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Related Experiment Videos

Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.

Pabak Sarkar1, Kaitlin A Davis1, Henry L Puhl1

  • 1Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.

Biophysical Journal
|March 30, 2017
PubMed
Summary

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Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) holoenzymes function as paired catalytic domains. This study engineered dimeric CaMKII to reveal its functional and structural behavior, confirming paired domains are the fundamental units.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Kinases are typically monomeric or dimeric, but CaMKII holoenzymes comprise 8-14 subunits arranged in pairs.
  • The functional unit of the CaMKII holoenzyme (independent subunits, catalytic pairs, or a single unit) remains unclear.

Purpose of the Study:

  • To investigate the functional and structural behavior of CaMKII holoenzymes.
  • To determine if CaMKII catalytic domains function as independent subunits or as pairs.
  • To compare the activity of wild-type (WT) CaMKII holoenzyme with engineered monomeric and dimeric forms.

Main Methods:

  • Utilized fluorescence correlation spectroscopy and homo-FRET analysis to characterize Venus-tagged CaMKIIα assembly mutants.
  • Identified and isolated a dimeric CaMKII mutant.

Related Experiment Videos

  • Compared CaM-activated structural and functional changes in dimeric CaMKII, WT-holoenzyme, and a monomeric mutant.

    • Main Results:

    • CaM activation increased hydrodynamic volume in both WT and dimeric CaMKII without changing subunit stoichiometry or catalytic domain homo-FRET.
    • The dimeric CaMKII mutant exhibited kinase activity comparable to WT holoenzyme for both exogenous substrates and endogenous autophosphorylation.
    • Spectroscopic analysis confirmed that paired catalytic domains are the fundamental functional units of CaMKII.

    Conclusions:

    • The fundamental functional units of CaMKII holoenzyme are paired catalytic domains.
    • Dimeric CaMKII mutants mimic the functional behavior of WT holoenzyme.
    • This study clarifies the quaternary structure-function relationship in CaMKII.