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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
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Dynamics and local ordering of spin-labeled prion protein: an ESR simulation study of a highly PH-sensitive site

Yun-Wei Chiang1, Yuki Otoshima, Yasuko Watanabe

  • 1Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan. ywchiang@mx.nthu.edu.tw

Journal of Biomolecular Structure & Dynamics
|September 24, 2008
PubMed

Insights

Mouse prion protein (moPrPC) dynamics at Valine 160 change with pH. A combination of site-directed spin labeling and spectral simulations reveals distinct mobile and immobile protein conformations, crucial for understanding prion structure.

Area of Science:

  • Biophysics
  • Structural Biology
  • Prion Biology

Background:

  • The pH sensitivity of mouse prion protein (moPrPC) at Valine 160 (V160) was previously identified using electron spin resonance (ESR) spectroscopy and site-directed spin labeling (SDSL).
  • Limited theoretical analysis existed to explain the molecular dynamics underlying the experimental ESR spectra.
  • Understanding protein dynamics is critical for elucidating protein function and misfolding pathways.

Purpose of the Study:

  • To theoretically analyze the molecular dynamics of moPrPC at V160 and other sites using ESR spectral simulations.
  • To investigate the pH-dependent changes in protein dynamics and local environment ordering.
  • To explore the coexistence of multiple protein conformations within the prion protein structure.

Main Methods:

  • X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites were analyzed.
  • Spectral simulations were performed using the stochastic Liouville equation (SLE) over wide pH and temperature ranges.
  • Analysis focused on identifying and quantifying different dynamic components (immobile and mobile) within the spectra.

Main Results:

  • Molecular mobility of the V160 spin label increased as pH decreased from 7.5 to 4.5.
  • Two distinct spectral components, 'immobile' (Im) and 'mobile' (Mb), were simultaneously present for V160.
  • The Im/Mb ratio increased with pH, indicating a shift towards a more mobile and disordered structure at lower pH, likely linked to increased beta-sheet content.

Conclusions:

  • The simultaneous presence of Im and Mb spectral components strongly suggests the coexistence of multiple protein conformations in moPrPC.
  • The observed pH-dependent dynamics correlate with changes in secondary structure, particularly beta-sheet content.
  • The combined SDSL and SLE approach provides a powerful method for dissecting complex protein dynamics and conformational heterogeneity.

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