Specific interactions between complementary macromolecules play a critical role in most biological processes, from signal transduction, to gene regulation and motor activities. Although such interactions have been studied with many biochemical and biophysical methods, a direct examination with single molecular pairs was only realized recently with the advent of the atomic force microscopy (AFM). However, our ability to study intermolecular interactions or recognition at great detail is still severely limited at present, because with the AFM technique, only a single rupture force, which is related to the maximum binding force, can be directly measured, due to the lack of control of the cantilever position and movement. The relationship of such measurements to those measured thermodynamically can only be inferred and is often far from clear. It has been recognized that this limitation may be technical in nature and might be eliminated with a properly designed apparatus. The primary objective of this project is to develop a force measuring apparatus based on the AFM, but incorporating a magnetic force to the force sensing cantilever, in order to control its position and motion. In this system, the cantilever should remain during the approach/retraction cycle of the measurement, allowing the determination of both the interacting force and the distance between the two molecules. Because the position of the cantilever does not move during the entire measurement, other problems due to the lateral swing of the cantilever which has the potential of introducing artifacts are also eliminated. To further improve the precision in position, the thermal motion of the cantilever will be compensated with a fast response coil, branched from the main feedback. When this technique is fully implemented, the entire force- distance curve of a molecular pair can be measured at sub-A intervals under physiolo gical conditions. A simple integration will yield the potential curve that can be directly compared with the parameters obtained with thermodynamic measurements, such as the binding free energy. This will provide unprecedented details of intermolecular interaction, which will be invaluable for our understanding of this important process, especially when carefully selected mutant proteins are studied in comparison.
