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Course outline. Introduction. Sizes. Organ Tissue Cell Molecule Atoms. A cell is an organization of millions of molecules Proper communication between these molecules is essential to the normal functioning of the cell
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Sizes Organ Tissue Cell Molecule Atoms • A cell is an organization of millions of molecules • Proper communication between these molecules is essential to the normal functioning of the cell • Structure provides an understanding of how molecules communicate
Nanotechnology • The creation of functional materials, devices and systems through control of matter at the scale of 1 to 100 nm, and the exploitation of novel properties and phenomena at the same scale. • Self-assembling highly functional molecular machines aimed at performing specific tasks. Often highly ordered repeated patterns of a single functional unit. • 1 nm = 0.0000000001 m or 0.0000001 mm
ribosomes make proteins in cells Molecular machines protein DNA mRNA
protein motors move material in cells ATP synthase rotor size: 10nm Molecular machines Nature, 386, 299 (1997)
Bacterial chemotaxis • Bacteria move using flagellar motors • Protein network directs movement based on external conditions and random motion: attractants/repellents • Simulate chemotaxis network (7 proteins)
Bacterial chemotaxis • Bacteria move towards chemical attractants and away from repellents • Process: • attractants/repellents bind to chemorecptors • chemoreceptors transmit information to a central processing system • central processing integrates many inputs and sends a signal to control flagellar motors • Interesting feature: adaptation of sensitivity [ Pfeffer ]
Movement of flagellar rotation • Bacteria swim by rotating flagella • Motor located at junction of flagellum and cell envelope • Motor can rotate clockwise orcounterclockwise CW CCW CW
Bacterial motor Bacterial motor and drive train. Above: Rotationally averaged reconstruction of electron micrographs of purified hook-basal bodies. The rings seen in the image and labeled in the schematic diagram (right) are the L ring, P ring, MS ring, and C ring. (Digital print courtesy of David DeRosier, Brandeis University.)
Biased random walk Bacteria • swim smoothly for 1sec (30 m) • tumble, change direction by an average of 60 deg
Tumbling frequency • Movement with respect to attractants: • increasing concentrations less tumbling • decreasing concentrations more tumbling • Temporal or spatial regulation? [Koshland/Macnab] • mix a bacterial suspension without attractant with solution containing attractant • tumbling suppressed within a second • bacteria swam for long distances in a straight line • solution has no spatial gradient temporal regulation! • Specifically, compares past second versus previous three seconds
Structure of the chemoreceptors ligand binding domain
Microtubule motor proteins Two main families of microtubule motor proteins carry out ATP-dependent movement along microtubules: • Kinesin: Most members of the kinesin family of motor proteins walk along microtubules toward the plus end, away from the centrosome (MTOC). • Dynein: The dyneins walk along microtubules toward the minus end (toward the centrosome). In each case there is postulated to be a reaction cycle similar (but not identical) to that of myosin. The motor domain undergoes conformational changes as ATP is bound and hydrolyzed, and products are released.
Kinesins • Kinesins are a large family of proteins with diverse structures. Mammalian cells have at least 40 different kinesin genes. • The best studied is referred to as conventional kinesin, kinesin I, or simply kinesin. • Some are referred to as kinesin-related proteins (KRPs). • Kinesin I has a structure analogous to but distinct from that of myosin. • There are 2 copies each of a heavy chain and a light chain.
Kinesin I • Each heavy chain of kinesin I includes a globular ATP-binding motor domainat the N-terminus. • Stalk domains of heavy chains interact in an a-helical coiled coil that extends from heavy chain neck to tail. • The coiled coil is interrupted by a few hinge regions that give flexibility to the otherwise stiff stalk domain.
Kinesin I • N-termini of the 2 light chains associate with the 2 heavy chains near the tail. The diagram above is over simplified. • Light chains at the N-terminus include a series of hydrophobic heptad repeats predicted to interact with similar repeats in the heavy chains near the tail region, in a 4-helix coiled coil.
Kinesin I • C-terminal tail domains of kinesin light chains include several "tetratrico peptide repeats" (TPRs). The 34 amino acid TPRs mediate protein-protein interactions. • Kinesin light chain TPRs are involved in binding of kinesins to cargo. • C terminal domains of heavy chains may also participate in binding some kinesins to cargo.
Cargo Cargoproteins bound by kinesins are diverse. • Some organelle membranes contain transmembrane receptor proteins that bind kinesins. Kinectin is an ER membrane receptor for kinesin-I. • Scaffolding proteins, first identified as being involved in assembling signal protein complexes, mediate binding of kinesin light chains to some cargo proteins or receptors. • Some membrane-associated Rab GTPases, that provide specificity for vesicle transport & fusion, are known to bind particular kinesins.
Cargo into contact with the motor domains. • In this folded over state kinesin exhibits decreased ATPase activity and diminished binding to microtubules. • This may prevent wasteful hydrolysis of ATP by kinesin when it is not transporting cargo. • In absence of cargo, the kinesin heavy chain stalk folds at hinge regions, bringing heavy chain tail domains
Cargo Unfolding of kinesin into its more extended active conformation is promoted by: • phosphorylation of kinesin light chains, catalyzed by a specific kinase, or • binding of cargo.
Kinesin transport Observations of conventional kinesin transporting elongated particles have demonstrated that cargo particles do not roll along the microtubule. Instead kinesin walks along, maintaining the orientation of a cargo particle.
Kinesin transport • Movement of the 2-headed kinesin is processive, meaning that it takes many steps without dissociating from a microtubule. A hand over hand reaction cycle involving the 2 heads has been proposed. • Myosin V, which transports vesicles along actin filaments, also exhibits processive movement.
Kinesin transport View an animation emphasizing the cycle of ATP binding, hydrolysis & product dissociation during processive movement of kinesin along a microtubule.
Flagella • Cilia & flagella are bounded by the plasma membrane. • A basal body, which is a single centriole cylinder, is at the base of each cilium or flagellum. • Cilia & flagella have a core axoneme, a complex of microtubules and associated proteins. Some distinctions: • Flagella are usually 1 or 2 per cell. They tend to have a rotary or sinusoidal movement. They may have additional structures outside the core axoneme • Cilia are usually many per cell. They tend to have a whip-like movement.
Flagella • An axoneme includes: • Nine doublet microtubules around the periphery. The A tubule of each doublet has attached dynein arms. • Two singlet central microtubules, surrounded by a sheath. • Nexin links & radial spokes. These provide elastic connections between microtubule doublets and between the A tubule of each doublet and the central sheath.
Flagella • Few mammalian cell types have motile cilia or flagella, including some respiratory epithelial cells and sperm cells. • Many mammalian cells have a single short non-motile primary cilium. • The photoreceptor structure of each retinal rod & cone cell develops from a non-motile cilium.
DNA secondary structures • Secondary structures are made of base pairs. They are stable with respect to free energy. • Nearest neighbor model (Zimm et al., 1964). Summing up stacking energies of adjacent base pairs and mismatched pairs • Folding problem (Zuker et al., 1981)
DNA secondary structures 3’ Base sequence (linear structure) Secondary structure folding 5’ 5’ 3’ TTC…GCA inverse folding
Thermo-dynamical model • Inverse folding problem (Hofacker et al., 1994). Optimization with the fold function for evaluation • Search for sub-optimal structures (Wuchty et al., 1999). Enumeration of (sub-optimal) structures whose energy is under mfe+d • Computation of the partition function (McCaskill, 1990). Computation of the frequency of a structure • Estimation of the energy barier between structures (Flamm et al., 2000).
DNA nanomachines • Various DNA nanomachine • DNA motor by B-Z transiton (Seeman et al., 1999) • molecular tweezers (Yurke et al., 2000) • three-state machine (Simmel et al., 2002) • PX-JX2 (Yan et al., 2002) • Hybridization inhibition by bulge loop (Tuberfield et al., 2003) • Designing DNA sequence with bistable structures (Flamm et al., 2001)
B-Z DNA nano-mechanical device Seeman, 1999
Yurke’s DNA tweezers http://news.bbc.co.uk/1/hi/sci/tech/873097.stm
Yurke’s DNA tweezers • Because the thermodynamic paths for opening and closing the molecular tweezers are different it is a thermodynamic engine. • It is a clocked molecular motor. • Biological molecular motors are catalysts that convert fuel to waist product. • Hence, DNA systems in which interactions are catalytically controlled are of interest in devising free running DNA motors.
