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Probing functional conformation-state fluctuation dynamics in recognition binding between calmodulin and target

Sunidhi Jaiswal1, Yufan He1, H Peter Lu1

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Single-molecule FRET reveals how calcium-activated calmodulin (CaM) interacts with the C28W peptide. The study details multi-step binding dynamics, showing the C-domain binds first, followed by the flexible N-domain.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Biochemistry

Background:

  • Protein conformational dynamics are vital for biological functions.
  • Understanding protein-protein interactions, like calmodulin (CaM) with plasma membrane Ca-ATPase (via C28W peptide), is crucial for cell signaling.
  • The dynamic details of CaM-C28W interactions remain unclear.

Purpose of the Study:

  • To investigate the real-time conformational dynamics of calcium-activated calmodulin (CaM) interacting with the C28W peptide using single-molecule FRET.
  • To elucidate the step-wise binding mechanisms and dynamic features of the CaM-C28W complex.
  • To provide a mechanistic understanding of CaM signaling and Ca-ATPase activation.

Main Methods:

  • Utilized single-molecule fluorescence resonance energy transfer (sm-FRET) spectroscopy and imaging.
  • Employed Cyanine3-labeled CaM (N-domain) and Dylight 649-labeled C28W peptide (N-domain).
  • Applied unique statistical approaches to analyze FRET efficiency trajectories.

Main Results:

  • Observed multiple binding steps with distinct dynamic features of loosely and tightly bound states.
  • Determined that the CaM C-domain binds to C28W first with higher affinity.
  • Identified that the CaM N-domain exhibits flexible, potentially switching binding states (selective/non-selective) while the C-domain remains bound.

Conclusions:

  • The study provides a mechanistic insight into CaM-C28W interaction dynamics, involving multi-state binding.
  • Reveals sequential binding of CaM domains to the C28W peptide, crucial for Ca-ATPase activation.
  • Demonstrates the utility of single-molecule spectroscopic analyses for studying protein dynamics and interactions.