MEMBRANE TRANSPORT

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MEMBRANE TRANSPORT BIOENERGETICS Richard
Neutze Richard.Neutze_at_chembio.chalmers.se Ph.
773 3974 http//www.csb.gu.se/neutze/bioenergetics
.html Aims Present a detailed analysis of the
mechanisms of energy transduction within the
cell. Emphasis placed on the interplay between
structure function for membrane proteins
governing energy transduction.
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• Tutorials
• Aim to provide skills resources for the
  literature project.
• Using structural citation data bases.
• Packages for structural analysis.
• Making figures.
• Writing scientific reports.
• Presentation of literature study.
• Literature study will
• Work in pairs.
• Choose one structure-related research article.
• Ask what new information concerning the
  functional mechanism of this protein has emerged
  from its structure?
• Write a report of not more than eight pages.
• Give a 15 minute presentation of the project.

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• Course Assessment
• 50 written exam.
• Wednesday December 12th - time we discuss now.
• A literature project.
• 40 written project.
• - Due Monday December 6th.
• 10 presentation.
• Thursday December 2nd.
• Grades 5, 4, 3 or fail.

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• Science the way it is!
• 1900 Physicists thought they were close to a
  theory of everything.
• Newton gravitation worked well.
• Electricity magnetism unified by Maxwell.
• Milky Way seemed to encompass everything.
• Within the first two decades of 20th century
• Quantum Mechanics.
• Theory of Relativity.
• Other galaxies discovered.
• 21st century the century for biology!
• Piecing together the jig-saw of life will
  uncover numerous remarkable phenomenon.

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• The Big Bang
• 14 billion years ago the universe began from a
  point.
• - Quantum fluctuations borrowed'' just enough
  from the vacuum to kick it all off.
• - Positive mass energy negative gravitational
  0!
• After 300 thousand years hydrogen condensed
  from a plasma.
• - Stars condensed from hydrogen.
• Inhomogeneities led to galaxy formation.
• 200 billion stars in a galaxy.
• 100 billion galaxies in the universe.

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• Star cycle
• Stars burn'' hydrogen
• Hydrogen fusion product is helium.
• Fusion products of helium hydrogen etc.
  heavier atoms (eg. carbon, oxygen, nitrogen etc).
  
• When stars burn out'' they collapse.
• Massive gravitationally driven heating.
• Heating drives supernova (huge explosions).
• More complex atoms created.

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• Solar system creation
• Began after one (or more) local supernova 4.6
  billion years ago.
• - Our sun a second or third generation star.
• Inner planets formed from collisions of
  moon-sized planetismals.
• Venus, Earth Mars received similar inventories
  of C, O H2O.
• Mars still has frozen water below the surface.
• Thin (not massive enough) CO2 atmosphere cold!
• Venus lost water due to a runaway greenhouse
  effect.
• - Water vapor in the high atmosphere is
  photolysed into H2 O, H2 lost to space.
• Thick CO2 atmosphere hot (500oC)!

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• Earth
• Most CO2 in carbonate materials (eg. limestone).
• - CO2 blanket much less no runaway greenhouse
  effect.
• - Water on the surface.
• Volcanic activity.
• - Recycles the biosphere.
• Magnetic field.
• - Protects against solar (ions) radiation.

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• Moon
• Pro-Earth struck by an inner planet Mars 4.5
  billion years ago.
• - Ejected molten mantel into the orbit.
• - Some coalesced into the moon.
• Gave the earth its spin tilt.
• - Heating cooling pattern (day/night seasons)
  critical.
• Exaggerated tides.
• - Powers plate tectonics volcanism.
• - Volcanism provides reducing cations recycles
  biosphere.
• Role in evolution?

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• Early bioenergetics
• First life about 3.8 billion years ago.
• Earliest replicating system possibly an RNA
  molecule.
• RNA world located in rock pores around volcanic
  springs.
• Lived on redox contrast of more oxidised
  atmosphere/ocean more reduced fluids in contact
  with volcanic magma.
• Dissolved suplhate provide oxidation power for
  organisms to react against hydrothermal fluids,
  eg. hydrogen methane.
• ATP addiction probably emerged from the RNA
  world.
• Ribozymes (catalytic RNA) can polymerise RNA-NTP
  molecules (NTP nucleotide tri-phosphates).
• 14 nucleotide addition with 97 fidelity was
  demonstrated.
• Early metabolism of RNA world based on an NTP.
• Triphosphate bond releases 10 kcal/mol is
  stable (10 -10/min).

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• Cell membranes
• RNA based life contained some cellular
  compartmentalization.
• Compartments keep replicase genomic RNA
  together.
• Evolutionary advantages remain with the organism
  which produced it.
• Cells in contemporary biology surrounded by
  amphiphatic lipids.
• In early life compartmentalization could be
  achieved by
• Organising centres (like a rybozome).
• Organisation along a surface.
• Passive compartmentalization within pores of
  rocks, or surface of fine particles.

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• DNA proteins
• RNA world invented of protein synthesis.
• Crowning achievement.
• Protein synthesis instructed catalysed by RNA.
• Rybosome has RNA _at_ amino-acid polymerisation
  active site.
• Proteins more versatile efficient in
  catalysis.
• DNA more chemically stable than RNA.
• - Allows larger genomes.
• DNA not catalytically active.
• - Forms a partnership with proteins.

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• Evolution of photosynthesis
• Accidental use of pigments where disequillibria
  easily exploited.
• Anoxygenic photosynthesis may have evolved from
  bacterium using infrared thermotaxis.
• Organism drifted into shallow water used
  sunlight.
• Later evolution of an oxygen generating complex
  exploiting Mn4O4 to Mn4O6 chemistry to generate
  O2 from H2O.
• 3.5 billion years ago cyanobacteria evolved.
• Probably a genetic exchange between
  interdependent green purple bacteria.
• Created an organism which could live freely on
  the planet wherever H2O, CO2 light available.
• Tremendous increase in the biosphere.

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• Rubisco carbon dating
• Rubisco is an ancient enzyme which fixes
  atmospheric CO2.
• When there is excess it prefers 12C to 13C.
• Measuring 12C to 13C ratio in rocks the standard
  method of carbon dating.
• Rubisco predates oxygenic photosynthesis.
• A qwerty enzyme.
• The qwerty keyboard - designed to avoid
  mechanical jamming far from optimal.
• Rubisco fixes CO2, but may also fix O2 (ie. undo
  the benefits of photosynthesis).
• This ineffeciency maintains reasonable CO2
  levels in the atmosphere.

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• Change of atmosphere
• Debate as to when the atmosphere became O2 rich.
• - Entire biosphere now rich'' in redox
  potential.
• Oxygenic photosynthesis appeared 3.5 billion
  years ago.
• Global oxygen production similar order of
  magnitude ever since.
• But levels depend on consumption also!
• Evidence that O2 levels increased 2.2 billion
  years ago.
• - Possibly due to complex eukaryotes.
• Biodiversity took off.

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• Four aeons
• Hadean
• - From formation of the planet to early life.
• Archaean
• - Prokaryotic life.
• Proterozoic
• - First eukaryotes.
• Phanerozoic.
• Life visible without need of a microscope.

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• Amphipathic molecules
• Solubility depends on favorable interactions
  with water.
• Hydrogen bonds.
• Ionic interactions.
• Polar compounds.
• Hydrocarbons are non-polar, non ionic cannot
  form H-bonds.
• - Hydrophobic not hydrophilic substances.
• Amphipathic molecules have a hydrophilic head
  group a
• hydrophobic tail.
• - Form mono-layers, micelles and bilayer vesicles.

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• Lipid bilayers
• Lipids have polar hydrophilic head''
  hydrophobic tail''.
• Form lipid-bilayer membranes.
• Most biological membrane lipids have two
  hydrocarbon tails.
• Divide different compartments of the cell.
• Allow non-polar molecules to move within the
  membrane.
• Prevent the diffusion of polar molecules across
  the membrane.
• Can with-stand 200 mV over 40Å (50 MV/m).
• Glycerophospholipids.
• - Major class of naturally occurring
  phospholipids (phosphate containing head-group).

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• Transport across membranes
• A cell is neither entirely open or closed to its
  surroundings.
• Interior of cell protected from toxic
  substances.
• Metabolites imported into the cell.
• Waste products extracted from the cell.
• Changes in cell environment detected.
• Inter-cellular signals transmitted.

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• Passive transport.
• - Accomplished by the random diffusion of
  molecules through the membrane (non-polar
  molecules only).
• Facilitated transport
• Ion pores, which define holes'' in the
  membrane.
• Carrier molecules, which form a hydrophobic
  casing.
• Both allow only specific ions to move through
  the membrane.

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• Active transport.
• - Integral membrane proteins use chemical or
  light energy to pump ions against a concentration
  gradient (eg. H or K).

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• Modern bioenergetics
• Biology harvests the energy content of light.
• - Photosynthesis ? chemical energy.
• ATP is the basic energy currency'' of the
  cell.
• Produced by energy-transducing membrane
  proteins.
• Protons are first pumped across a cell membrane.
• ATP-synthase harvests the proton concentration
  gradient to generate ATP from ADP Pi.

Chemical energy to light Light to
chemical energy
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• Mitochondria
• Found within eukaryotic cells.
• Primary producer of ATP.
• - Basic workhorse'' of the cell.
• Typically 0.7 to 1.0 mm long.
• Outermembrane has porins allowing free access of
  small particles.
• Approximately 500 mg/ml of the inner membrane is
  protein.

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• Respiratory chain of mitochondria
• Membrane proteins transfer e- from NADH or
  Succinate to O2.
• Electrons enter at complex I or II.
• Complex I, III and IV pump protons.
• Complex IV reduces O2 to H2O.
• Complex V (ATP-synthase) uses the
  proton-gradient to generate ATP from ADP Pi.

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• ATPsynthase
• ATPsynthase harvests the H gradient to
  regenerate ATP from
• ADP Pi.
• Back diffusion of H causes membrane portion to
  rotate.
• Mechanically coupled to the soluble portion.
• Maintains the ATP/ADP ratio 10 orders of
  magnitude from equilibrium.
• Excess ATP used as the energy currency of the
  cell.
• Mechanism conserved throughout biology.

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• Summary of lecture 1
• Universe 14 billion years old.
• Earth/solar system 4.5 billion years old.
• First life appeared 3.5 billion years ago.
• ATP addiction inherited from RNA world.
• Overwhelming source of ATP is highly a conserved
  process in energy-transducing membranes.
• To understand the bioenergetics must understand
  proton translocation processes.