Functional tuning of molecular motors
Final Report Abstract
In the human body, a spectrum of highly specialized motor proteins needs to be available at the right locations with their motor activity only activated when functionally required. We hypothesize that this logistic problem is alleviated by modulation of molecular properties by the local environment, like local phosphate- or Mg++-concentration, pH, or local differences in the activity of specific kinases and phosphatases. Each of these mechanisms can result in particular modulation of molecular properties of motor proteins and thus may tune motile function of a myosin molecule to different local requirements. In the case of class 5 myosins, which show processive movement moving hand over hand along their actin tracks without dissociation, we were able to demonstrate that the length of such processive runs can be tuned by the free Mg2+-ion concentration. Tuning by free Mg2+- ion concentration in the physiological range results in single 36nm steps at low concentrations (< 1 mM) and 50 and more consecutive steps at higher free Mg2+ resulting in processive runs that extend over several µm. This tuning of processivity is related to a reduced rate of ADP-release from the active site, as free Mg2+-ion concentration increases. The concomitant increase in the time during which the myosin head domain remains strongly bound to the actin filament was verified by following binding and release of fluorescently labelled nucleotide (Cy3-EDA-ATP), one molecule at a time, on individual myosin molecules by TIRF-microscopy. Trying to elucidate the steps around ADP release and the second substep in the working stroke of class 5 myosins, we found that ADP is released prior to the second substep. Morover, the stiffness of the myosin head domain increases substantially (up to 3-fold) with the second substep. Thus, while the 2nd substep reduces intramolecular elastic distortion during the processive stepping of vertebrate myosin 5a, the increased stiffness of the head domain with the substep maintains or even increases the intramolecular forces that are essential for the coordination of the two heads of a myosin 5 molecule to safeguard processive stepping in the movement of cargo along actin filaments.
Publications
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(2008) Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II. J Biol Chem 283: 3773-3781
Amrute-Nayak M, Antognozzi M, Scholz T, Kojima H, Brenner B
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(2009) Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance. Biophys J 97: 806-824
Seebohm B, Matinmehr F, Kohler J, Francino A, Navarro-Lopez F, Perrot A, Ozcelik C, McKenna WJ, Brenner B, Kraft T
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(2009) Force-generating cross-bridges during rampshaped releases: evidence for a new structural state. Biophys J 96: 1430-1446
Radocaj A, Weiss T, Helsby WI, Brenner B, Kraft T
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(2009) The mechanism of pentabromopseudilin inhibition of myosin motor activity. Nat Struct Mol Biol 16: 80-88
Fedorov R, Böhl M, Tsiavaliaris G, Hartmann FK, Taft MH, Baruch P, Brenner B, Martin R, Knölker H- J, Gutzeit HO, Manstein DJ
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(2010) Targeted optimization of a protein nanomachine for operation in Biohybrid devices. Angew. Chem. Int. Ed. 49: 312-316
Amrute-Nayak M, Diensthuber RP, Steffen W, Kathmann D, Hartmann FK, Fedorov R, Urbanke C, Manstein DJ, Brenner B, Tsiavaliaris, G
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(2012) Tau Protein Diffuses along the Microtubule Lattice. J Biol Chem 287: 38559-38568
Hinrichs MH, Jalal A, Brenner B, Mandelkow E, Kumar S, Scholz T
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(2013) Familial hypertrophic cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes. J Mol Cell Cardiol 57: 13-22
Kraft T, Witjas-Paalberends ER, Boontje NM, Tripathi S, Brandis A, Montag J, Hodgkinson JL, Francino A, Navarro-Lopez F, Brenner B, Stienen GJ, van der Velden J
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(2013) Lipoteichoic acid from Staphylococcus aureus directly affects cardiomyocyte contractility and calcium transients. Mol Immunol 56: 720-728
Mutig N, Geers-Knoerr C, Piep B, Pahuja A, Vogt PM, Brenner B, Niederbichler AD, Kraft T
