Measuring Compressional Resistance in Large Surface Molecules

Abstract

A class of receptors on leukocytes, termed the non-catalytic tyrosine phosphorylated receptors (NTRs), play important roles in the interactions of these cells with their environment, including between lymphocytes and antigen-presenting cells. These receptors share common features, including ligands that are attached to external surfaces, and short ectodomains (i.e., the cell-external domain) compared to large surface molecules (LSM) that regulate their function. This size difference is hypothesized to play a functional role - depending on the compressional resistance of LSMs, it could lead to emergent properties such as pressure on the NTR-ligand bond, segregation of large and small surface molecules, and coalescence of comparably-sized NTR binding partners. Although much effort has focussed on determining the flexibility of lipid bilayers, the biophysical properties of surface proteins remain poorly understood.We have developed an in situ method based on Forster resonance energy transfer (FRET) and Bayesian statistics to elucidate the mechanical properties of surface protein ectodomains. Using this method we investigated CD148, an abundantly expressed and functionally important LSM. Our initial results show that its ectodomain is stiff enough to put pressure on nearby NTR-ligand complexes and lead to segregation at cell-cell interfaces, but not stiff enough to preclude binding. These results support a functional role for LSM ectodomain mechanics.The modular nature of our FRET assay will allow it to be applied to other surface molecules. The long-term goal of this work is to manipulate structural aspects of relatively well characterised ectodomains and establish a method to relate their structural elements to biophysical properties.