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The presence of non-native helical structure in the unfolding of a beta-sheet protein MPT63

Amrita Kundu1, Sangeeta Kundu1, Krishnananda Chattopadhyay1

  • 1Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India.

Protein Science : a Publication of the Protein Society
|December 21, 2016
PubMed

Insights

Mycobacterium tuberculosis protein MPT63, a beta-sheet protein, unexpectedly forms helical structures during unfolding. This intermediate state is prone to aggregation, challenging native conformation assumptions.

Area of Science:

  • Protein folding and biophysics
  • Structural biology
  • Tuberculosis research

Background:

  • MPT63 is a major secreted protein from Mycobacterium tuberculosis.
  • It possesses immunogenic properties and is linked to virulence.
  • Native MPT63 is characterized as a beta-sandwich protein with minimal helical content.

Purpose of the Study:

  • To investigate the equilibrium unfolding transition of MPT63.
  • To characterize the structural properties of MPT63 intermediates during unfolding.
  • To explore the relationship between local interactions and native conformation during protein folding.

Main Methods:

  • Circular Dichroism (CD) spectroscopy (Far UV-CD)
  • Fourier-Transform Infrared (FTIR) spectroscopy
  • Fluorescence Correlation Spectroscopy (FCS)
  • Molecular Dynamics (MD) simulations
  • Site-directed mutagenesis (Gly25Ala mutant)

Main Results:

  • MPT63 exhibits a strong propensity for helix formation in early unfolding intermediates, despite its native beta-sheet structure.
  • Low-pH intermediates show enhanced helical content and significant structural contraction.
  • MD simulations confirm the presence of intermediates with increased helical characteristics and exposed hydrophobic surfaces, indicating aggregation propensity.
  • The Gly25Ala mutant further supports the hypothesis by increasing non-native helical propensity.

Conclusions:

  • The early folding pathway of MPT63 involves intermediates with non-native helical structures.
  • Local interactions during folding may not always align with the final native conformation.
  • These findings provide insights into the complex folding mechanisms of beta-sheet proteins and potential implications for M. tuberculosis virulence.
Keywords:
fluorescence correlation spectroscopymolecular dynamics simulationprotein aggregationprotein foldingsingle molecule fluorescence

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