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Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA

Sergei Khrapunov1, Huiyong Cheng, Subray Hegde

  • 1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

The Journal of Biological Chemistry
|November 4, 2008
PubMed

Insights

Mycobacterium tuberculosis MfpA protein, a pentapeptide repeat protein (PRP), confers antibiotic resistance. A novel time-dependent renaturation method successfully refolds MfpA into its native structure, unlike standard protocols.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Pentapeptide repeat proteins (PRPs) are a newly identified protein fold.
  • Mycobacterium tuberculosis MfpA is a PRP that confers fluoroquinolone antibiotic resistance by inhibiting DNA gyrase.
  • MfpA's structure mimics duplex DNA, suggesting a role in topoisomerase regulation.

Purpose of the Study:

  • To investigate the solution structure and refolding of MfpA.
  • To understand the biophysical properties of the pentapeptide repeat fold.
  • To explore MfpA's role in antibiotic resistance and topoisomerase activity.

Main Methods:

  • Fluorescence spectroscopy
  • Circular Dichroism (CD) spectroscopy
  • Analytical centrifugation
  • Protein refolding studies (urea, guanidinium)

Main Results:

  • A unique CD spectrum characteristic of the pentapeptide repeat fold was identified.
  • Standard refolding methods (dialysis, dilution) resulted in non-native MfpA aggregates.
  • A novel "time-dependent renaturation" protocol successfully yielded MfpA with native secondary, tertiary, and quaternary structure.

Conclusions:

  • The pentapeptide repeat fold possesses unique secondary structure elements.
  • MfpA refolding requires a specific time-dependent protocol for achieving native structure.
  • This study provides insights into PRP structure, function, and potential for antibiotic resistance mechanisms.

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