Advances in atomic force microscopy for the study of biological systems.

摘要

This thesis deals with the development of novel techniques for the improved study of biological systems using atomic force microscopy (AFM). Two separate areas are covered in this work: (i) solution nanomanipulation and (ii) the use of AFM to measure protein-protein interactions. The nanoManipulator combines AFM with a computer assisted interface to allow manipulations of nanoscopic objects. Solution nanomanipulation of biological macromolecules is limited by the poor reliability of the imaging technique. Solution imaging using resonant techniques for AFM is greatly simplified by driving the cantilever directly. I have taken advantage of the temperature-sensitive bending properties of metal-coated silicon nitride cantilevers to induce vibrations in the lever using a modulated laser. The capabilities of this novel technique are demonstrated by the manipulation of DNA on a mica surface under solution.; The second area of research deals with the use of AFM as a tool to study protein-protein interactions. I have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. I have developed a method to determine the molecular weight of a protein based on its volume when the protein is imaged with the AFM. Based on the measured volume, the fraction of monomers as dimers was determined for DNA helicase UvrD, and the dissociation constant (K_d) for the helicase was calculated. I determined a K_d for UvrD of 1.4 μM, which is in good agreement with published K_d data obtained from analytical ultracentrifugation (AUC) studies.; The volume analysis method can also be used to study the assembly state of proteins that cannot be studied using other techniques. Techniques such as AUC require high concentration of protein. Often times the protein does not remain soluble at these higher concentrations. The volume analysis method described in this thesis allows for much lower protein concentrations to be used. I present preliminary K_d data for UvrD in solution conditions which until now were unknown.