Muscle Biophysics Summer School , 2018, Budapest In vitro mechanics of molecular motors Pasquale Bianco PhysioLab, BIO, University of Florence, Italy
Brief history of single-molecule Imaging and Manipulation 1976: Fluorescence image of single antibody molecule 1986: J. Spudich, T. Yanagida, in vitro motility assay 1991: J.Spudich, T.Yanagida, J.Molloy, single myosin mechanics 1994: T.Yanagida, single ATP turnover in myosin 1994: K.Svoboda, S. Block, single kinesin mechanics 1996: C.Bustamante, D.Bensimon, DNA overstretch (B-S) transition 1996: T.Ha, S.Weiss, single pair FRET 1997: W.E. Moerner, GFP blinking 1997: M.Kellermayer, M.Rief, L.Tskhovrebova, mechanical unfolding of titin 1998: Kinosita, F1F0 ATPase stepping kinetics 1998: J. Fernandez, genetic polyprotein mechanics 2001: J.Liphardt, C.Bustamante, RNA hairpin mechanics 2004: J.Fernandez, single-protein refolding
Methods of mechanical manipulation Glass micropipette Microfabricated AFM cantilevers laser beam deflection pulled glass micropipette Cantilever reference methods beam Δ x cantilever microfabricated bending = F/k silicon cantilever Δ z pedestal Optical tweezers Flow field Magnetic tweezers magnetic field photon field Stokes drag Force field methods latex bead magnetic bead moveable micropipette
Molecular manipulators are picotensiometers Cantilever Photon field F = K Δ x Virtual spring F = K Δ x Δ x ≈ nanometer scale K ≈ 0.1 - 10 pN/nm F ≈ picoNewton scale
How to manipulate individual molecules? 1. Atomic force microscope Single Molecule Force Spectroscopy photodetector laser cantilever molecule 140 Force (pN) 120 100 Δ F 80 Δ x 60 Δ x Force = k Δ x 0.22 0.24 0.26 0.28 0.3 0.32 0.34 End-to-end Extension ( µ m) length
How the Optical Tweezers works Incoming Laser light beam P 1 Microscope objective Refractile microsphere F F Gradient force Refractile F= Δ P/ Δ t EQUILIBRIUM P 2 microbead Scattering Δ P force Photon field Δ x ≈ nanometer scale K ≈ 0.15 pN/nm F = K Δ x F ≈ picoNewton scale Virtual spring
Direct Force Measurement Measuring the change of light momentum Objective: partially filled with incoming laser beam Outgoing laser beam: Objective: Integrated intensity partially filled with And position monitored incoming laser beam Outgoing laser beam: Integrated intensity And position monitored Smith et al, Science 271, 795, 1996.
The DLOT enhances the axial trapping stability Refractile microbead Laser 1 Laser 2 Microscope objective Microscope objective In the Dual-beam optical tweezers utilizing counter-propagating beams, DLOT , two microscope objectives face each other and focus two separate laser beams to the same spot. Since the scattering force due to reflection is approximately the same for each laser, these forces cancel and the axial trap stability is greatly enhanced. Dual-beam optical tweezers are therefore able to generate higher trapping forces for a given laser power and can be constructed with lower NA microscope objectives. Red lines represent light reflected at the surface
The force calibration depends on the value of Δ X θ = angular deflection of the beam n 1 = refractive index of the medium R L = focal length of the lens Δ X = linear distance of the angular deflection c = speed of light W = intensity of the laser Δ X/ R L = n 1 sin θ F trap = (n 1 W/c) sin( θ ) F trap = (W/c)*( Δ X/R L ) Direct measurement of the angular intensity distribution of the laser as it enters and leaves the trap, determines the change in the momentum flux of the light beam, which is equal to the externally applied force on the particle; the force calibration becomes independent of particle’s size, shape, refractive index, viscosity of the medium, etc …
Viscous drag forces are compared to light-momentum sensor output Bead diameter ( µ m) (Smith, S.B., Y. Cui, and C. Bustamante, Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol, 2003. 361: p. 134-62)
A light dynamometer! Stokes’ force (pN) force (pN) Stokes law F f = − 6 πη rv
Dual-beam counter-propagating optical tweezers Trapping Trapping Fluorescence laser 1 laser 2 emission Bright field Fluorescence illumination excitation Working range: Force 0-200 pN, resolution ∼ 0.3 pN; Movement 0-75.000 nm, resolution ∼ 0.3 nm; rise time ≤ 2 ms
DLOT implementations: 1. Temperature control in the range 4-45 °C. 2. Integration of the fast nano-positioner into a micro-positioner to provide centimeter movement for transport of particles in a multi-compartment chamber. 3. Development of a fast force and length feedback (force steps complete within 2 ms). 4. Measurement of Intracellular Calcium Signal. Copper jackets X-Y-Z nanopositioner X-Y micropositioner for temperature control
Characteristics of the temperature control flow chamber copper jackets for temperature control circulation fluids Laser 2 Laser 1 objective objective circulation fluids + thermocouple - Detail of the copper jacket (Mao et al. (2005), Biophys. J.89:1308_1316) Temperature control in the range 4-40 ° C A) The temperature in the chamber, measured by a miniaturized thermocouple recovered the set value within 7-8 s B) Power spectrum of force fluctuations for an optically trapped polystyrene bead of 3.28 µ m diameter: gray trace, bath on; black trace, bath off. Acquisition time, 15 s at 15 kHz.
Force and length clamp mode command DLOT Σ Driven output (+/- piezo movement ) x ( piezo ) - Piezo position x 0 - x Δ F k * x x = Δ ( light ) + k Force Trap stiffness x ( piezo ) - L x x = − ( piezo ) ( bead ) x ( bead ) Length +
Schematics of force-driven reactions increase in force
How to grab individual molecules? Design of molecular handles microbead ~ 1 µ m molecule ~ 10 nm
Handling individual molecules I. 1. Non-specific adsorption AFM cantilever Layer of molecules Surface 2. Sequence-specific antibodies Ab molecule Titin’s I-band segment bead 3. Non-covalent tags Biotin/streptavidin His-tag/Ni-NTA GST-tag cantilever tip streptavidin biotin myosin subfragment-1 actin filament Streptavidin dimer binds 4 biotins (tetrameric protein purified from Electrostatic interaction Glutathion-S-transferase Streptomyces avidinii ) Strength controlled with His n length Conjugated to protein of interest Biotin : vitamin derivative Works under denaturing conditions Binds glutathion specifically, strongly Binding specific and strong, K d ~10 -14
Handling individual molecules II. 4. Covalent cross-linkers Specific surface chemistries EDC: 1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide Carboxy- and amino-reactive 5. Au-S bond Gold-coated AFM cantilever Gold surface Au SH groups (usually vicinal cysteines) S Covalent bond Protein with terminal vicinal cysteines 6. Photoreactive cross-linkers Non-specific Photoreactive N 3 -group UV illumination
Handling individual molecules III. 7. DNA handles Molecular dimensions Can be made specific via cloning techniques Provides mechanical fingerprint 8. Recombinant polyprotein Protein of Genetic polymer of known protein domain (titin I27) I27 interest Repetitive sawtooth force pattern Provides mechanical fingerprint 9. Functionalized carbon nanotube High aspect ratio High Young modulus Chemical activation difficult
A synthetic nanomachine based on the fast myosin isoform of skeletal muscle Pertici et. al. (2018) Nature Communication , in press
Biological motility is due to motor proteins that use the free energy of the hydrolysis of ATP to generate force and reciprocal displacement between the motor and polar filamentous structures (tracks) formed by the polymerisation of the globular proteins actin and tubulin. A B - + + - Motors function as rowers when Motors function as porters , they are fixed to a substratus when they carry intracellular and powers the sliding of the cargoes walking along their track tracks Rowers are organised in array Porters are processive motors: to generate steady force and they walk long distances along sliding by cyclic interaction their track. This is possible for with their track. The duty ratio a single motor because it is a is <<0.5 and reduces with dimer with a duty ratio ≥ 0.5. increase in sliding velocity
Background In each half-sarcomere, myosin motors are mechanically coupled by their attachment to the thick filament and this collective motor, not accessible to investigations using single molecule mechanics, is the functional unit that accounts for (H.E. Huxley, J. Biophys. Biochem. Cytol., 1957 ) the power output of the striated muscle. thick (myosin) filament ELC Myosin Heavy Chain RLC myosin II motor thin (actin) filament S2 LMM S1 95 nm HMM Cell studies cannot give the details of the motor coupling mechanism, being complicated by the large ensemble of motor proteins and filaments and by the hardly distinguishable role of cytoskeleton proteins. Single molecule studies on purified proteins suffer from the intrinsic limit that they cannot detect the function emerging from the motor ensemble and its architecture in the half-sarcomere.
From cell to molecule measurement Laser focus FORCE Optical TRANSDUCER trap molecule LENGTH Latex TRANSDUCER bead moveable micropipette
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