Abstract
Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique—constant-deflection AFM—in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.