BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 13: Molecular Devices Last time: biological strategies for inorganic templating by organic materials Biomimetic organic template materials Biomimesis of bone Today: molecular devices Reading: V. Vogel, ‘Reverse engineering: Learning from proteins how to enhance the performance of synthetic nanosystems,’ MRS Bull. Dec. 972-978 (2002) Overview to date Current Road Map of the course: ? Started with degradable synthetic polymers – structural and controlled release materials ? Discussed modifying degradable materials for biological recognition ? Moved to controlled release devices fabricated from degradable polymers ? Next, hydrogel materials for drug delivery, tissue engineering, and lab-on-a-chip applications o Structure, what are they made of o Theory of gel swelling for neutral and ionic gels ? Biomineralization: approaches used by biology and how we are trying to mimic them o Future materials for hard tissue engineering ? So far, largely looking at ‘macroscopic’ materials o Materials from which micron-sized or larger scaffolds, drug delivery devices and gels are fabricated ? Moving to smaller length scales: molecules and aggregates of molecules, we come to some new applications o Performing molecular-level functions o Delivering molecular cargos to cells (labeling or treating cells) Application areas we’ll focus on: ? Molecular devices o (Length scale of one or a few molecules) o Single-molecule switches o Molecular motors ? Nano- to micro-scale drug carriers and detection reagents o (Length scale of supramolecular aggregates to many-molecule aggregates) ? Drug targeting Molecular Devices Current Approaches to Molecular Devices based on Protein-polymer hybrids ? 3 examples we’ll discuss: 1. Use synthetic polymers to control ‘on’ and ‘off state of a protein 2. Use engineered surfaces to direct the function of proteins 3. Use engineered proteins to build nano-motorized devices on surfaces Lecture 13 – Hybrid macromolecules 1 of 13 - - BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Single-molecule switches ? Using LCST polymers as the basis of a molecular switch 1 o LCST polymers show sharp volume change at the transition temperature as they transform from swollen coil to globule Dehydration allows water to disorder ( ) 'S = S dehydrated - S hydrated > 0 Poly(N-isopropylacrylamide) ordered water molecules (minimize water-hydrophobe contacts) entropically-driven (Wu and Wang, 1998) 2-4 ? A temperature-sensitive streptavidin mutant o Chime animation of streptavidin with biotin bound to tetrameric pockets: http://www.chem.uwec.edu/Webpapers2001/barkacs/Pages/Steptavidin.html - T LCST R h,0 Hydrodynamic radius (related to <r 2 0 > 1/2 ) ~R h,0 /3 T (?C) (Ding et al. 2001) o Blockade of access to biotin-binding pocket is dependent on the size of the biotinylated target: ? Small protein G is not sterically blocked by the hydrated PDEAAm chain ? Large biotinylated IgG can’t access pocket even when PDEAAm chain is collapsed o Varying the length of the thermally-responsive chain allows the degree of binding blockade to be tuned (Figure 4) Lecture 13 – Hybrid macromolecules 2 of 13 Poly(N,N-diethylacrylamide): Mutatation introducing cysteine-(CH 2 -CH) n C=O near binding pocket dehydrates with increasing temperature- analogous to PEG- PPO-PEG triblock copolymers N H C 3 2 C H H C - -2 C H 3 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 size-selective blockade of streptavidin binding Polymer switch shows pocket: ? Also the basis for triggered release switches o Expose biotin-loaded conjugates to successive cycles of T < T LCST through T > T LCST ? 4 cycles ‘kick out’ all bound biotin All bound biotin released by 4 temperature cycles: (Ding et al., 1999) T LCST 37?C 4?C = 24?C ? Mechanisms for controlling access by large or small ligands o Small ligands have access to binding pocket next to immobilized chain blocked when chain is collapsed, but can access the pocket when the chain is hydrated ? Conversely, if biotin binds in the pocket, collapse of the chain can eject the bound small ligand Lecture 13 – Hybrid macromolecules 3 of 13 B B BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Larger protein ligands are always prevented from accessing the pocket next to the immobilized chain ? Selective access occurs for the second binding pocket 20 ? away- when the chain is collapsed it does not prevent access to the second pocket, but when hydrated, long chains can prevent Mechanisms of switch operation: access to the neighboring pocket and block protein binding ? Fabrication of capture and release devices 3,5 o Conjugation to magnetic micro- or nano-spheres ? Affinity purification QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. Applications: ? Affinity purification 1) QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. 3) B B QuickTime? and a Graphics decompressor are needed to see this picture. B B B B Conjugation to magnetic microspheres/nanospheres B B B 2) B B B B ? Cell-surface labeling QuickTime? and a Graphics decompressor are needed to see this picture. ? Responsive drug release QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. ? Generality of concept o Switch temperature can be tuned by copolymerizing with more hydrophilic monomers such as hydroxyethyl methacrylate Lecture 13 – Hybrid macromolecules 4 of 13 - - BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Other temperature-responsive polymers that could be used: ? Poly(N-isopropylacrylamide) o pH-responsive switches ? copolymers of PNIPAAm and AAc Copolymerization allows switch Switches can also be synthesized for temperature to be varied: light or pH triggering: -(CH 2 -CH) n - C=O N 2 C H H C - 2 C- H C 3 H 3 HEMA DEAAm Molecular Motors 6 ? Engineering principles on which macroscopic engines/motors are based fail at the nano-scale ? How to miniaturize controlled force-generating devices cell-manipulating devices, nanobots, etc.? o Molecular motors driven by single-molecule fuels, photons, etc. 7 Protein motors used by nature for force generation and motion ? Motor proteins convert chemical energy into mechanical force via conformational changes o Generation of protein motion along guide-wires: protein filaments o Driven by energy released on hydrolysis of adenosine 5’-triphosphate o Myosin and kinesin are two examples of ubiquitous motor proteins found in eukaryotic cells ? Kinesin o Motor protein translates along 25nm-diameter rigid rods (microtubules, up to 100 μm in length possible in vitro) o Transport of molecular cargos through cells ? Small membrane organelles or protein complexes ? E.g. encapsulated neurotransmitters from nerve cell nucleus to the synapse to excite neighboring cells o Coordination of two heads allows continuous ‘walking’ along microtubules with 80 ? steps ? Efficient processive motion allows long-range transport by one or a few motor proteins ? Motion is directional toward ‘plus’ end of microtubule ? Myosins o Motor protein moves along actin filaments o Enables contractile cell functions such as cell motility and muscle contraction ? Operates in a large array of motors to produce large-scale motions/forces Lecture 13 – Hybrid macromolecules 5 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o ~100 ? per ATP hydrolysis step o Two heads act independently of one another- single stroke then release from actin polymer ? Can’t continuously march along polymer by itself Myosin kinesin Muscle motor protein, transport along transport along microtubules actin fibers QuickTime? and a Video decompressor are needed to see this picture. QuickTime? and a Video decompressor are needed to see this picture. Limitations for use in bioengineering applications: ￥unidirectional motion ￥Controlling orientation of ? car?cable?and ? (Vale and Milligan, 2000) ? Energy source for these molecular motors o ATP hydrolysis cycle linked to conformational change cycle o Energy gained by binding ATP moves kinesin neck linker from rearward-facing position forward to dock against catalytic core of head ? Motion of one neck pulls other free from previous microtubule binding site and throws it forward to the next site (~80 ?) o Origin or directionality: myosin goes opposite direction from kinesin ? Directionality comes from conformation matching of head to polymers in one direction combined with time sequence of head release from polymer ? Both motors have an upstroke on binding ATP, downstroke on hydrolysis x Kinesin neck docks onto head on upstroke x Myosin: tight binding of head to polymer in ATP-free state o Forward motion on upstroke when head releases from polymer x Kinesin: tight binding in ATP-bound state o Reverse motion on downstroke when head releases from polymer Lecture 13 – Hybrid macromolecules 6 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 SHOW SCHEMATICALLY ON BOARD: Some of Matt Lang’s work? Engineering devices for nanoscale assembly using nature’s motors (Hess et al. 2001) ? Question: How can we manipulate, move, and assemble objects with nanoscale sizes? E.g. individual proteins, nanocrystals, etc. o AFM probe tip- one by one- too slow to be really useful in biosensors, lab-on-a-chip or other materials applications o Alternatives? ? Surfaces with microtubule nano-cargo carriers (Hiratsuka et al. 8 ) o Discovered that kinesin molecules adsorbed to a surface could be used to drive random motion of microtubules in 2D ? Researchers sought to use photolithographically patterned surfaces to gain control over motion and develop nano-carriers with directed motion Lecture 13 – Hybrid macromolecules 7 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Random transport of microtubules over randomly oriented surface-bound kinesin molecules: QuickTime? and a éVélépébéN decompressor are needed to see this picture. (Hiratsuka et al, 2001) o Simple approach: tracks etched in a photoresist, exposing glass ? Kinesin motors adsorbed randomly onto exposed glass under conditions where adsorption to resist was minimal (high ionic strength and 0.1% tween surfactant present during adsorption) ? Circular tracks: x Tracks confine motion of microtubules approximately linearly forward or backward x No arrowheads: microtubules walk in both directions around circles x With arrowheads: microtubules on inside track move counter-clockwise, microtubules on outer track move clockwise o Arrowheads act as directional rectifiers, moving against direction of arrow, high probability of microtubule striking wall and reversing direction as it jumps to new set of kinesin molecules o Steady motion observed up to 2 hrs in the presence of ATP 20 μm Lecture 13 – Hybrid macromolecules 8 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Vogel, 2002) ? We don’t yet understand the physicochemical principles controlling molecular motor speed, unloading/loading of cargo A molecular rotor built from ATPase ? Question: how do we create engines to provide piconewton forces for nanodevices? o One answer: engineered materials based on protein motor-based engines ? Work of the group of Carlo Montemagno at UCLA (Dept. of Bioengineering) 9-13 ? F 1 fragment of adenosine triphosphate synthase (F 1 -ATPase) o Role of this protein in cell o Rotary motion during ATP hydrolysis as J subunit transitions between 3 equidistant positions around the ATPase complex ? No-load rotational velocity of ~17 revolutions per second o Generates > 80 pN?nm work o Approximately 100% efficient! o ~10 nm diameter Lecture 13 – Hybrid macromolecules 9 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 F 1 fragment of adenosine triphophate synthase (F 1 -ATPase) ? Montemagno’s group prepared mutants of this protein to use as ATP-fueled molecular motors for nanodevices 750-1400 nm ? ? ? 80 nm Hybrid ATPase Fluorescent microsphere axle?of the motor feet?for immobilization of motor feet?bind Ni-capped posts (Soong et al., 2000) (Bachand et al., 2000) Lecture 13 – Hybrid macromolecules 10 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 QuickTime? and a GIF decompressor are needed to see this picture. ATP-driven motors ? Creating motors with a chemical on/off switch 13 o Mutated ATP binding face of ATPase to contain a 3-amino acid Zinc ion binding domain (3 histidines) o Mutant protein binds zinc and zinc blocks action of motor ? Classical allosteric enzyme inhibition o Chelation of zinc returns motor to active state Turning nanorotors addition of Zn ++ and Zn ++ sensitivity of motor: on and off by a Zn ++ chelator: ? Assembling these hybrid proteins into molecular devices Lecture 13 – Hybrid macromolecules 11 of 13 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Key need for device design: controlled placement of motors on surfaces Combining the hybrid Fluorescent microsphere molecular motor with engineered materials as a step toward nanodevices Hybrid ATPase (Bachand et al., 2000) References 1. Wu, C. & Wang, X. H. Globule-to-coil transition of a single homopolymer chain in solution. Physical Review Letters 80, 4092-4094 (1998). 2. Ding, Z., Fong, R. B., Long, C. J., Stayton, P. S. & Hoffman, A. S. Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield. Nature 411, 59-62 (2001). 3. Bulmus, V., Ding, Z., Long, C. J., Stayton, P. S. & Hoffman, A. S. Site-specific polymer-streptavidin bioconjugate for pH-controlled binding and triggered release of biotin. Bioconjug Chem 11, 78-83 (2000). 4. Shimoboji, T., Ding, Z., Stayton, P. S. & Hoffman, A. S. Mechanistic investigation of smart polymer-protein conjugates. Bioconjug Chem 12, 314-9 (2001). 5. Ding, Z. et al. Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate. Bioconjug Chem 10, 395-400 (1999). 6. Vogel, V. Reverse engineering: Learning from proteins how to enhance the performance of synthetic nanosystems. Mrs Bulletin 27, 972-978 (2002). 7. Vale, R. D. & Milligan, R. A. The way things move: looking under the hood of molecular motor proteins. Science 288, 88-95 (2000). 8. Hiratsuka, Y., Tada, T., Oiwa, K., Kanayama, T. & Uyeda, T. Q. Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks. Biophys J 81, 1555-61 (2001). 9. Montemagno, C. Biomolecular motors: Engines for nanofabricated systems. Abstracts of Papers of the American Chemical Society 221, U561-U561 (2001). 10. Montemagno, C. & Bachand, G. Constructing nanomechanical devices powered by biomolecular motors. Nanotechnology 10, 225-231 (1999). 11. Bachand, G. D. et al. Precision attachment of individual F-1-ATPase biomolecular motors on nanofabricated substrates. Nano Letters 1, 42-44 (2001). 12. Soong, R. K. et al. Powering an inorganic nanodevice with a biomolecular motor. Science 290, 1555-1558 (2000). 13. Liu, H. Q. et al. Control of a biomolecular motor-powered nanodevice with an engineered chemical switch. Nature Materials 1, 173-177 (2002). Lecture 13 – Hybrid macromolecules 12 of 13