Motor Proteins- Experiments

Biochemistry 4000

Dr. Ute Kothe

Switch-based mechanism of kinesin

Kikkawa M, Nature 2001

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)

Switch-based mechanism of kinesin

ADP-form: yellowATP-like form: red

Kikkawa M, Nature 2001

Questions:

1. Describe the structural differences of kinesin in the ADP- and ATP-like form.

2. Compare the switch mechanism of kinesin to that of G proteins.

3. What is another name of helix 4?

4. Discuss the problem a monomeric kinesin has to be processive.

AMPPCP

Cryo-EM of KIFA + Microtubules

Kikkawa M, Nature 2001

KIF1A-AMPPNP-Microtubule22/15 Å resolution

KIF1A-ADP-Microtubule22 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.

Mechanism of the kinesin step

Carter NJ, Nature 2005

Experimental Description:To examine stepping in more detail we used the nowclassicalsingle-bead optical-trap arrangement in which a singlemolecule of kinesin is attached to a spherical bead about 1 mm indiameter. The bead is optically trapped at the focus of an infraredlaser19 and the trap is steered so as to bring the kinesinwithin range ofan immobilized microtubule. The kinesin then walks along themicrotubule, tending to pull the bead out of the trap, while thetrap applies an opposing force tending to restore the bead to the trapcentre.Within the range of interest, the trap acts like a spring obeyingHooke’s law. By accurately tracking the bead, the stepwise motions ofthe attached kinesin molecule can be observed. We measured inparticular the effect of extreme backward loads on the pattern ofStepping.

Question:Draw a schematic picture of the experimental set-up and label all important components.

Mechanism of the kinesin step

Carter NJ, Nature 2005

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

Mechanism of the kinesin step

Carter NJ, Nature 2005

Questions:

1. Describe your observations for each of the three experiments.

2. Interpret your observations.

In vitro selection of an RNA ligase

Seelig, Nature 2007

Question:

1. Describe the principle of mRNA display for in vitro selection of proteins.

2. Explain the selection strategy for a RNA ligase (protein enzyme) depicted in Fig.a. Provide a rational for each step.