Structure and Folding Thermodynamics of MfpA, a Pentapeptide Repeat Protein From mycobacterium Tuberculosis

Abstract

The Mycobacterium tuberculosis protein MfpA confers bacterial resistance to the antibiotic fluoroquinolone. MfpA is a dimer in solution and in the crystal. The C-terminal α helices of two monomers form the dimer interface. The shape and distribution of the negative charge on the surface of MfpA mimics those of DNA (1). We quantitatively explored the unfolding, refolding and aggregation of MfpA as a function of temperature, urea concentration and the anionic surfactant SDS by circular dichroism (CD) and intrinsic fluorescence. These analyses reveal a structural transition followed by aggregation of intermediate conformers; the intramolecular and intermolecular interactions occur almost simultaneously. Stacking of phenylalanine side chains stabilize the N-terminal portion of MfpA's pentapeptide thus expanding on the motif of DNA mimicry. The high Arrhenius activation energy of aggregate formation rationalizes the nature of the kinetic trap shown earlier (2) that facilitates aggregate formation. Although secondary structure contents can not be calculated accurately for α/β proteins from their CD spectra (3), the increased α-helical content and a long-wavelength shift of the fluorescence emission maximum show intramolecular secondary and tertiary structure changes along the structural transition of MfpA. Overall, the unfolding and refolding of MfpA in solution is described by the ‘double funnel’ energy landscape where the ‘native’ and ‘aggregation’ funnels are separated by the high kinetic energy barrier that is not overcome during in vitro refolding.[1] S. Hegde, M. Vetting, S. Roderick, L. Mitchenall, A. Maxwell, H. Takiff and J. Blanchard, Science 308, 1480-1483 (2005).[2] S. Khrapunov, H. Cheng, S. Hegde, J. Blanchard, and M. Brenowitz, J. Biol. Chem. 283, 36290-36299 (2008).[3] S. Khrapunov, Anal. Biochem. 389, 174-176 (2009)