|
|
|
 Force Probe Molecular Dynamics |
|
|
|
|
Simulation of Atomic Force Microscope Rupture Experiments
|
|
Helmut Grubmüller and Berthold Heymann
The force required to rupture the streptavidin-biotin complex has been calculated by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. The simulations suggest a multiple-pathway rupture mechanism, which involves five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute although conformational motions are observed.
This simulation study [1,2] aimed at a microscopic interpretation of single molecule atomic force microscope (AFM) experiments carried out by Herman Gaub and co-workers at the University of Munich, in which unbinding forces between individual protein-ligand complexes have been measured. In particular we asked, what interatomic interactions cause the experimentally observed unbinding forces.
|
| |
|
The top of the figure sketches a typical AFM rupture experiment: receptor molecules are fixed via linker molecules to a surface (left); in the same way, ligand molecules are connected to the AFM cantilever (right). When pulling the cantilever towards the right, the pulling force applied to the ligand can be measured. At the point of rupture of the ligand-receptor complex the measured force abruptly drops to zero so that the rupture force can be measured.
The bottom of the figure sketches our computer experiment: in the course of an molecular dynamics (MD) simulation of the ligand-receptor complex the ligand is pulled towards the right with a `computer spring', while the receptor (drawn as a ribbon model, but simulated at atomic detail) is kept in place. From the elongation of the `spring' the pulling force during the unbinding process is computed, and, thereby, a `force profile' is obtained. The rupture force is interpreted as the maximum of this force.
Both the AFM rupture experiments as well as our simulation studies focussed on the streptavidin-biotin complex as a model system for specific ligand binding. Streptavidin is a particularly well-studied protein and binds its ligand biotin with high affinity and specificity.
|
| |
|
One of the results of an MD rupture simulation is the pulling force as a function of time or cantilever position. From this profile, a rupture force was computed, which, after correction for the difference in pulling velocity between the AFM experiment and our simulation, compared well with the AFM experimental value. In our simulation, the force profile displayed a multitude of relative force maxima mirroring the complexity of the energy landscape traversed by the biotin on its way out of the binding pocket. We attributed the peaks of this force profile to the rupture and formation of individual hydrogen bonds and water bridges shown in the snapshots of the figure to the right, which characterize the main steps of the rupture process.
Two unexpected features of biotin unbinding are predicted by our simulations: One is that the rupture of the initially very strong hydrogen bonds between the ligand and the residues of the binding pocket does not entail immediate unbinding (first snapshot). Rather, the complex is stabilized by a transient network of water bridges and other transient hydrogen bonds, which form during the unbinding process (snapshots 2 and 3). Only after subsequent rupture of these hydrogen bonds the maximum force - the rupture force - is reached, and the biotin rapidly moves out of the entry of the binding pocket (snapshots 4).
As another feature we observed, towards the end of the unbinding process, a second force maximum, which we attribute to a strong transient hydrogen bond and several water bridges between biotin and the entry of the binding pocket (snapshot 5). Crossing of that second barrier, which cannot yet be resolved in the AFM experiment, completes the unbinding process.
In summary, our simulations provided detailed insight into the complex mechanisms of streptavidin-biotin rupture. They attribute the binding force to a network of hydrogen bonds between the ligand and the binding pocket and show that water bridges substantially enhance the stability of the complex. Good agreement with experimental results was obtained. Further `force simulations' of various systems, e.g., an antigen-antibody complex, are in progress.
|
| |
|
Whereas previous experiments and simulation studies referred only to bound/unbound states and the associated kinetics, the recent AFM rupture experiments have provided a new and complementary perspective on ligand binding by focussing at atomic details of binding/unbinding pathways: The former were described in terms of binding free energies as thermodynamic quantities, which are independent of the particular reaction pathway; the latter relate to forces, which actually depend on details of the unbinding reaction path and, therefore, can provide new insights into these details.
To enable an atomic interpretation of the AFM experiments, we have developed a molecular dynamics technique to simulate these experiments [1]. From such `force simulations' rupture models at atomic resolution were derived and checked by comparisons of the computed rupture forces with the experimental ones. In order to facilitate such checks, the simulations have been set up to resemble the AFM experiment in as many details as possible.
|
| |
|
MPG-movies
|
|
Below you'll find two movies showing part of a 1 nanosecond molecular dynamics simulation of the rupture process. Three numbers are shown at the bottom of each of the movies, indicating the elapsed time in picoseconds (left side), the distance in angstrom the pulling `spring' has traversed (middle), and the actual force in piconewton measured with the `spring' using Hooke's law (right side).
The frames were drawn with MolScript v1.4 (P.J. Kraulis, J.Appl.Cryst (1991) 24, 946-950) and Raster3D (Bacon and Anderson (1988) J.Molec.Graphics 6, 219-220, Merritt and Murphy (1994) Acta Cryst. D50, 869-873).
To trade off quality and file size, four mpeg-files are available for each of the two movies, which differ in resolution and sequence length.
|
| |
|
A global view of the rupture process:
The protein streptavidin is sketched with red ribbons; the ligand biotin is drawn as a yellow ball-and-stick model. The biotin is pulled towards the right using a harmonic potential (symbolized by a spring).
|
| |
|
A detailed view of the binding pocket:
Only the ligand and the residues involved in ligand binding are shown.
As above, biotin is pulled towards the right.
|
| |
|
Another detailed view of the binding pocket:
Streptavidin and biotin are now drawn as a space-filling model.
Biotin is pulled towards the right.
|
| |
|
References:
|
|
- H. Grubmüller, B. Heymann, and P. Tavan. Science 271:997-999 (1996)
[pdf]
- M. Eichinger, B. Heymann, H. Heller, H. Grubmüller, and P. Tavan. Conformational dynamics simulations of proteins. In P. Deuflhard, J. Hermans, B. Leimkuhler, A. E. Mark, S. Reich, and R. D. Skeel, editors, Lecture Notes in Computational Science and Engineering (Vol 4). Computational Molecular Dynamics: Challenges, Methods, Ideas, pages 78-97, Springer (1998).
[pdf]
|
| |
|
|
|
Top