Home › Biochemistry › Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
Biochemistry JoVE (Open Access) Citable · DOI

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

DOI: 10.3791/52142-v
What you'll learn
  • ✓ Measure association and dissociation rate constants for mantATP using stopped-flow fluorescence
  • ✓ Determine dissociation kinetics of mantADP in kinesin–microtubule interactions
  • ✓ Interpret fluorescence data to reconstruct ATP turnover cycle transitions
  • ✓ Apply labeled nucleotide methodology to analyze motor protein mechanics
Protocol

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

Difficulty
advanced
Total time
~4–6 hours per sample set (including instrument setup, stopped-flow runs, and data analysis)
Biosafety
BSL-1

Steps

1
Measure mantATP association and dissociation kinetics

Use stopped-flow fluorescence spectrophotometry to monitor real-time binding and release of mantATP (fluorescently labeled nucleotide) to kinesin motor domain in the presence of microtubules. Record changes in intrinsic fluorescence intensity to calculate rate constants.

▶ 01:13
2
Measure mantADP dissociation rate constant

Apply stopped-flow methodology to determine the dissociation rate of mantADP from the kinesin–microtubule complex by monitoring fluorescence decay upon nucleotide exchange. Quantify kinetic parameters from transient kinetic traces.

▶ 04:18
3
Analyze and interpret ATP turnover cycle results

Compile individual rate constants from mantATP and mantADP measurements to reconstruct the complete nucleotide-dependent cycle of kinesin–microtubule interaction. Integrate kinetic data to reveal mechanistic transitions.

▶ 05:19
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