High-speed Force Spectroscopy Research Articles

Mechanics of Adhesion Molecules Probed by Molecular Dynamics and High-Speed Force Spectroscopy

Leukocytes travel at high velocities with the blood flow and slow down within milliseconds by interacting with the vessel wall via adhesion molecules [1]. This fast formation and rupture of bonds is crucial during the early steps of the immune response. One of the essential leukocyte adhesion complexes is the pair formed by the integrin αLβ2 and intercellular adhesion molecule-1 (ICAM-1) [2]. While the unbinding response is well known [3], little or none attention has been given to possible unfolding of the 5 immunoglobulin-like domains of ICAM-1 during the unbinding process. The main goal of this work is to determine the molecular mechanisms of the unfolding of leukocyte adhesion molecules at high loading rates. For that, we combined high-speed force spectroscopy (HS-FS), allowing mm/s pulling rates and µs time resolution [4,5], and all-atom steered molecular dynamics (SMD) simulations at overlapping rates providing an atomic description of the process supported by experimental results. HS-FS measurements and SMD simulations allowed us to determine the forces required to unfold ICAM-1. Experiments and simulations showed good agreement indicating that domain 3 unfolds first at forces lower than the unbinding forces of αLβ2/ICAM-1 at similar loading rates. This suggests that ICAM-1 partially unfolds before complex rupture, regulating leukocyte adhesion by buffering the applied force, working as a shock nanoabsorber.

Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations.

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.

α-Helix Unwinding as Force Buffer in Spectrins.

Spectrins are cytoskeletal proteins located at the inner face of the plasma membrane, making connections between membrane anchors and the actin cortex, and between actin filaments. Spectrins share a common structure forming a bundle of 3 α-helices and play a major role during cell deformation. Here, we used high-speed force spectroscopy and steered molecular dynamics simulations to understand the mechanical stability of spectrin, revealing a molecular force buffering function. We find that spectrin acts as a soft spring at short extensions (70-100 Å). Under continuous external stretching, its α-helices unwind, leading to a viscous mechanical response over larger extensions (100-300 Å), represented by a constant-force plateau in force/extension curves. This viscous force buffering emerges from a quasi-equilibrium competition between disruption and re-formation of α-helical hydrogen bonds. Our results suggest that, in contrast to β-sheet proteins, which unfold in a catastrophic event, α-helical spectrins dominantly unwind, providing a viscous force buffer over extensions about 5 times their folded length.

High-Speed Force Spectroscopy of Lipid Bilayer Rupture

Supported Lipid Bilayers (SLBs) are simple biological membrane models widely used for fundamental studies. Atomic Force Microscopy (AFM)-based Force Spectroscopy is an ideal technique to investigate the mechanical properties of SLBs at the nanoscale, their elastic constants but also their resistance to failure. The mechanical rupture of the lipid bilayer by the AFM tip has been used extensively as a hallmark of its mechanical stability, usually by measuring the forces required to break the bilayer probed at different velocities. Being a stochastic process, rupture forces generally increases linearly with the logarithm of the loading rate, defining a characteristic dynamic force spectrum. Thanks to the miniaturization of the cantilever and piezoelectric elements, High-Speed AFM (HS-AFM) allows reaching tip velocities in the millimeter per second range with microsecond time resolution (1), covering a wider dynamic force spectrum. Here, we apply HS-AFM force spectroscopy to access six decades of indentation velocities on supported lipid bilayers. We also assess the possible contribution of the cantilever resonance frequency to the force spectrum. The preliminary dynamic force spectrum of DOPC can be interpreted with a single energy barrier although possibly followed by an outer state. The outcomes allow us to evaluate the validity of general theoretical developments applied in the field (2). 1.Rico F, Gonzalez L, Casuso I, Puig-Vidal M, & Scheuring S (2013) High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations. Science 342(6159):741-743. 2.Butt HJ & Franz V (2002) Rupture of molecular thin films observed in atomic force microscopy. I. Theory. Physical Review E 66(3).

Dynamics of breaking intermolecular bonds in high-speed force spectroscopy.

Atomic force microscope based single-molecule force spectroscopy provides a description of a variety of intermolecular interactions such as those occurring between receptor molecules and their ligands. Advances in force spectroscopy have enabled performing measurements at high-speeds and sub-microsecond resolutions. We report experiments performed on a biotin-avidin system that reveal that the measured force decreases with the loading rate at high rates. This result is at odds with the established Bell-Evans theory that predicts a monotonic increase of the rupture force with the loading rate. We demonstrate that inertial and hydrodynamic forces generated during the breaking of the bond dominate the measured force at high loading rates. We develop a correction factor to incorporate those effects into the Bell-Evans theory. The correction is necessary to obtain accurate values of the intermolecular forces at high speeds.

High-Speed Force Spectroscopy for Single Protein Unfolding.

Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain.

High-Speed Force Spectroscopy Unbinds Streptavidin-Biotin at the Velocity of Molecular Dynamics Simulations

The forced disruption of the (strept)avidin-biotin complex by atomic force microscopy (AFM) and other techniques opened the field and established the basis of single molecule force spectroscopy (1-3). Steered molecular dynamics (SMD) simulations provided atomic description of the unbinding process (4). However, the maximum experimental AFM velocities were typically of 10 µm/s, while SMD simulations were performed in the range of 1 m/s to 10 m/s. Recent development of high-speed force spectroscopy (HS-FS) using HS-AFM allowed velocities in the mm/s range (5). We have applied HS-FS to probe the binding strength of the streptavidin-biotin complex at velocities up to ∼8 mm/s, paralleled by SMD simulations. The experimental dynamic force spectrum of the unbinding process is compared with SMD simulations. The combination of HS-FS and SMD at overlapping velocities provides an atomic description of the unbinding process measured by experiment.1. Merkel, R., P. Nassoy, A. Leung, K. Ritchie, and E. Evans. 1999. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397:50-53.2. Florin, E. L., V. T. Moy, and H. E. Gaub. 1994. Adhesion Forces Between Individual Ligand-Receptor Pairs. Science 264:415-417.3. Lee, G. U., D. A. Kidwell, and R. J. Colton. 1994. Sensing Discrete Streptavidin Biotin Interactions with Atomic-Force Microscopy. Langmuir 10:354-357.4. Grubmuller, H., B. Heymann, and P. Tavan. 1996. Ligand binding: Molecular mechanics calculation of the streptavidin biotin rupture force. Science 271:997-999.5. Rico, F., L. Gonzalez, I. Casuso, M. Puig-Vidal, and S. Scheuring. 2013. High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations. Science 342:741-743.

Bringing Force Probe Molecular Dynamics Simulations Closer to Experiments

The interaction between Streptavidin and Biotin is one of the strongest known non covalent bonds in nature. For this reason, many experimental and computational studies have been done on this complex, which revealed many details on binding and unbinding of biotin, which turned out to be an astonishingly complex process.However, taken all these studies together and despite considerable technical advances over the past years, there is still a time scale gap remaining, i.e., the loading rates possible in force spectroscopy experiments and those achievable in molecular dynamics (MD) simulations hardly overlap.Recent high speed force spectroscopy (HS-FS) experiments by Scheuring offer the possibility to reach pulling velocities up to 5 mm/s.Here we present extensive MD simulations covering pulling velocities from 0.5 mm/s to 50 m/s, thus providing an overlap of one order of magnitude with experimental time scales. Further, to resemble the AFM experiment as closely as possible in the simulations, we modeled the PEG-linker between Biotin and the cantilever as a wormlike chain and chose a spring constant of the pulling potential to match the stiffness of the cantilever in HS-FS experiments done by Scheuring.Over the whole interval, agreement of the probability distributions of unbinding forces is seen. Taken together, these data cover unbinding time scales over 10 orders of magnitude, which offers a unique opportunity to compare against several proposed transition state treatments of this process.

High-Speed Atomic Force Microscopy: Integration with Optical Microscopy and High-Speed Force Spectroscopy

High-speed atomic force microscopy (HS-AFM, [1]) offers unique novel possibilities to study single molecule dynamics [2,3]. Here we present two HS-AFM developments, first the integration of optical microscopy into HS-AFM for imaging membrane proteins on cells, and second high-speed force spectroscopy (HS-FS) for fast protein unfolding studies:HS-AFM is a powerful tool for studying structure and dynamics of proteins. So far, however, HS-AFM was restricted to well-controlled molecular systems. Here, we integrate optical microscopy (OM) into HS-AFM, allowing bright field and fluorescence microscopy, without loss of HS-AFM performance. This hybrid HS-AFM/OM setup [4] allows positioning the HS-AFM tip on an optically identified zone of interest on cells. We present movies at 960ms per frame displaying aquaporin-0 array and single molecule dynamics on intact eye lens cells. This hybrid setup allows HS-AFM imaging on cells ∼1000 times faster than conventional AFM/OM setups, and first time visualization of unlabeled membrane proteins on a eukaryotic cell under physiological conditions.The mechanical unfolding of muscle protein titin by AFM is a landmark experiment in single molecule biophysics. Molecular dynamics simulations offered an atomic level mechanistic description of the process. However, experiment and simulation differ in pulling velocity by orders of magnitude. We have developed HS-FS [5] to unfold titin at velocities reached by simulation (∼4 mm/s). Our results show that an intermediate state in a small βeta-strand pair dynamically unfolds and refolds, buffering pulling forces up to ∼100pN. Furthermore, our data indicates that the distance to the transition state of domain unfolding is much larger than previously estimated, but in better agreement with atomistic predictions.[1] Ando, et al., PNAS,2001,98(22):12468-12472.[2] Kodera, et al., Nature,2010,468(7320):72-76.[3] Casuso, et al., Nature Nanotechnology,2012,7(8):525-529.[4] Colom, et al., Nature Communications,2013,4:DOI:10.1038/ncomms3155.[5] Rico, et al., submitted,2013.

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.

Strength of Non-Covalent Biomolecular Interactions Probed at the Microsecond Timescale

We have measured strength of non-covalent interactions at the microsecond timescale by using a recently developed high-speed force spectroscopy technique. The resulting loading rates narrow the gap between time scales of experimental methods and molecular dynamics simulations substantially. Measurements on biotin-streptavidin complexes provide direct experimental verification of forces predicted by steered molecular dynamics simulations [1]. This technique uses a T-shaped atomic force microscope cantilever with its tip placed offset from the longitudinal axis [2,3].