The Effect of Tethering on the Chemical Kinetics of Single Bonds
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The Effect of Tethering on the Chemical Kinetics of Single Bonds Melanie Nguyen-Duong, Karl-Wilhelm Koch1 and Rudolf Merkel ISG-4, Research Center Jülich, D-52425 Jülich, Germany 1 Biochemistry, Faculty V, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany
ABSTRACT
We investigated the force induced dissociation of single specific bonds between protein A and immunoglobulin G. In these experiments both proteins were tethered to microbeads. Piconewton forces were applied by means of an ultrasoft spring. Upon bond formation the thermal fluctuations of this soft spring were reduced. This resulted in a sizeable contribution to the free enthalpy of bonding. Including this effect we calculated the relation between the lifetime of bonds connecting both microbeads and the lifetime of the bond between free molecules. We found that the lifetime depended on the stiffness of the force probe. Moreover, the lifetime was reduced by almost two orders of magnitude. Both predictions were verified in our experiments.
INTRODUCTION
Biomolecular recognition and binding is of stunning selectivity, which is of paramount importance for living organisms. For example, specialized cell surface proteins detect and recognize specific motifs on molecules that come into contact with the cell. Some of these surface receptors sense invading pathogenic cells which triggers the immune response. Others recognize the native environment of the cell and mediate e.g. the adhesion of cells to other cells or to some physiological substrates. In many physiological processes like wound healing or phagocytosis such adhesive contacts must withstand mechanical forces. In most relevant cases, bioadhesion is caused by a multitude of different molecular species acting in parallel. This complicates the interpretation of measured yield strengths of living cells enormously. These complications are avoided in mechanical experiments on single molecular bonds which became possible in recent years [1-5]. In these studies on single bonds it became apparent that mechanical strength of adhesion and the influence of force on the chemical kinetics of single molecular bonds are intimately related. The dissociation rate depends exponentially on force. This surprising finding can be nevertheless straightforwardly understood from classical chemical kinetics as follows [6-9]. In all basic theoretical approaches to the dissociation of bimolecular complexes an exponential dependence of the dissociation rate, kr, on the activation energy is found:
⎛ ∆G ∗ ⎞ ⎟⎟ k r = α exp⎜⎜ − k T B ⎝ ⎠
(1)
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Here kB denotes Boltzmann's constant and T the temperature in absolute units. ∆G* is the free enthalpy of activation, see figure 1. Different theoretical approaches differ in the expression given for the constant α [8,9]. However, the exponential dominates the behavior in all cases.
Figure 1: Left: Scheme of Gibbs' free enthalpy, G, along the reaction path. Right: The action of an external mechanical force. In this case the free enthalpy (solid lin
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