Non Linear Effects of Macromolecular Crowding on the Mechanical Unfolding of Proteins

Abstract

Macromolecules can occupy a large fraction of the volume of the cell and this crowded environment affects many properties of the proteins inside the cell, such as unfolding forces, thermal stability and rates of folding. Although much is known about molecular crowding, it is not well understood how the relative size of the crowder affects the resistance of a protein to mechanical stress.An atomic force microscope-based single molecule method was used to measure the effects of crowding on the mechanical stability of I-27. Crowding was provided by the addition of Dextran of three different molecular weights 6kDa, 10 kDa and 40 kDa, with concentrations varying from zero to 300 g/L in a pH neutral buffer solution at room temperature. The forces required to unfold biomolecules were found to increase when a high concentration of crowder molecules was added to the buffer solution and the force required to unfold a domain reached a maximum value when the crowder size was 10 kDa, a size comparable to that of the protein. To model the effect of Dextran on the energy barrier Ogston's Theory as well as Scaled Particle Theory were employed, neither of which was completely satisfactory at describing the results. We hypothesize that the composition of Dextran may play a role in the deviation of the predicted behavior with respect to the experimental data.