Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

Yao Song, Jay L. Zweier, Yong Xia

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the Kd of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P <0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Issue number6 50-6
StatePublished - 2001


  • Calmodulin binding affinity
  • Electron paramagnetic resonance spin trapping
  • Heat shock protein 90 modulation
  • Nitric oxide synthase regulation
  • Tryptophan fluorescence

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)


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