Protein–protein unbinding induced by force: single-molecule studies

https://doi.org/10.1016/S0959-440X(03)00039-3Get rights and content

Abstract

Experiments in which two specifically interacting protein molecules are dissociated by external force have yielded new insights into mechanisms involved in cell adhesion, leukocyte rolling, regulation of integrin activity, antigen–antibody interactions and other protein-mediated reactions contingent upon molecular recognition. Another important aspect of force-induced protein–protein unbinding studies is the new information being gleaned about the thermodynamics and kinetics of bond rupture.

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:

  • of special interest

  • ••

    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.

References (63)

  • S. Chen et al.

    Selectin receptor-ligand bonds: formation limited by shear rate and dissociation governed by the Bell model

    Proc. Natl. Acad Sci. USA

    (2001)
  • Hanley W, McCarty O, Jadhav S, Tseng Y, Wirtz D, Konstantopoulos K: Single-molecule characterization of...
  • S. Uemura et al.

    Kinesin-microtubule binding depends on both nucleotide state and loading direction

    Proc. Natl. Acad Sci. USA

    (2002)
  • P.P. Lehenkari et al.

    Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy

    Biochem. Biophys. Res. Commun.

    (1999)
  • M. Benoit et al.

    Discrete interactions in cell adhesion measured by single-molecule force spectroscopy

    Nat. Cell Biol.

    (2000)
  • G. Kada et al.

    Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1)

    Ultramicroscopy

    (2001)
  • K. Kawaguchi et al.

    Equilibrium and transition between single- and double-headed binding of kinesin as revealed by single-molecule mechanics

    Biophys. J.

    (2003)
  • R. Merkel

    Force spectroscopy on single passive biomolecules and single biomolecular bonds

    Phys. Rep. Rev. Sect. Phys. Lett.

    (2001)
  • P. Hinterdorfer et al.

    Detection and characterization of single biomolecules at surfaces

    J. Biotechnol.

    (2001)
  • Howard J: Mechanics of Motor Proteins and the Cytoskeleton. Sunderland, Massachusets, USA: Sinauer Associates Inc;...
  • G.I. Bell

    Models for the specific adhesion of cells to cells

    Science

    (1978)
  • E. Evans

    Probing the relation between force-lifetime-and chemistry in single molecular bonds

    Annu. Rev. Biophys. Biomol. Struct.

    (2001)
  • D. Bartolo et al.

    Dynamic response of adhesion complexes: beyond the single-path picture

    Phys. Rev. E.

    (2002)
  • G. Hummer et al.

    Free energy reconstruction from nonequilibrium single-molecule pulling experiments

    Proc. Natl. Acad. Sci. USA

    (2001)
  • C. Jarzynski

    Nonequilibrium equality for free energy differences

    Phys. Rev. Lett.

    (1997)
  • J. Liphardt et al.

    Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski’s equality

    Science

    (2002)
  • B. Isralewitz et al.

    Steered molecular dynamics and mechanical functions of proteins

    Curr. Opin. Struct. Biol.

    (2001)
  • K.C. Chang et al.

    The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion

    Proc. Natl. Acad Sci. USA

    (2000)
  • B. Heymann et al.

    Dynamic force spectroscopy of molecular adhesion bonds

    Phys. Rev. Lett.

    (2000)
  • D.E. Hyre et al.

    Early mechanistic events in biotin dissociation from streptavidin

    Nat. Struct. Biol.

    (2002)
  • Chen AL, Moy VT: Single-molecule force measurements. In Atomic Force Microscopy in Cell Biology. Edited by Jena BP,...
  • Cited by (100)

    • An integrated model for bead-based immunoassays

      2020, Biosensors and Bioelectronics
    View all citing articles on Scopus
    View full text