Single-Molecule Protein Unfolding and Refolding Using Atomic Force Microscopy

Over the past few years, atomic force microscopy (AFM) became a prominent tool to study the mechanical properties of proteins and protein interactions on a single-molecule level. AFM together with other mechanical, single-molecule manipulating techniques (Bustamante et al., Nat Rev Mol Cell Biol 1:130–136, 2000) made it possible to probe biological molecules in a way that is complementary to single-molecule methods using chemicals or temperature as a denaturant (Borgia et al., Annu Rev Biochem 77:101–125, 2008). For example, AFM offered new insights into the process of protein folding and unfolding by probing single proteins with mechanical forces. Since many proteins fulfill mechanical function or are exerted to mechanical forces in their natural environment, AFM allows to target physiologically relevant questions. Although the number of proteins unfolded with AFM continually increases (Linke and Grutzner, Pflugers Arch 456:101–115, 2008; Zhuang and Rief, Curr Opin Struct Biol 13:88–97, 2003; Clausen-Schaumann et al., Curr Opin Chem Biol 4:524–530, 2000; Rounsevell et al., Methods 34:100–111, 2004), the total number of proteins studied so far is still relatively small (Oberhauser and Carrion-Vazquez, J Biol Chem 283:6617–6621, 2008).