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Related Experiment Videos

A fluorescent probe designed for studying protein conformational change.

Bruce E Cohen1, Arnd Pralle, XiaoJie Yao

  • 1Howard Hughes Medical Institute and Departments of Physiology and Biochemistry, University of California, San Francisco, CA 94143-0725, USA. bcohen@itsa.ucsf.edu

Proceedings of the National Academy of Sciences of the United States of America
|January 20, 2005
PubMed
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Researchers developed aminophenoxazone maleimide (APM), a novel red fluorescent probe, to better track protein conformational changes. APM offers improved spectral properties for studying protein motions in biological systems.

Area of Science:

  • Biochemistry
  • Biophysics
  • Molecular Biology

Background:

  • Fluorescence is valuable for studying protein dynamics due to its sensitivity and kinetic resolution.
  • Existing probes often lack suitability for reporting environmental changes in specific protein residues.
  • Advances in microscopy and protein purification enable observation of fluorescence changes in eukaryotic membrane proteins.

Purpose of the Study:

  • Introduce a new red Cys-reactive probe, aminophenoxazone maleimide (APM), for monitoring protein conformational changes.
  • Design APM with improved chemical and spectral properties for enhanced reporting of protein motion.
  • Evaluate APM's utility in tracking functional transitions of membrane proteins.

Main Methods:

  • Synthesized and characterized the novel red Cys-reactive probe, aminophenoxazone maleimide (APM).

Related Experiment Videos

  • Assessed APM's spectral properties, including polarity-dependent shifts in excitation and emission maxima.
  • Applied APM to study voltage-sensing conformational changes in the Shaker potassium channel and aqueous positioning in the beta2 adrenergic receptor.

    • Main Results:

    • APM exhibits favorable characteristics: compact size, uncharged nature, and a short linker for precise side-chain motion tracking.
    • APM displays significant polarity-dependent spectral shifts (Stokes shift, excitation/emission maxima).
    • APM successfully detected conformational changes in the Shaker potassium channel, outperforming rhodamine maleimide at a challenging site.
    • APM's spectral properties revealed subtle differences in aqueous environments within the beta2 adrenergic receptor.

    Conclusions:

    • Aminophenoxazone maleimide (APM) is a versatile red fluorescent probe for studying protein conformational dynamics.
    • APM's spectral properties provide a sensitive readout of local protein structure and motion.
    • APM demonstrates superior performance in specific applications compared to existing probes, enhancing the study of membrane protein function.