodulin and have activities that are altered by increases in [Ca2+]. Thisstudy will delineate the nature of the CaM/Ca 2+interactions and modulation of myosin V and VI function. This projectwill utilize in vitro expression and functional assays that will enable structure/function studies of recombinant myosin Vand VI. We hypothesize that at resting cellular [Ca 2 ] the lever arm of myosin V has optimal compliance for itsprocessive movement. Changes in [Ca 2 ] alter that compliance by changing the interactions of the CaMs with the IQmotifs of the lever arm. The IQ motifs are not likely all equivalent in these interactions, nor are the [Ca 2 ]dependencyof the interactions. We will perform the following experiments to address these issues: a. kinetic, motility and TIRFanalyses of myosin V in presence of Ca 2+with different number of CaMs/ELCs; b. determine which CaMs are lost atelevated [Ca 2+] by introducing disulfide cross-links between individual CaMs and the heavy chain; c. use disulfidelocking of all but one of the CaMs to map lever arm distortions in myosin V during processive movement; d. obtain EMimages of single- and double-headed myosin V constructs in presence and absence of [CaZ+]; and, e. determinerelationship between lever arm compliance and processivity. The large step size of myosin VI is difficult to reconcile with the lever arm hypothesis. We propose thepossibility that there is a CaM arm connected to a flexible linker to enable the large, diffusive steps. The followingexperiments will address this and the effect of [Ca z+] on the CaM arm: a. ascertain CaM affinities as a function ofcalcium for each of the CaM binding sites; b. use labeled CaM/ELC mutants to enable monitoring of CaM orientationand movement; c. examine double-headed and truncated myosin VI using electron microscopy and AFM in differentnucleotide states with and without actin.
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