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Principles of Operation of Nanodevices and Micromachines of the living cell Metro Anatomy and Physiology Fall 2016 Stanley Misler <[email protected] Review "What is Life?“ a. The basic unit of life is the cell, a nanodevice-based, multi-tasking chemical reactor with unique abilities to self replicate via genetic information (DNA), self regulate and adapt to environmental changes. 1. 2. 3. 4. Nanodevices are found (a) within cell membranes (lipid bilayer), (b) loose in the watery cytoplasm; or (c) clustered in organelles & working together as micromachines Nanodevices are synthesized de novo and then, after varying time, are degraded; these processes require input of energy and a nearly error free genetic coding program Biological nanodevices are specially folded during their synthesis (i) to recognize specific ligands (often small molecule) that can enter into a crevice in the molecule or (ii) to change configuration in the presence of light, and electricity or mechanical pull. Most important take home message: Protein nanodevices respond to stimuli with a small and reversible stepwise changes in conformation over micro- to millisec. Protein nanodevices include: (a) enzymes = protein catalysts that speed up chemical reactions but are not destroyed in the process; (b) cell surface receptors acting as switches turning an extracellular chemical stimulus into a cell response; (c) the cytoskeleton assembling and disassembling tracts on which material is quickly transported from region to region; (d) channels that allow ions and water to move down chemical gradients; (e) chaparones to aid in protein folding; (f) extracellular matrix serving as protein adhesives anchoring cell to tissue support (body glue); and (g) motors that rotate or step Organelle micromachines include (i) vesicles with varying contents; these can fuse with the cell membrane (exocytosis) and release their contents or pinch off the cell membrane for retrieval (endocytosis) often with cargo inside and (ii) mitochondria that break down metabolite monomers and store bond energies ATP Nanodevices are most often specially folded proteins amino acid sequence of polypeptide chain by amide linkages Folding formation of polypeptide chains Multiple polypeptide chains interact to form a complete complex protein Cell cytoplasm and organelles are surrounded by lipid bilayer membranes “Fluid mosaic” model of membrane: Proteins may be attached to (A) or embedded into one side of lipid bilayer (B) or else traverse the bilayer (C) Proteins can be solubilized from membrane by detergents and rendered inactive in situ by proteases Functions of lipid bilayer membranes: (i) Compartmentalization (ii) Scaffold for enzymes (iii) Selective permeability barrier: The more lipid soluble a chemical species is the more likely it is to transfer across a membrane (iv) Signal transduction for response to external stimuli (v) Lipid rafts for anchoring of protein clusters (vi) Self sealing A. protein enzyme acting as a catalyst Enzymes speed up what would otherwise be slow but “spontaneous” reaction from substrate state (A+B) to product state (P+Q) which has lower free energy (a) Enzymes allow the binding of substrate, via multiple weak attractions, to crevices serving as “active site” within the protein. This produces subtle changes in the configuration of the whole protein allowing the stretching and breaking of covalent bonds within the substrate. (b) Looked at it another way, binding of substrate to active site lowers the critical activation energy barrier that must be surmounted to make the transition state (X or activated substrate/enzyme complex) more stable (see middle). Realistically reactions usually involve several transition states (see right) e.g., Enzyme (urease) vs. metallic catalyst (Pt) 1. Enzymes have much higher reaction rates than metallic catalysts thus speeding up time to reach equilibrium concentrations of reactants and products. To get enzyme to function against equilibrium, or in a situation where product has more energy than substrate, requires input of energy (coupling to ATP hydrolysis). 2. Enzymes work at milder, more physiological temperatures, pressures and substrate concentrations than metallic catalyst. 3. Enzymes have greater reaction specificities due to need for substrate to snugly fit in enzyme pocket. This leads to fewer errors of function and fewer unwanted side products 4. Enzymes often need cofactors where vitamins are often serve as cofactor precursors. 5. Enzymes have large capacity for regulation by: (a) D availability = balance of synthesis vs. degradation; (b) D activity = increases by covalent modification (addition of phosphate = phosphorylation) and feedback inhibition by end products down the pipe line 6. Enzymes often need assistance of the insertion of water to make a transition state possible Inhibition of one substrate reactions Multienzyme complexes Ex. In muscle, particles of glycogen are stored within a cluster of enzymes that work sequentially (handing off substrate from one enzyme to another) for coordinated breakdown of glycogen to lactate B. Ion channels : Motivation for their study How ions might cross a lipid bilayer membrane of consistency of olive oil (into which ions cannot dissolve) Ion channels 1. Ion channels are complex multisubunit intramembrane proteins spanning the cell membrane. Enclosed within these proteins are transmembrane conduction pores which selectively allow transfer of specific ions down their concentration gradients at a rate at > 10-fold greater free diffusion in solution. 2. Access to the conducting pore can be open or closed (channel gating). The rate of opening vs closing is determined by transmembrane voltage, extracellular or intracellular molecules that bind to the channel, membrane stretch, or extent of phosphorylation of channel. 3. Channel gating by these variables contribute to a multitude of cellular functions including: (a) membrane excitability (action potential and synaptic potential) (b) neurotransmitter or hormone release, both over ms; (c) initiation of second messenger cascades (e.g., requiring Ca entry) over s to min; (d) cell volume regulation over min; (e) regulation of cell division and apoptosis (programmed cell death) over min to h 4. Changes in channel expression or folding are responsible for many diseases such as cystic fibrosis, epilepsy (seizures), and heart arrhythmias 5. Ion pumps also are proteins that also contain a pore but ion access to or release from the pore only happens when ATP is hydrolyzed Pictorial model of channel architecture seen in crosssection: not a featureless barrel open at both ends. a. Channels contain ion selective central pores that selectively transport ion down their concentration gradients without much or any accompanying movement of water, b. Opening of channel by voltage step is based on reorientation of voltage sensor in response to change in electric field. This uncrosses the criss-crossed tails opening the vestibule (or undressing chamber) at the cytoplasmic side of protein allowing the vestibule to fill with solvated ions c. In the case of well studied K selective channels, K+ ions shed their waters of hydration and single denuded K ions move from the vestibule (undressing chamber) into the selectivity filter due to attraction to a charge dipole buried nearby in the protein. The wall of the selectivity filter is lined with carboxyl sites (C=O) that together form local ion cages. Ions attracted into selectivity filter jump from one caging region to another, powered by the electrochemical gradient; in the case of K there are many more ions near inner region of vestibule than in extracellular fluid. Ion selectivity is based on the size of the ion relative to the diameter of the pore as well as on the affinity of ions binding to the C=O cage. d. With prolonged exposure to depolarizing Vc, a ball-on-chain type charged structure get sucked into vestibule resulting in self blockade or inactivation Combining ion channels & ion pumps for complex function Segregation in time -> Segregation in space -> Excitable cell (nerve, muscle) Salt & H2O transporting cell (kidney or gut) 1. Trans-(kidney or gut) C. Cell surface receptors (top) vs. cytosolic receptors (bottom) in triggering a cell response Ligand binding to G-protein coupled receptor initiation of cascade-like response Amplification: 1-2 adrenaline molecules bind to a bAR (beta adrenergic receptor) which then interacts promiscuously with several GTP binding (or G) proteins which each release an a subunit. Free a subunits each interact with one AC (adenylate cyclase) that in turn produces several cyclic AMP molecules (cAMPs). cAMPs binds to receptor subunits of a cytosolic protein kinase A (PKA), releasing the catalytic subunit (C). C in turn phosphorylates (adds phosphate to) specific residues on many members of a species of target proteins (in this case a voltage activated Ca channel) to enhance their function (here Ca channel stays open longer in response to voltage activation) D. Molecular Motors Getting “pull” out of a cycle of ATP binding/hydrolysis Power stroke: Hydrolysis of ATP allows attachment of flexible hinge molecule to more rigid structure and motion of hinge at flexion point Straightening of hinge and pull on rigid structure with release of PO4 Displacement of ADP by ATP produces molecular detachment Summary: Nanodevices are combined to produce micromachines with complex functions and pumps t Cartoons of nanodevices making life possible Action potential attachment to Contractile tension