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Motor Proteins - Experiments

Motor Proteins - Experiments. Biochemistry 4000 Dr. Ute Kothe. Switch-based mechanism of kinesin. Crystal Structure of KIF1A: monomeric, processive kinesin Neck linker exchanged for the one from conventional kinesin FHA = fork-head-associated domain, might bind microtubule

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Motor Proteins - Experiments

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  1. Motor Proteins- Experiments Biochemistry 4000 Dr. Ute Kothe

  2. Switch-based mechanism of kinesin • Crystal Structure of KIF1A: • monomeric, processive kinesin • Neck linker exchanged for the one from conventional kinesin • FHA = fork-head-associated domain, might bind microtubule • PH = pleckstrin homology domain, cargo binding • ADP-bound (light blue / yellow) • AMPPCP-bound (dark blue / red) Kikkawa M, Nature 2001

  3. Switch-based mechanism of kinesin AMPPCP • Questions: • Describe the structural differences of kinesin in the ADP- and ATP-like form. • Compare the switch mechanism of kinesin to that of G proteins. • What is another name of helix a4? • Discuss the problem a monomeric kinesin has to be processive. ADP-form: yellow ATP-like form: red Kikkawa M, Nature 2001

  4. Cryo-EM of KIFA + Microtubules KIF1A-AMPPNP-Microtubule 22/15 Å resolution KIF1A-ADP-Microtubule 22 Å resolution Question: After determination of the cryo-EM structure, the crystal structure of KIFA in the AMPPCP and AMP-form, respectively, have been fitted into the electron density. A 20° clockwise rotation of kinesin upon changing the nucleotide state was detected. Discuss the quality of the fit and the interpretation of these data. Kikkawa M, Nature 2001

  5. Mechanism of the kinesin step Experimental Description: To examine stepping in more detail we used the nowclassical single-bead optical-trap arrangement in which a single molecule of kinesin is attached to a spherical bead about 1 mm in diameter. The bead is optically trapped at the focus of an infrared laser19 and the trap is steered so as to bring the kinesinwithin range of an immobilized microtubule. The kinesin then walks along the microtubule, tending to pull the bead out of the trap, while the trap applies an opposing force tending to restore the bead to the trap centre.Within the range of interest, the trap acts like a spring obeying Hooke’s law. By accurately tracking the bead, the stepwise motions of the attached kinesin molecule can be observed. We measured in particular the effect of extreme backward loads on the pattern of Stepping. Question: Draw a schematic picture of the experimental set-up and label all important components. Carter NJ, Nature 2005

  6. Mechanism of the kinesin step Black: trap & microtubule remain fixed, kinesin walks away from trap centre Red: on reaching 4 pN, the microtubule is moved rapidly pulling the kinesin to 14pN Blue: on reachin 4 pN, the microtubule is moved rapidly towards the kinesin applying a large negative (assisting) force to the kinesin Carter NJ, Nature 2005

  7. Mechanism of the kinesin step • Questions: • Describe your observations for each of the three experiments. • Interpret your observations. Carter NJ, Nature 2005

  8. In vitro selection of an RNA ligase • Question: • Describe the principle of mRNA display for in vitro selection of proteins. • Explain the selection strategy for a RNA ligase (protein enzyme) depicted in Fig.a. Provide a rational for each step. Seelig, Nature 2007

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