Protein stretching V: two forms of carbonic dehydratase detected by force microscopy

Abstract.

Two conformers of the human carbonic anhydrase II (HCA II) counterpart, bovine carbonic anhydrase II (CAB), which has 259 amino acid residues, were detected using the force mode of an atomic force microscope (AFM). Genetically engineered CAB (named CAB253Cys) was prepared by adding a cysteine residue at its N-terminus as well as replacing Glu253 with Cys to sandwich the protein molecule between silanized surfaces of silicon wafer and functionalized tip in the AFM. The genetically inserted Cys253 was located upstream of the protein’s previously established knot structure, such that the protein could be stretched without forming the “knot”. Two distinct types of force-extension curves were observed when the protein was stretched using the force-extension mode of the AFM. One represents a native-like state and the other a rather relaxed conformation relative to the native one. The relaxed CAB conformation was not affected by the addition of enzyme’s specific inhibitor p-aminomethylbenzenesulfonamide, indicating that, in this state, the protein lacks its active site. As the AFM was able to identify two newly found conformers of CAB by mechanical means, results of this study indicate that AFM is a useful means to describe multiple competent conformations for a variety of biologically important proteins.