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Related Concept Videos

Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...

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Pull-down of Calmodulin-binding Proteins
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Published on: January 23, 2012

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.

Brian D Slaughter1, Jay R Unruh, E Shane Price

  • 1Department of Chemistry, 1251 Wescoe Hall Drive, University of Kansas, Lawrence, Kansas 66045-7582, USA.

Journal of the American Chemical Society
|August 25, 2005
PubMed
Summary

Single-molecule FRET reveals calmodulin (CaM) unfolds via intermediate states, not a simple two-state process. Apo and Ca(2+)-bound CaM exhibit distinct intermediate conformations during denaturation by urea.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Calmodulin (CaM) is a crucial calcium-signaling protein.
  • Understanding CaM's conformational dynamics is vital for its function.
  • Denaturation studies reveal protein folding/unfolding pathways.

Purpose of the Study:

  • To characterize the denatured and partially denatured states of CaM.
  • To investigate the unfolding pathway of apo and Ca(2+)-bound CaM.
  • To identify intermediate conformational states during CaM denaturation.

Main Methods:

  • Single-pair fluorescence resonance energy transfer (spFRET) measurements.
  • Utilizing a doubly labeled CaM mutant (CaM-T34C-T110C).
  • Employing the denaturant urea at varying concentrations.

Main Results:

  • Multiple CaM conformational states were observed dependent on urea concentration.
  • CaM denaturation does not follow a two-state model, indicating intermediate states.
  • Distinct intermediate conformations were identified for apoCaM and Ca(2+)-CaM.

Conclusions:

  • CaM unfolds through at least one intermediate state.
  • A stepwise, sequential domain unfolding model for CaM is proposed.
  • The presence of distinct intermediates highlights Ca(2+)-dependent structural changes.