Single molecule force spectroscopy using polyproteins

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. In this tutorial review we introduce the reader to single molecule force spectroscopy using the atomic force microscope (AFM), and explain the two main modes of operation of the AFM for force spectroscopy: force-extension and force-clamp. We introduce the approach of using polyproteins to obtain a clear mechanical fingerprint for monitoring the response of proteins to an applied mechanical force. In addition, we provide an informative and representative review of recent research on proteins using single molecule force spectroscopy. We focus on areas which have made a significant contribution to the single molecule protein folding field and highlight emerging areas of research which have wider implications for the general scientific community.