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

Sergei Khrapunov, Huiyong Cheng, Subray Hegde, John S. Blanchard, Michael D. Brenowitz

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose β-strands and turns within the right-handed quadrilateral β-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.

Original languageEnglish (US)
Pages (from-to)36290-36299
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number52
DOIs
StatePublished - Dec 26 2008

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Mycobacterium tuberculosis
Microbial Drug Resistance
Proteins
DNA Gyrase
Protein Denaturation
Fluorescence Spectrometry
Fluoroquinolones
Anti-Bacterial Agents
Centrifugation
Denaturation
Dialysis
Fluorescence spectroscopy
Urea
Dilution
Monomers
DNA

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA. / Khrapunov, Sergei; Cheng, Huiyong; Hegde, Subray; Blanchard, John S.; Brenowitz, Michael D.

In: Journal of Biological Chemistry, Vol. 283, No. 52, 26.12.2008, p. 36290-36299.

Research output: Contribution to journalArticle

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