Atomic force microscope force spectroscopy
The ability to pull on or stretch individual molecules with subnanometer-scale precision provides a way to directly investigate the underlying physical properties of the biological molecules involved in life’s processes. This ability is realized when performing what are called “force spectroscopy” experiments (Figure 3.1), which can be used to reveal the structural and mechanical properties of those biological molecules. During these experiments, how much force is required to extend or deform an individual molecule to a specific length (Figure 3.1b) is recorded while that molecule is being mechanically stretched by an instrument such as an atomic force microscope (Figure 3.1a) or an “optical tweezer” apparatus. These measurements can be thought of as qualitatively similar to “stress-strain” measurements of macroscopic materials by tensometer, brought down to the level of single molecules. Very sensitive measurements of the “force versus extension” characteristics of biomolecules subjected to forces smaller than ~100 pN can be performed with an optical tweezer apparatus (Chapter 5), but force spectroscopy using an atomic force microscope (AFM) has a functional range of approximately 10–10,000 pN, making it ideal, for example, to characterize the forces that hold biological molecules in their functional threedimensional (3D) conformations; to identify internal molecular transitions that occur when a biomolecule is subjected to moderate or (biologically) large forces; or to determine the mechanical strength of binding or interactions between two biological molecules, all of which can occur across this range of forces. These experiments can reveal structural and physical details about biomolecules, which are largely hidden from other methods of biochemical characterization (Fisher et al. 2000; Sotomayor and Schulten 2007; Muller 2008; Oberhauser and Carrion-Vazquez 2008; Alegre-Cebollada et al. 2010; Noy 2011; Marszalek and Dufrene 2012; Puchner and Gaub 2012; Scholl et al. 2013a; Schuler and Hofmann 2013).
