scholarly journals Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap

2013 ◽  
Vol 104 (1) ◽  
pp. 4-5
Author(s):  
Ziad Ganim
Keyword(s):  
Vibrational Spectroscopy ◽  
Single Molecule ◽  
Optical Trap

scholarly journals The mechanochemistry of the kinesin-2 KIF3AC heterodimer is related to strain-dependent kinetic properties of KIF3A and KIF3C

2020 ◽  
Vol 117 (27) ◽  
pp. 15632-15641
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Force Sensitivity ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent ◽  
Transport Potential

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs and determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1= 11 s−1) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.

scholarly journals Measurement of hindered diffusion in complex geometries for high-speed single-molecule experiments

2020 ◽  
Author(s):  
Tobias F. Bartsch ◽  
Camila M. Villasante ◽  
Ahmed Touré ◽  
Daniel M. Firester ◽  
Felicitas E. Hengel ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Single Molecule ◽  
High Speed ◽  
Optical Trap ◽  
Glass Coverslip ◽  
Hydrodynamic Coupling ◽  
Hindered Diffusion ◽  
Protocadherin 15 ◽  
Human Hearing ◽  
Infinite Wall

AbstractIn a high-speed single-molecule experiment, a protein is tethered between two substrates that are manipulated to exert force on the system. To avoid nonspecific interactions between the protein and nearby substrates, the protein is usually attached to the substrates through long, flexible linkers. This approach precludes measurements of mechanical properties with high spatial and temporal resolution, for rapidly exerted forces are dissipated into the linkers. Because mammalian hearing operates at frequencies reaching tens to hundreds of kilohertz, the mechanical processes that occur during transduction are of very short duration. Single-molecule experiments on the relevant proteins therefore cannot involve long tethers. We previously characterized the mechanical properties of protocadherin 15 (PCDH15), a protein essential for human hearing, by tethering an individual monomer through very short linkers between a probe bead held in an optical trap and a pedestal bead immobilized on a glass coverslip. Because the two confining surfaces were separated by only the length of the tethered protein, hydrodynamic coupling between those surfaces complicated the interpretation of the data. To facilitate our experiments, we characterize here the anisotropic and position-dependent diffusion coefficient of a probe in the presence of an effectively infinite wall, the coverslip, and of the immobile pedestal.

scholarly journals Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

2019 ◽  
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Slow Step ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Cargo Transport ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent

AbstractKIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo-and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s−1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.Significance StatementKinesins are important long-range intracellular transporters in neurons required by the extended length of the axon and dendrites and selective cargo transport to each. The mammalian kinesin-2, KIF3AC, is a neuronal heterodimer of fast and slow motor polypeptides. Our results show that KIF3AC has a single observed stepping rate in the presence and absence of load and detaches from the microtubule rapidly under load. Interestingly, both KIF3A and assisting loads accelerate the kinetics of KIF3C. These results suggest that KIF3AC is an unconventional cargo transporter and its motile properties do not represent a combination of alternating fast and slow step kinetics. We demonstrate that the motile properties of KIF3AC represent a mechanochemistry that is specific to KIF3AC and may provide functional advantages in neurons.

scholarly journals Characterization of the Interactions of Arg-Gly-Asp- and Ala-Gly-Asp-Val-Containing Peptides with the Platelet Integrin αIIbβ3

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1350-1350
Author(s):  
Rustem I. Litvinov ◽  
Olga Kononova ◽  
Dmitry S. Blokhin ◽  
Vladimir V. Klochkov ◽  
John W. Weisel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Graphics Processing Units ◽  
Optical Trap ◽  
Umbrella Sampling ◽  
Binding Energies ◽  
Dynamic Force ◽  
Contact Duration ◽  
Α Subunit ◽  
Integrin Αiibβ3 ◽  
Open Conformation

Abstract Binding of soluble fibrinogen to the activated open conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. Paradoxically, however, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3. Moreover, the Aα chain RGD motifs make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. The interaction of αIIbβ3 with a variety of RGD- and γC-12 peptides has been studied extensively, but the characteristics of their binding to the active open and inactive closed conformations of αIIbβ3 are largely unknown. Here, we have used both experimental and computational approaches to compare the two-dimensional kinetics, thermodynamics and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. First, we employed optical trap-based single-molecule nanomechanical measurements to determine the probability of peptide binding to αIIbβ3 as a function of the time the peptides interact with αIIbβ3. In the optical trap-based experimental system, a microscopic bead coated with either cRGDFK or γC-12 is trapped in a fluid chamber by a focused laser beam and moved in an oscillatory manner to touch a stationary pedestal coated with αIIbβ3. When the peptide on the bead interacts with αIIbβ3 on the pedestal, tension is generated when the bead is displaced from the laser focus until the αIIbβ3-peptide bond ruptures. The percentage of binding/unbinding events at a particular time of contact duration enabled us to plot the force-free binding probability as a function of contact duration. From these curves, we extracted first-order binding rates: 0.61×10-14 cm2/s for γC-12 and 0.71×10-14 cm2/s for cRGDFK and unbinding rates: 2.05/s and 1.53/s for the γC-12 and cRGDFK, respectively. Corresponding binding affinity constants were 0.3×10-14 cm2 and 0.46×10-14 cm2 for the γC-12 and cRGDFK, respectively, indicating that cRGDFK binds to αIIbβ3 somewhat tighter than γC-12. These measurements were then followed by computational modeling using NMR-determined 3D solution structures of cRGDFK and γC-12, as well as the crystallographically resolved αIIbβ3 conformers of the αIIbβ3 headpiece (PDB entries: 3ZDX and 3ZE2). Docking analysis confirmed that both cRGDFK and γC-12 bound to the αIIbβ3 conformers at a site located in the interface between the α subunit β-propeller domain and the β subunit βI domain. Estimated docking energies revealed that the closed αIIbβ3 conformation is almost as reactive towards the peptides as the open conformation. Next, we probed the strength of the αIIbβ3-peptide interactions and determined their comparative binding energies and the dynamics of interatomic contacts by performing dynamic force-ramp unbinding in silico using all-atom Molecular Dynamics simulations in implicit solvent on Graphics Processing Units. Force was increased linearly at a pulling rate of 2×104 μm/s and applied to specified Cα-atom of the peptides. Analysis of the molecular force profiles and the number of ligand-receptor binding contacts revealed that both peptides interact strongly with αIIbβ3, forming stable bimolecular complexes that dissociate in the 60-120 pN range through several competing unbinding pathways that display multi-step kinetics and distinct bond lifetimes. We profiled the Gibbs free energy (ΔG) of the αIIbβ3-peptide complexes as a function of the separation distance between the open form of αIIbβ3 headpiece and the peptide using the Umbrella Sampling technique. The results showed that the energies of ligand binding to the αIIbβ3 headpiece are ΔG~16 kcal/mol for cRGDFK and ΔG~13 kcal/mol for γC-12, meaning that the overall thermodynamic stability of the αIIbβ3-peptide complex is higher for cRGDFK than for γC-12. Thus, these results provide a kinetic and thermodynamic explanation for previous observations that the affinity of RGD for αIIbβ3 is greater than AGDV and support our hypothesis that the RGD motifs preferentially support the interaction of αIIbβ3 with immobilized fibrinogen and fibrin. Disclosures Weisel: Bayer: Research Funding.

On Nonlinear Control of Optical Traps Using Pulling Trajectory Tracking for Single-Molecule Experiments

2011 ◽  
Author(s):  
Daniel G. Cole
Keyword(s):  
Single Molecule ◽  
Optical Trap ◽  
Static Condition ◽  
Optical Force ◽  
Optical Traps ◽  
Pi Control ◽  
Force Estimation ◽  
Integral Control ◽  
Servo Tracking ◽  
Additional Feedback

This article explores nonlinear position plus integral (PI) feedback for controlling an optical trap used in single-molecule experiments. In general, nonlinearities in the spatial dependence of the optical force complicate feedback control for optical traps. Furthermore, the extension of a molecule creates an additional feedback path that puts constraints on the PI control gains. The nonlinear PI control presented here is shown to provide all of the benefits of integral control: disturbance rejection, servo tracking, and force estimation. The ability of nonlinear PI control to lower the measurement SNR is evaluated. Finally, constraints on the pulling rate are given to ensure the system trajectory remains in a quasi-static condition, stable, and the bead remains held in the trap.

Measurement of the Lifetime of Bonds Between αIIbβ3 and Fibrinogen Using Constant Unbinding Forces Generated by Optical Tweezers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 254-254
Author(s):  
Rustem I. Litvinov ◽  
Henry Shuman ◽  
John Weisel ◽  
Joel S. Bennett
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Time Course ◽  
Tensile Force ◽  
Optical Trap ◽  
Compressive Force ◽  
Binding Interface ◽  
Fibrinogen Binding ◽  
Interacting Surfaces ◽  
Coated Surfaces

Abstract We have shown that the distribution of rupture forces between individual αIIbβ3 and fibrinogen molecules displays at least two components that differ in kinetics, loading rate dependence, and susceptibility to activation and inhibition of the integrin. This suggests that the binding and unbinding of αIIbβ3 and fibrinogen is a complex multistep process that depends on the conformational state of both αIIbβ3 and fibrinogen, the duration of their interaction, and environmental factors such as externally-applied shear force. To directly test these possibilities, we quantified the lifetime of bonds stabilizing individual αIIbβ3-fibrinogen complexes with a novel nanoscale laser tweezers-based system that uses an optical trap to apply a constant unbinding force to single-molecule protein-protein interactions. When a ligand-coated bead is brought into repeated contact with a receptor-coated silica pedestal using an optical trap, the signal parameters that are measured correspond to both compressive and rupture forces. To measure bond lifetimes, the amplitude of the generated tensile force signal must remain constant throughout the lifetime of the bond. This can be accomplished by incorporating an analog feedback circuit within the optical system. The system also enables us to control the time of contact between interacting surfaces, the magnitude of compressive force during contact, the magnitude of the tensile force, and the time of protein-protein separation when binding occurs. To quantify the forced unbinding of fibrinogen molecules covalently bound to latex beads and αIIbβ3 molecules covalently attached to silica microspheres, we measured the distribution of bond lifetimes obtained under constant tensile force, mimicking the effect of hydrodynamic blood flow on an adherent platelet. We found that the separation times of the αIIbβ3- and fibrinogen-coated surfaces varied, indicating that the interactions occurring at the interface had heterogeneous kinetic and thermodynamic properties. Discrimination of specific αIIbβ3-fibrinogen binding events versus non-specific interactions was based on comparison of bond lifetime distributions in the absence and presence of abciximab or eptifibatide, specific inhibitors of fibrinogen binding to activated αIIbβ3. We found that the separation times of the αIIbβ3- and fibrinogen-coated surfaces were bimodal, with specific integrin-fibrinogen interactions lasting more than 2s under a constant tensile force of 50 pN. Varying the time of contact between αIIbβ3 and fibrinogen from 0.1s to 2.0s at the same unbinding force revealed that the bond lifetimes increased as the duration of contact between that interacting surfaces increased, suggesting that stability of αIIbβ3-fibrinogen interactions is time-dependent. Because these measurements mimic the binding/unbinding parameters and the time course of the αIIbβ3-fibrinogen interactions under conditions of shear, they are relevant to physiological processes of fibrinogen-mediated platelet adhesion and platelet aggregation. Taken together, our data suggest a model for fibrinogen binding to αIIbβ3 in which the initial interaction is followed by reorganization of the binding interface, thereby enhancing the strength and stability of fibrinogen binding.

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