An experimental approach to probe conformational changes in proteinstructure using a biotin derivative followed by mass spectrometry
The biotinylation patterns of two soluble model proteins (BSA and CA II) were analyzed under different conditions. In a first step these proteins were biotinylated with an increasing molar excess of a biotin derivative. In this case increases in biotin concentration led to an increase in the number...
|PDF Full Text
No Tags, Be the first to tag this record!
|The biotinylation patterns of two soluble model proteins (BSA and CA II) were analyzed under different conditions. In a first step these proteins were biotinylated with an increasing molar excess of a biotin derivative. In this case increases in biotin concentration led to an increase in the number of biotinylated lysine residues until a point of saturation was reached. A further increase in biotin concentration did not result in additional lysine residues being biotinylated. The biotinylation pattern was reproducible and under any given conditions, only specific lysine residues are biotinylated.
After showing that the biotinylation of proteins is specific and reproducible, the same approach was used to map artificially induced conformation changes in BSA and CA II. These proteins were subjected to elevated temperatures that induce conformation changes. The biotinylation pattern of these proteins at room temperature and after exposure to 80°C was compared. For BSA, heating results in the biotinylation of two additional lysine residues. This is most likely due to conformation changes in the protein structure induced by the high temperature. In the case of CA II there was no difference in the biotinylation pattern of the protein at room temperature and at 80°C.Having established that biotinylation patterns can be used to reliably detect conformational changes in protein structure, the biotinylation pattern of Band III (a transmembrane protein of the human erythrocyte plasma membrane) in infected and noninfected erythrocyte was analyzed. These experiments confirmed previous observations that this protein undergoes a conformational change upon infection. The biotinylation pattern was different between RBC and IRBC in respect to a single lysine residue which is biotinylated only in RBC. These results suggest that, upon invasion by P. falciparum, the Band III protein undergoes conformation changes which are probably important for the survival of the parasite within the host cell.