Motor Proteins - Experiments

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Motor Proteins- Experiments

• Biochemistry 4000
• Dr. Ute Kothe

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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
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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
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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
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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 Hookes 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
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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
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Mechanism of the kinesin step

• Questions
• Describe your observations for each of the three
  experiments.
• Interpret your observations.

Carter NJ, Nature 2005
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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