Protein Stretching II*1: Results for Carbonic Anhydrase

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Abstract
The force curve measurement mode of an atomic force microscope was used to record the force required to stretch a protein molecule that was covalently sandwiched through gold-thiol bonds between a mica substrate and a silicon nitride tip, both coated with gold. In one experiment, 8 ±1 out of 20 lysyl residues of bovine carbonic anhydrase B were randomly derivatized to give free thiols, and grafted on an atomically flat gold (111) surface on mica. Force curves taken on the surface covered with protein molecules using a gold coated tip occasionally showed a large downward deflection indicating trapping and subsequent stretching of protein molecules between the tip and the substrate. In another experiment, the same protein was genetically engineered so that cysteine residues were introduced at both the amino and the carboxyl terminus. Force curves taken in a similar manner as in the first experiment indicated almost complete extension of a linear polypeptide chain. The result was explained in terms of extension of a pseudo-three-dimensional gel in both cases, with additional stretching of a linear chain in the second case.