Solution Structure and Refolding of the Mycobacterium tuberculosis Pentapeptide Repeat Protein MfpA
| Autor: | Subray S. Hegde, John S. Blanchard, Sergei Khrapunov, Huiyong Cheng, Michael Brenowitz |
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| Rok vydání: | 2008 |
| Předmět: |
Protein Denaturation
Protein Folding Protein Conformation Molecular Sequence Data Biology Biochemistry Pentapeptide repeat DNA gyrase Protein Structure Secondary Protein structure Bacterial Proteins Urea Denaturation (biochemistry) Amino Acid Sequence Molecular Biology Protein secondary structure Guanidine Monomeric GTP-Binding Proteins Sequence Homology Amino Acid Circular Dichroism Drug Resistance Microbial Mycobacterium tuberculosis Cell Biology Protein tertiary structure Protein Structure Tertiary Spectrometry Fluorescence DNA Topoisomerases Type I Protein Structure and Folding Protein quaternary structure Protein folding |
| Zdroj: | Journal of Biological Chemistry. 283:36290-36299 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m804702200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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