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). This chapter aims at giving protocol-like instructions for people who are actually getting started using AFM to study mechanical protein unfolding or refolding. The instruction includes different approaches to produce polyproteins or modular protein chains which are commonly used to screen for true single-molecule AFM data traces. Also, the basic principles for data collection with AFM and the basic data analysis methods are explained. For people who want to study proteins that unfold at small forces or for people who want to study protein folding which also occurs typically at small forces (<30 pN), an averaging technique is explained, allowing to increase the force resolution in this regime. For topics that would go beyond the scope of this chapter - as, for example, the details about different cantilever calibration methods - references are provided.