Dynamic force spectroscopy of parallel individual Mucin1–antibody bonds

Abstract

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.atomic force microscopy ͉ multivalency ͉ radioimmunmotherapy ͉ binding affinity I nteractions between biological molecules drive a vast variety of cellular processes and span a wide range of strength and complexity. Multivalent interactions where several binding units combine to produce superior binding strength play an important role in adaptive immune response (1) and intercellular adhesion (2), as well as in the mechanism of action of many pharmaceuticals (3). Clinical researchers have used multivalency as an affinity-enhancing approach (4, 5) in a variety of immunotherapies and imaging techniques to target specific tissues (6, 7).Linking several molecules into a large multivalent binding construct also creates bulky agents that exhibit reduced tissue penetration and have a higher probability of accumulation in liver (8). Therefore, a better understanding of the multivalent binding is necessary for the creation of optimized agents that balance binding efficiency and molecular size. Quantitative characterization of multivalent interactions is also important for understanding the basic biophysics of complex molecular systems.The last decade saw an explosion of interaction force measurement techniques that allowed researchers to measure and apply molecular level stresses (9-11). Atomic force microscopy (AFM) probes ligand-receptor interactions by simply pulling off the ligand from the receptor using external force (12). Kinetic approaches to the binding force measurements, such as dynamic force spectroscopy ...

Nanomechanics of Hemichannel Conformations

“…5B). Considering the relatively constant tether stretching distance in all single rupture curves, single unbinding events with higher forces would probably be attributed to ruptures of multiple parallel antibody-antigen bonds (43). In addition to the single rupture events as described above, ruptures with multiple unbinding 4 F. Liu, F. Terán Arce, S. Ramachandran, and R. Lal, unpublished data.…”

Unified Model of Dynamic Forced Barrier Crossing in Single Molecules

“…(7). This may explain why a vast amount of experimental investigations [5,6,10,11,13,22,23] have matched well against the phenomenological theory. This also underscores the importance of samplingḟ over several orders of magnitude, otherwise fitting Eq.…”

“…15.Aa Continued progress in magnetic [1], optical [2] and mechanical [3] manipulation has enabled experimentalists to study the microscopic states of systems ranging from single molecule magnets to individual biological complexes. A particularly common technique is found by steadily increasing a force field until a change in state is observed (such as a switch in domain polarity [4] or the dissociation of an intermolecular bond [5]). The power of this technique relies on theoretical developments which indicate that repeating such a measurement over a range of loading rates can enable the normally hidden details of the intrinsic energy landscape to be determined [6][7][8].…”

“…Furthermore, by taking the largeḟ limit of Eq. (7) we find the model frequently used for analyzing single molecule bond rupture data [5,6,11,22], f ≈ k B T x ‡ ln(e −γ /X). Therefore, all of the simple model landscapes (defined by b) converge to a universal spectrum given by the phenomenological Bell theory.…”

Enhanced stochastic fluctuations to measure steep adhesive energy landscapes

“…Hundreds of approach-retract cycles were carried out to provide sufficient data to fit to the dissociation model. The force spectrum and corresponding load rates were plotted in a force vs. logarithm of loading rate plot (57). Furthermore, unbinding forces from quasi-equilibrium traces using inherent drift at ∼10 pN/s were included.…”