Transcript Nanodevices

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.
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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