Protein–protein unbinding induced by force: single-molecule studies
Introduction
Protein–protein unbinding studies are a part of the major field of investigation termed receptor–ligand interactions or molecular recognition. During the past decade, important new information about receptor–ligand interactions at the single-molecule level has complemented conclusions based on conventional methods, which measure the properties of large ensembles of molecules or observe the behavior of whole cells. The main advantages of studying individual receptor–ligand pairs are: minimized cooperative and/or clustering effects; the possibility of probing conformational transitions of individual molecules, such as activation/inactivation; revealing the structural and functional heterogeneity of seemingly identical molecules; knowing the number of molecules involved in reactions; quantifying directly the magnitudes and working distances of forces in ligand–receptor interactions to elucidate the relationships between molecular structure and the thermodynamics of bond dissociation. Watching individual events and distributions rather than observing average values may reveal rare but physiologically important functional fluctuations 1.•, 2..
The study of protein–protein interactions has been dominated by a static viewpoint, such that the emphasis is on molecules in solution under equilibrium conditions, whereas their real-life biological interactions generally occur on surfaces under nonequilibrium conditions; the latter is the focus of the papers summarized in this review. The study of the mechanics of protein interactions is necessary to understand the many cellular functions and properties, such as rolling, motility, adhesion, deformability and so on, that are mediated by specific receptor and ligand molecules, and controlled by mechanical forces produced by either external (shear flow) or internal (cytoskeletal rearrangement, motor proteins) sources. In addition, the study of protein–protein unbinding by an applied force has turned out to be a precise and unique tool for analyzing protein structure and function, as well as mechanisms of their regulatory changes [3].
Section snippets
Theory
The results of mechanical rupture (pulling) experiments have been analyzed using two distinct theoretical methods [4]. The first, called the energy landscape model, is based on Kramers’ rate theory and leads to general predictions about the distribution of rupture forces [5•]. In this model, it is assumed that an applied load changes the energy of the transition state as well as the equilibrium of the bound and unbound states, thereby altering the kinetics of association and dissociation [6].
Principles and methodology
Force-induced receptor–ligand unbinding studies are always performed at an interface. Molecular binding and rupture result from controlled touching and separation of two surfaces, one bearing receptors and another coated with ligand. The different techniques used to perform these kinds of experiments differ from each other mainly by the surfaces to which the proteins are bound, as well as by the methods of generating, sensing and measuring mechanical forces. The techniques used during the past
Applications
The strength of cell attachment to substrata and/or to another cell is a good example of how the mechanical characteristics of single molecules determine cell function. That is why integrins, selectins and cadherins, which mediate cellular interactions, were among the first proteins studied at the single-molecule level using force-induced unbinding methodology. Cell adhesion and aggregation are strongly influenced by the mechanical plasticity of cells, by the direction and rate of applied
Conclusions and perspectives
Most of the results of those experiments listed in Table 1 that were carefully designed and executed show that rupture forces for adhesion proteins are characteristic of each ligand–receptor pair. The usual range for typical proteins appears to be ∼100 pN +/− 50 pN, but there may be exceptions. Although loading rates under most physiological conditions have not been determined, it is likely that these forces were measured with loading rates that cells might really experience, for example in
Update
Recently, Kawaguchi et al. [63••] have used unbinding force distribution studies to reveal more mechanistic details of kinesin motility.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
We thank James Torbet for critical comments on the manuscript. Our research was supported by grants HLBI 57407, 30954, NIAMS AR45990 from the National Institutes of Health, and NSF BIR95-12,950. We thank Joel S Bennett for his role in much of this research.
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