Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA.
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 beta-strands and turns within the right-handed quadrilateral beta-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.
Khrapunov
Sergei
S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Cheng
Huiyong
H
Hegde
Subray
S
Blanchard
John
J
Brenowitz
Michael
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004352:Drug Resistance, Microbial; D019791:Guanidine / Q000737:chemistry; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D014508:Urea; C445685:MfpA protein, Mycobacterium smegmatis; D020559:Monomeric GTP-Binding Proteins; D004264:DNA Topoisomerases, Type I; D019791:Guanidine