Figure 5
CarD has the tendency to form higher order oligomers in solution. (a) MALDI-TOF based intact mass spectrum analysis of CarD (50 μM) shows the presence of monomeric (17.687 kDa), dimeric (35.521 kDa) and trimeric (53.400 kDa) species. (b) Chemical cross-linking experiments performed using BS3 demonstrates that CarD (50 μM) has a tendency to form dimers, and higher order oligomers in solution. Lane 1, CarD in absence of BS3; lane 2, and 3, CarD in presence of 30-fold and 50-fold molar excess of BS3 respectively. L denotes the molecular weight ladder used as a standard. (c) CarD samples (50 μM) were incubated at 4, 20, 30, 40, 50, and 60 °C for 30 min and resolved in the native-PAGE (10%). Native-PAGE analysis suggests the formation of higher order oligomers with increase in temperature. (d) SDS-PAGE analysis of the thermally induced higher order oligomers of Mtb CarD (50 μM). L denotes the molecular weight ladder. UH and H represent the unheated and heated protein samples of CarD, indicating the formation of SDS-resistant dimers and higher order oligomers in the gel. The un-cropped images of the gels shown in b–d are provided in Supplementary Fig. 7a–c.
