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Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers

Sergei Khrapunov1, Michael Brenowitz

  • 1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA. khraps@einstein.yu.edu

Biophysical Chemistry
|May 25, 2011
PubMed

Insights

Mycobacterium tuberculosis MfpA protein stabilizes its structure through phenylalanine stacking, mimicking DNA to resist fluoroquinolone antibiotics. Its denaturation involves dimer dissociation and aggregation, guided by a double funnel energy landscape.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • MfpA from Mycobacterium tuberculosis is a pentapeptide repeat protein (PRP).
  • MfpA is hypothesized to confer fluoroquinolone resistance by mimicking DNA.
  • Understanding MfpA's stability and denaturation is crucial for antibiotic resistance research.

Purpose of the Study:

  • To investigate the structural basis of MfpA's DNA mimicry and stability.
  • To elucidate the denaturation and refolding mechanism of MfpA.
  • To explore the role of dimerization in MfpA's structural integrity.

Main Methods:

  • Application of the Lumry-Eyring model to spectral measures of MfpA denaturation.
  • Analysis of MfpA's secondary and tertiary structure under denaturing conditions.
  • Thermodynamic analysis of MfpA aggregation and refolding pathways.

Main Results:

  • Phenylalanine side chain stacking stabilizes the MfpA N-terminus, enhancing DNA mimicry.
  • MfpA denaturation involves dimer dissociation into monomers, followed by aggregation.
  • MfpA maintains significant secondary and tertiary structure under denaturation, with dimerization stabilizing its fold.

Conclusions:

  • MfpA's stability is attributed to its pentapeptide repeat fold, stabilized by dimerization and a high energy barrier to aggregation.
  • The denaturation-refolding pathway follows a 'double funnel' energy landscape, with limited in vitro refolding due to the high aggregation barrier.
  • These findings provide insights into bacterial drug resistance mechanisms and protein stability.

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