Comparative biochemical analysis of three members of the Schistosoma mansoni TAL family: Differences in ion and drug binding properties.
Bottom Line: The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms.Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional.Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.
Affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK; Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK.Show MeSH
Mentions: SmTAL1 was partially protected from limited proteolysis by chymotrypsin by PZQ, CPZ, W7, TFP and, possibly, thiamylal (Fig. 3). Similar results were observed with subtilisin (Supplementary Figure S2). This method has been previously used with calmodulin-like proteins from F. hepatica demonstrating that, as expected, TFP and W7 interacted with FhCaM1 (which differs from human calmodulin by just two amino acid residues) [55,56]. The same drugs, except thiamylal, caused a significant (p < 0.05; unpaired t-test with Welch's correction) increase in the melting temperature of the protein compared to the DMSO control (Fig. 3; Table 1). These data suggest that the drugs interact with SmTAL1 increasing its stability towards both proteolysis and thermal denaturation as a consequence. Of the drugs tested, only W7 protected SmTAL2 from limited proteolysis by trypsin or chymotrypsin (Fig. 3; Supplementary Figure S2). As noted above, it was not possible to carry out DSF analysis on this protein. Both TFP and thiamylal protected SmTAL3 from proteolysis by trypsin (Fig. 3). CPZ, TFP and W7 (but not PZQ or thiamylal) caused a significant (p < 0.05; unpaired t-test with Welch's correction) decrease in the melting temperature of SmTAL3 (Fig. 3; Table 1). A decrease in the melting temperature can arise from the drug binding to partially folded states and thus destabilising the overall population of protein molecules . The situation with SmTAL3 is clearly more complex than with SmTAL1 where the results from limited proteolysis and DSF were broadly in agreement. However, the two techniques measure different consequences of ligand binding and a negative result does not provide evidence for a lack of a drug–protein interaction. Furthermore, it is possible for an interacting drug to show a positive result in only one of two experiments; indeed this has been observed previously with FhCaBP3 using limited proteolysis and fluorescence quenching . While Tm values report on the protein's overall stability, proteolytic digestion reports on events in the immediate vicinity of the protease cleavage site. Therefore, it is not surprising to observe that TFP causes loss of overall stability as measured by DSF, while locally rigidifying the polypeptide backbone (or sterically hindering access to the protease) in limited proteolysis experiments.
Affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK; Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK.