Conformational Changes in Surface-Immobilized Proteins Measured Using Combined Atomic Force and Fluorescence Microscopy.

Abstract

Biological organisms rely on proteins to perform the majority of their functions. Most protein functions are based on their physical motions (conformational changes), which can be described as transitions between different conformational states in a multidimensional free-energy landscape. A comprehensive understanding of this free-energy landscape is therefore of paramount importance for understanding the biological functions of proteins. Protein dynamics includes both equilibrium and nonequilibrium motions, which typically exhibit a wide range of characteristic length and time scales. The relative probabilities of various conformational states in the energy landscape, the energy barriers between them, their dependence on external parameters such as force and temperature, and their connection to the protein function remain largely unknown for most proteins. In this paper, we present a multimolecule approach in which the proteins are immobilized at well-defined locations on Au substrates using an atomic force microscope (AFM)-based patterning method called nanografting. This method enables precise control over the protein location and orientation on the substrate, as well as the creation of biologically active protein ensembles that self-assemble into well-defined nanoscale regions (protein patches) on the gold substrate. We performed AFM-force compression and fluorescence experiments on these protein patches and measured the fundamental dynamical parameters such as protein stiffness, elastic modulus, and transition energies between distinct conformational states. Our results provide new insights into the processes that govern protein dynamics and its connection to protein function.