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Atomic Force

Microscopy of Biological Samples

P.L.T.M. Frederix, B.W. Hoogenboom, D. Fotiadis, D.J. Müller, and A. Engel Abstract The atomic force microscope (AFM) allows biomolecules to be observed and manipulated under native conditions. It produces images with an outstanding signal-to-noise ratio and addresses single molecules while the sample is in a buffer solution. Progress in sample preparation and instrumentation has led to topographs that reveal subnanometer details and the surface dynamics of biomolecules. Tethering single molecules between a support and a retracting AFM tip produces force–extension curves, giving information about the mechanical stability of secondary structural elements. For both imaging and force spectroscopy, the cantilever and its tip are critical: the mechanical properties of the cantilever dictate the force sensitivity and the scanning speed, whereas the tip shape determines the achievable lateral resolution. Keywords: atomic force microscopy, biological molecules, imaging, native membranes, protein dynamics, single-molecule force spectroscopy.

Introduction The number of resolved three-dimensional (3D) structures of biological molecules and complexes imaged at atomic resolution is increasing every year. These structures are obtained from large ensembles of molecules in the same configuration. Occasionally, it has been possible to arrest biomolecules in different configurations and consequently resolve the structure of the different states. However, it is still difficult to assess the dynamic changes in these nanomachines during their functional cycles. In order for researchers to observe biomolecules at work, the biomolecules must reside in a physiological environment in which they can carry out their cycles. For soluble proteins, this is a physiological buffer, while membrane proteins also need to be embedded in a lipid bilayer. The atomic force microscope (AFM)1 is still the only instrument that provides subnanometer spatial resolution and that can be operated with the sample in solution. This makes it an important characterization tool for biological samples, despite the limitation that it can only provide information on the structure and dynamics of the sample surface. Progress has been achieved by optimizing sample preparation2–5 and image MRS BULLETIN/JULY 2004

acquisition methods6,7 and by continuous development of the instrumentation8–11 (see sidebar article). Topographs of the surfaces of biomolecules acquired by AFM reveal the objects in their most native state. The high signalto-noise ratio provided by this technique allows submolecular features to be discerned on single biomolecules. Moreover, structural variations at their surfaces can be detected that correspond to conformational changes of the molecule at work. In addition, the AFM stylus is a nanotool that allows single molecules to be manipulated—for example, biomolecules can be tethered between the stylus and the support and unfolded as the stylus-to-support distance is increased. The forces and associated dis