Measuring Protein Mechanics by Atomic Force Microscopy

INTRODUCTION

Proteins are frequently subjected to mechanical forces in vivo. Because these forces affect a wide range of biological activities, it is important to develop methods that directly investigate the mechanical properties of these molecules. Force spectroscopy of individual proteins (modular or single-domain) allows one to characterize their entropic elasticity under low stretching forces and to determine their persistence length by fitting the worm-like chain (WLC) model to the force-extension curve. At higher stretching forces, it is possible to mechanically unravel these proteins, study their mechanical strength, and examine their unfolding and refolding properties. This provides data on their rates of mechanical unfolding and refolding, as well as on the position of the transition state along the unfolding reaction coordinate, which is defined as the extension of the protein module along the direction of the applied force. Thus, single-protein force spectroscopy may generate a plethora of interesting data that cannot be obtained using traditional biophysical methods. The atomic force microscope (AFM) is an excellent force spectrometer for probing the mechanical properties (e.g., length and tension) of individual proteins. The following protocol describes the use of AFM for measurements of protein mechanics under the constant extension rate regime.