Optimierung molekularer Motoren mit Hilfe von gelenkter Evolution
Final Report Abstract
While we were not successful with the directed evolution approach to obtain myosin constructs with artificial neck regions that are functional in vivo, our rational engineering approach to generated constructs that can be used as processive and tunable motors for nanotechnological applications succeeded. Our recent contribution in the Journal Angewandte Chemie Int. Ed. was published as “hot paper” for its importance in a rapidly evolving field of nanotechnology and received wide recognition in the molecular motors and nanotechnology fields. In summary, the use of a multifaceted approach that combined computer assisted structure based protein engineering, fast solution kinetics, microscopy-based functional assays, and X-ray structure determination has allowed us to: • describe the conditional processive mechanism of a class 5 myosin, thus providing first experimental evidence for the physiological relevance of free Mg2+-ions in tuning myosin motor activity; • obtain elementary insights into the molecular mechanisms of processivity by generating an artificial processive myosins from a minimal set of functionally independent building blocks. • engineer the active sites to specifically increase or decrease the dependence of ADP-release rate on the concentration of free Mg2+-ions • solve the atomic structures of the motor domains of Dictyostelium myosin-5b and myosin-2 with engineered loop-2 and a mutation in switch-2, respectively. The engineering of artificial myosins has been a continuation of our previous work to generate myosins with altered substrate specificity, increased or decreased enzymatic and motile activity, or reversed directionality of movement. Here, we defined the minimal molecular requirements for a double-headed motor to move processively along F-actin and elucidated important aspects of the mechanism of Mg2+-coordination and ADP-release in myosins. The results provide further evidence how our “tool-box for structure-based engineering” can be used to fine-tune the properties of a molecular motor. Our results have implication in regard to the ease with which the functional states of motor protein-based biohybrid devices can be altered and the extent to which the useful lifetime of such devices can be prolongated. The simple parametric control that is possible with our engineered motors is important ongoing work aimed at the development of biohybrid microdevices with assembly and sensing functions.
Publications
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„Molekulare Aspekte der Krafterzeugung, Bewegung und Funktion von Myosinen mittels Strukturaufklärung & Proteinengineering“. PhD, Leibniz Universität Hannover
Daniela Kathmann
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„Struktur-Funktionsanalyse prozessiver und nicht-prozessiver Myosine“. PhD, Leibniz Universität Hannover
Ralph Diensthuber
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(2008) Dictyostelium myosin-5b is a conditional processive motor. J. Biol. Chem 283, 26902- 26910
Taft, M.H., Hartmann, F.K., Rump, A., Keller H., Chizhov, I. , Manstein, D.J., and Tsiavaliaris, G.
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(2008). Mechanism of switch-2 mediated Mg2+-sensing in the myosin motor domain. 48th annual meeting of the American Society of Cell Biology, San Francisco
Diensthuber, R.P., Hartmann, F.K., Fedorov, R., Manstein, D.J., and Tsiavaliaris, G.
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(2010) Targeted optimization of a protein nanomachine for operation in biohybrid devices. Angew. Chem. Int. Ed. Engl., 49, 312- 316
Amrute-Nayak, M., Diensthuber, R.P., Steffen, W., Kathmann, D., Hartmann, F.K., Fedorov, R., Urbanke, C., Manstein, D.J., Brenner, B., and Tsiavaliaris, G.
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(2010). Switch-2 dependent modulation of the myosin power stroke. 54th annual meeting of the American Biophysical Society
Kathmann, D., Diensthuber, R.P., Hartmann, F.K., Fedorov, R., Manstein, D.J., and Tsiavaliaris, G.
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(2010). Targeted optimization of a molecular motor for controlling movement in biohybrid devices. 54th annual meeting of the American Biophysical Society
Amrute-Nayak, M., Diensthuber, R.P., Steffen, W., Kathmann, D., Hartmann, F.K., Fedorov, R., Urbanke, C., Manstein, D.J., Brenner, B., and Tsiavaliaris, G.
