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Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

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Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
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A Ca2+-sensing molecular switch based on alternate frame protein folding.

Margaret M Stratton1, Diana M Mitrea, Stewart N Loh

  • 1Department of Biochemistry & Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.

ACS Chemical Biology
|October 25, 2008
PubMed
Summary

We developed a new method, alternate frame folding (AFF), to engineer allosteric control in proteins. This technique creates binding-induced conformational changes, enabling biosensor development even for molecules lacking natural structural shifts.

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Area of Science:

  • Protein engineering
  • Biochemistry
  • Molecular biology

Background:

  • Biosensor development typically requires large protein conformational changes upon ligand binding.
  • Many proteins do not naturally exhibit significant structural changes when binding to their substrates.
  • This limits the scope of existing biosensor design strategies.

Purpose of the Study:

  • To present a general method, alternate frame folding (AFF), for engineering allosteric control into ligand-binding proteins.
  • To enable the creation of biosensors from proteins that do not naturally undergo large conformational changes.
  • To demonstrate the broad applicability of AFF for designing novel molecular sensors.

Main Methods:

  • The AFF approach involves duplicating a portion of a protein's amino acid sequence to create an alternative folding "frame".
  • This design leads to two mutually exclusive structures: the wild-type and a circularly permuted variant.
  • Ligand binding energy is harnessed to shift the equilibrium between these two conformations.

Main Results:

  • The AFF method was successfully applied to calbindin D(9k), converting it into a calcium ion (Ca2+) sensor.
  • Despite minimal structural differences between Ca2+-free and Ca2+-bound calbindin, AFF induced a detectable conformational change.
  • This change was verified using covalently attached fluorescent probes, demonstrating the system's sensitivity.

Conclusions:

  • Alternate frame folding (AFF) provides a versatile strategy for engineering allosteric control and creating biosensors.
  • The AFF approach can be applied to any protein to induce binding-dependent conformational changes.
  • This method holds significant potential for developing genetically encoded biosensors for diverse small molecules.