Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature.

Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.