Simulation of Atomic Force Microscopy Rupture Experiments

The force required to rupture the streptavidin-biotin complex has been studied by computer simulations. The computed forces agree well with those obtained by single molecule atomic force microscope (AFM) experiments. The simulations suggest a multiple pathway rupture mechanism, which depends on the applied loading rate. Binding forces and specificity are attributed to a hydrogen bond network between biotin and streptavidin.

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Multiple simulation studies [Izrailev et al. 1997; Grubmuller et al. 1996; Sieben et al. 2012] aimed at a microscopic interpretation of single molecule AFM experiments, in which unbinding forces between individual protein-ligand complexes have been measured. We asked, what interactions cause the experimentally observed unbinding forces particulary.

The top of Figure 1 sketches a typical AFM rupture experiment: On one side, the molecule of interest is attached to a surface via a linker. On the other side, it is attached to a cantilever by a second linker. When the surface is moved away from the cantilever (indicated by the blue arrow), the cantilever bends and a force can be recorded. At the moment of rupture, the force will abruptly drop down to zero.

For the computer simulations (bottom part of Fig. 1), the complex was fixed on the far end side of the binding site. The cantilever was replaced by a harmonic potential (sketched as a grey spring in the bottom figure) with the same stiffness as the cantilever in the experimental setup. In the latest simulations (Video 4), not only a monomeric streptavidin-biotin complex was simulated but the whole tetramer and the linker between the cantilever and the ligand was modeled by a worm like chain potential with the same characteristics as the PEG linker in the experiments. In contrast to the experimental setup, the center of the harmonic potential was moved away com the complex.

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.

The computer simulations and AFM experiments were in good agreement and allowed us to describe rupture forces for more than 11 orders of magnitude of loading rates. The combination of the two techniques provided detailed insight into the complex mechanisms of streptavidin-biotin unbinding showing a heterogeneity of unbinding pathways depending on the applied loading rate.


Below, you'll find three 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 [Kraulis 1991] and Raster3D [Anderson et al. 1988].

The forth movie illustrates the unbinding process at low loading rates.

A global view of the rupture process (Movie 1, mpg)
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). more
A detailed view of the binding pocket (Movie 2, mpg)
Only the ligand and the residues involved in ligand binding are shown. As above, biotin is pulled towards the right. more
Another detailed view of the binding pocket (Movie 3, mpg)
Streptavidin and biotin are now drawn as a space-filling model. Biotin is pulled towards the right. more

Streptavidin-Biotin undbinding at low loading rates (Movie 4, avi)
Illustration of the undinding process of the Biotin ligand, depicted in stick represenation, from its binding partner streptavidin, depicted in red cartoon representation. Total simulation time scale was T=1.5 ms but only the last 6 ns of the unbinding event are shown when biotin leaves the binding pocket. In the top right corner, the recorded force is shown. In the lower left corner, the time is shown. more


Rico, F.; Russek, A.; Gonzalez, L.; Grubmüller, H.; Scheuring, S.: Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and molecular dynamics simulations. Proceedings of the National Academy of Sciences of the United States of America 116 (14), pp. 6594 - 6601 (2019)
Sieben, C.; Kappel, C.; Zhu, R.; Wozniak, A.; Rankl, C.; Hinterdorfer, P.; Grubmüller, H.; Herrmann, A.: Influenza virus binds its host cell using multiple dynamic interactions. Proceedings of the National Academy of Sciences of the United States of America 106 (34), pp. 13626 - 13631 (2012)
Eichinger, M.; Heymann, B.; Heller, H.; Grubmueller, H.; Tavan, P.: Conformational dynamics simulations of proteins. Springer, Berlin (1998)
Grubmüller, H.; Heymann, B.; Tavan, P.: Ligand binding: Molecular mechanics calculation of the streptavidin-biotin rupture force. Science (5251), pp. 997 - 999 (1996)


Izrailev, S.; Stepaniants, S.; Balsera, M.; Oono, Y.; Schulten, K.
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Biophysical Journal 72, 1568 (1997)
J. P. Kraulis
MOLSCIRPT - A Program to produce both detailed and schematic plots of protein structures.

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B. Bacon, W. F. Anderson
A fast algorithm for rendering space-filling molecule pictures.
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