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Molecular Features and Properties of Mycobacterial Proteins Linked to Tuberculosis Pathogenesis

By Dariush Ilghari

Abstract

The Mycobacterium tuberculosis genome codes for 11 pairs of CFP-10/ESAT-6 proteins (Esx family) as well as the apparatus required for secretion of these proteins. The core machinery for the secretion of ESAT-6 and CFP-10 is encoded by their surrounding genes. Recent studies also identified a distant region, the Rv3612c-Rv3616c operon, which is essential for the CFP-10/ESAT-6 secretion. Constructs carrying Rv3613c, Rv3614c, Rv3615c and Rv3616c coding regions were produced and used to express the corresponding proteins. However, only Rv3614c and Rv3615c were expressed using an E. coli-based expression system. Analysis using a range of spectroscopic techniques on the purified proteins revealed that both Rv3615c and Rv3614c contain stable secondary structure, but little if any stable tertiary structure and exist in a molten globule-like state. This suggests the proteins probably undergo folding upon binding with possible functional partners. Yeast-two hybrid studies showed no intermolecular interaction between the proteins encoded by the Rv3616c-Rv3612c operon, perhaps suggesting the formation of a higher order multi-protein complex. \ud Together with CFP-10 and ESAT-6, Rv0287 and Rv0288 are the members of the Esx family which are clearly implicated in M. tuberculosis pathogenesis. The expression vectors carrying Rv0287 and Rv0288 coding regions were constructed and used to express the proteins. Analysis using a range of spectroscopic techniques on the purified proteins showed that Rv0288 contains up to 30 % helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, Rv0287 has been found to form an unstructured, random coil polypeptide. The work reported here also shows that Rv0287 and Rv0288 form a tight 1:1 complex which is predominantly helical. Furthermore, the Rv0287-Rv0288 complex was found to be significantly more stable to thermal denaturation than CFP-10-ESAT-6. The high resolution solution structure reported here reveals that both proteins, Rv0287 and Rv0288, adopt an elongated helix-turn-helix hairpin structure in which the proteins lie antiparallel to each other, forming a stable four helix bundle. Comparison of the CFP-10-ESAT-6 and Rv0287-Rv0288 complexes also revealed that the overall backbone fold for the complexes is very similar although they display significantly different surface features

Publisher: University of Leicester
Year: 2009
OAI identifier: oai:lra.le.ac.uk:2381/9543

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