Unbinding Force Research Articles

Single molecule force spectrometer with magnetic force control and inductive detection

Mechanical investigations with single molecules have opened a rich spectrum of new experiments, ranging from molecular recognition over polymer elasticity and DNA unzipping to protein unfolding. Here we present the design of a novel instrument, where the force is applied to the molecule via magnetic fields acting on a magnetic cantilever for atomic force microscopy. The performance of this instrument was demonstrated by measuring the binding force of biotin/avidin complexes. A new and technically very appealing detection scheme for measurements of unbinding forces was introduced, which consists of picking up the induced voltage pulse caused by a discontinuous change of the magnet position upon bond rupture.

Direct force measurements of insulin monomer-monomer interactions.

Direct measurement of the forces involved in protein-protein and protein-receptor interactions can, in principle, provide insight necessary for the advancement of structural biology, molecular biology, and the development of therapeutic proteins. The protein insulin is illustrative in this respect as the mechanisms of insulin dimer dissociation and insulin-insulin receptor binding are crucial to the efficacy of insulin medications for the control of diabetes. Insulin molecules, modified with a photochemically active azido functionality on specific residues, were attached to force microscope tips and opposing mica surfaces in configurations that would either favor or disfavor dimer formation. Force curve measurements performed in buffer solution revealed the complexity of the insulin monomer-monomer interaction with multiple unbinding events occurring upon tip retraction, suggesting disruption of discrete molecular bonds at the monomer-monomer interface. Furthermore, the force curves exhibit long-range unbinding events, consistent with considerable elongation of the insulin molecule prior to dissociation. The unbinding forces observed in this study are the result of a combination of molecular disentanglement and dimer dissociation processes.

Scanning-probe-based science and technology.

Scanning-probe-based science and technology.

Strength and lifetime of the bond between actin and skeletal muscle α-actinin studied with an optical trapping technique

The force required to break the bond between skeletal muscle actin and α-actinin (unbinding force) was measured at the level of individual molecules with an optical trapping technique. An actin filament, to the barbed-end of which was attached a gelsolin-coated polystyrene bead, was bound to α-actinin molecules adsorbed to a nitrocellulose-coated glass surface (≈ 1 α-actinin molecule per 1 μm actin filament). The filament-bound bead was held by the optical trap and the force was applied to break the bond by pulling the bead. The unbinding force ranged from 1.4 to 44 pN. The average magnitude of the force was ≈ 18 pN. As the probability of the bond breakage has been suggested to be governed by the magnitude of the external force, the relationship was studied between the magnitude of the unbinding force and the time required to break the bond (unbinding time). The unbinding time ranged from ≈ 0.1 to ≈ 20 seconds, and tended to become shorter as the unbinding force became larger. The unbinding time seemed to be classifiable into two major groups: one group having a time value of 1 sec or less and the other having a time value ranging from several to 20 seconds. This suggests the existence of at least two classes of the actin-actinin bonds.

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Mechanical and functional properties of single motor molecules.

We sumrnarize the mechanical and functional properties of a motormolecule of muscle studied with a new type of an in vitro motility assay system and a multi-imaging microscope system. First, supercoiling of an actin filament is shown, suggesting that the filament moves forward like a right-handed screw. Second, we describe the unbinding force and the lifetime of a rigor bond between a single myosin molecule and an actin filament measured by using optical tweezers. From these results, the mechanism of sliding and force generation of the motor molecule is discussed.

Unbinding force of a single motor molecule of muscle measured using optical tweezers.

The unbinding and rebinding of motor proteins and their substrate filaments are the main components of sliding movement. We have measured the unbinding force between an actin filament and a single motor molecule of muscle, myosin, in the absence of ATP, by pulling the filament with optical tweezers. The unbinding force could be measured repeatedly on the same molecule, and was independent of the number of measurements and the direction of the imposed loads within a range of +/- 90 degrees. The average unbinding force was 9.2 +/- 4.4 pN, only a few times larger than the sliding force but an order of magnitude smaller than other intermolecular forces. From its kinetics we suggest that unbinding occurs sequentially at the molecular interface, which is an inherent property of motor molecules.

Intermolecular Forces and Energies Between Ligands and Receptors

The recognition mechanisms and dissociation pathways of the avidin-biotin complex and of actin monomers in actin filaments were investigated. The unbinding forces of discrete complexes of avidin or streptavidin with biotin analogs are proportional to the enthalpy change of the complex formation but independent of changes in the free energy. This result indicates that the unbinding process is adiabatic and that entropic changes occur after unbinding. On the basis of the measured forces and binding energies, an effective rupture length of 9.5 +/- 1 angstroms was calculated for all biotin-avidin pairs and approximately 1 to 3 angstroms for the actin monomer-monomer interaction. A model for the correlation among binding forces, intermolecular potential, and molecular function is proposed.

Adhesion Forces Between Individual Ligand-Receptor Pairs

The adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin, desthiobiotin, or iminobiotin was measured. Under conditions that allowed only a limited number of molecular pairs to interact, the force required to separate tip and bead was found to be quantized in integer multiples of 160 +/- 20 piconewtons for biotin and 85 +/- 15 piconewtons for iminobiotin. The measured force quanta are interpreted as the unbinding forces of individual molecular pairs.