A Tale of a Tail - making Sense of the Shape of Rupture Force Distributions in Single Protein Dissociation Measurements

Abstract

Atomic force microscopy has been used extensively to measure dissociation in protein systems at the single molecule level. The basic theory, based on a thermal activation and the deformation of the barrier through the applied force, results in a probability distribution for the bond rupture force which is skewed to the left, i.e. towards lower applied force. However, the majority of published measurements show a distinct tail to the right, i.e. the force histograms are skewed towards high force. This “mystery tail” has been variously ignored, or assigned to either “hidden” multiple attachment events or heterogeneity in the bonding. Multiple attachments can be minimized by using double tethers and discarding any force curves that show too short a contour length for the tether (corresponding to non-specific binding), or clearly show multiple rupture events in the same force curve. However, even with these precautions, multiple ruptures can occur at the same location and appear as a single unbinding events. We have developed a methodology to estimate the frequency of such “hidden” multiple rupture events. Using this methodology, we have used the avidin-biotin model system, and prepared samples with varying densities of active sites. This way we controlled the probability of multiple bonding events. From these measurements we were able to determine the relative importance of hidden multiple rupture events versus bond heterogeneity in explaining the unexpected high force tail in rupture force histograms.