Nanomechanics of Biomaterials using Atomic Force Microscope

Abstract

Understanding mechanical properties of biological elements such as cells, proteins, and deoxyribonucleic acid (DNA) is very important in order to understand their function. Various techniques such as nanoindentation, Optical Tweezers (OT), Magnetic Tweezers (MT) and Atomic Force Microscope (AFM) are widely used for studying the mechanical properties of biomaterials. AFM has been used previously for imaging of cells, bacteria and proteins as well as for protein-protein interaction, antigen-antibody interaction, ligand-receptor interaction and protein-drug interaction. Protein-drug interaction is very well studied using fluorescence, Ultraviolet-Visible (UV-Vis) absorption, and circular dichroism techniques. We have studied interaction of Chloramphenicol with titin I27 protein using Fluorescence and AFM. It has been found out from the fluorescence study that the drug binds to the protein resulting in the formation of protein-drug complex. Alteration in mechanical properties of proteins at different drug concentrations is studied using AFM. AFM data shows that the drug binds to the protein at 40 µM of drug concentration thereby resulting in an increment of unfolding force (by 25 pN) and decrease in persistence length. Therefore, the drug is mechanically stabilizing the protein. However, chemical stability of the protein is checked by equilibrium denaturation experiment. The denaturation experiment shows the increase in free energy of stabilization for the protein-drug complex with respect to protein only. Therefore, the drug stabilizes the protein mechanically and chemically.

In another study, mechanical properties of mouse embryonic stem cells (mESCs) is also studied. Previously, it was shown that loss of clathrin heavy chain results in loss of clathrin mediated endocytosis (CME) and thereby shows loss of pluripotency. However, the mechanical properties on loss of clathrin heavy chain was not known. The results shows that the mESCs lacking clathrin shows greater cellular stiffness in comparison to wild type cells. Also, treatment of mESCs with actin depolymerizing agents shows similar values of cellular stiffness as that of wild type cells.