Mechanical biochemistry of proteins one molecule at a time

Abstract

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work.

FIGURE 1. Pulling proteins with the AFM and effect of a mechanical force on the unfolding/folding reaction

A, schematic of the sequence of events during the stretching of a multimeric protein. Stretching the ends of the protein sequentially unfolds the domains, generating a saw-tooth pattern in the force-extension relationship (bottom) that reveals the mechanical properties of the protein (modified from Ref. 11). B, effect of a mechanical force on the free energy diagram of a protein that unfolds following a two-state model (folded (f) and unfolded (u)). The dashed gray curve represents the process in the absence of an applied force. An applied force (black dashed line) tilts the energy diagram of the process, decreasing the barrier to the transition state, ‡ (ΔG^‡(F) < ΔG₀^‡) (red curve). The application of force also lowers the energy of the unfolded state relative to that of the folded state (ΔG(F) < 0). The mechanical reaction coordinate is x (modified from Ref. 3).

FIGURE 2. Model system in protein nanomechanics: titin I27 domain

Upper, force-extension curve obtained after stretching an I27 polyprotein. The thin lines correspond to fits to the worm-like chain (WLC) model. Lower, mechanical architecture of the I27 module. The schematic representation of an I27 polyprotein shows patches of backbone hydrogen bonds in both zipper (dashed gray lines) and shear (AB, A′G; solid green lines) mechanical topologies.