A novel single-molecule study to determine protein-protein association constants

Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfu lly used the imaging capabilities of the AFM to determine protein-protein a ssociation constants. We have developed a method to measure the molecular w eight of a protein based on its volume determined from AFM images. Our volu me determination method allows for rapid, accurate analysis of large protei n populations. On the basis of the measured volume, the fraction of monomer s as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K-d) for the helicase was calculated. We determined a K-d for Uvr D of 1.4 muM, which is in good agreement with published K-d data obtained f rom analytical ultracentrifugation (AUC) studies. Our method provides a rap id method for determining protein-protein association constants.