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Quantifying cooperative multisite binding in the hub protein LC8 through Bayesian inference.

Aidan B Estelle1, August George2, Elisar J Barbar1

  • 1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, United States of America.

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This study reveals positive cooperativity in LC8 protein binding using advanced Bayesian isothermal titration calorimetry (ITC) analysis. This finding advances understanding of multi-site protein interactions and improves ITC data interpretation methods.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Multistep protein-protein interactions are crucial for biological processes.
  • Characterizing these interactions is challenging, especially intermediate binding steps.
  • Isothermal titration calorimetry (ITC) analyses often use simplified models for complex binding events.

Purpose of the Study:

  • To develop and apply advanced Bayesian ITC methods for quantifying thermodynamic parameters in multi-site binding interactions.
  • To investigate the binding mechanism of the essential hub protein LC8 and its client peptides.
  • To address challenges in ITC data analysis, including protein concentration uncertainty and parameter identifiability.

Main Methods:

  • Utilized Bayesian approaches to analyze isothermal titration calorimetry (ITC) data.
  • Employed a two-site binding model to quantify thermodynamic parameters for protein interactions.
  • Developed a system-agnostic heat map for assessing practical parameter identifiability in ITC experiments.

Main Results:

  • Identified positive cooperativity in LC8 binding to client peptides with high confidence.
  • Observed limited evidence of cooperativity in the NudE dimer and dynein intermediate chain interaction.
  • Highlighted a mathematical ambiguity in concentration determination affecting binding parameter precision in ITC.
  • Demonstrated that parameter identifiability in ITC is dependent on binding parameters and experimental conditions.

Conclusions:

  • Positive cooperativity in LC8 binding likely drives the formation of functional saturated induced-dimer structures.
  • The study provides a robust foundation for analyzing multi-site binding interactions using ITC.
  • Best practices for Bayesian analysis of ITC experiments are outlined to improve accuracy and reliability.