Mechanical chaperonin : Mechanical folding of proteins at the single molecular level

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The purpose of the study is to use the atomic force microscope (AFM) to extend a single protein molecule after sandwiching it between the crystalline substrate and the tip of the AFM. We chose carbonic dehydratase as a model enzyme and derivatized it so that it had cysteine residues at its N- and C-termini. Extending the protein using the AFM revealed that the protein was pulled to about 20 nm and further application of the extending force abruptly broke the 3D structure of the protein. After the rupture of the 3D structure, the protein behaved as a randomly coiled chain. The process may be regarded as a mechanical denaturation of a protein molecule. Retraction of the distance between the tip and substrate relaxed the protein to resume certain structural elements which may or may not be parts of the native 3D structure. The time allowance in this experiment was in the order of 100 ms and we are extending the time to 10 s so that more structure formation should be observed. A mutant protein that would not form the "knot" structure when pulled from the two ends revealed that the knot formation was indeed a factor to characterize the mechanical behavior of the wild type enzyme. We also conducted experiments on a synthetic polypeptide that forms alpha-helix at a lower pH.