A COMBINED IN VITRO AND IN SILICO APPROACH TO ESTIMATE THE MOLECULAR ARRANGEMENT WITHIN A FIBRONECTIN FIBER

摘要

It has become apparent that the extracellular matrix (ECM) is a powerful modulator of cell behavior. Fibronectin (Fn) is of particular interest because it is a requisite cell adhesion molecule for development and wound healing and it is a promiscuous binding partner for many soluble signaling molecules. It was recently shown that the binding affinity of some molecules is dependent on the strain state of the Fn [2,3], reinvigorating our interest in the molecular mechanism of Fn fiber extension. The tertiary structure of the approximately 30 Fn type III domains of the protein has been shown to be capable of unfolding in single molecule force spectroscopy experiments, although evidence that unfolding occurs in Fn fibers has been indirect and has not been quantified. Nevertheless, unfolding of Fn molecules predicts a possible mechanism of strain dependant binding in Fn matrix and commensurate strain feedback to attached cells, contributing to the cellular mechanotransduction toolbox [3]. In order to address these questions a technique was developed to quantify FnIII module unfolding within Fn fibers. The strain dependent unfolding of FnIII modules was measured on a per molecule basis using a cysteine shotgun type labeling approach with a novel calibration technique. This was contrasted with a simulation of Fn fibers based on experimentally determined molecular properties.