Cellular Machines at TU Dresden | Flashcards & Summaries

Lernmaterialien für Cellular Machines an der TU Dresden

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TESTE DEIN WISSEN

Explain the structure and functional principles of the bacterial flagellar motor!


Lösung anzeigen
TESTE DEIN WISSEN
  • flagella are driven by rotary electric motor
    1. 40-100 protons per rotation
    2. 2 protons per step
    3. transfer of one proton has same energy as hydrolysis of ATP
    4. rotates with up to 170 Hz; typically a few 100 Hz with attached flagellum
    5. can switch direction in quarter turn; clutch
      • counterclockwise (CCW): filaments bundle --> swimming
      • clockwise (CW): flagella de-bundle --> tumbling 
      • reversal frequency depends on CheY concentration and phosphorylation state influenced by methylation of the receptor (adaptation) (normal reverssl rate approx. 1s)
  • = self-assembled protein complex around 50 nm diameter and molecular mass of around 11 kDa
  • rotor rotates relative to cell
    1. MS ring: around 26 copies of the FliF proteins --> stepwise rotation
    2. C ring: site of torque generation; inside is export machinery for flagellar assembly
  • stator is anchored in cell wall
    1. stator complex in E.coli: MotA and MotB in H+-driven motors
    2. stator complex in Vibrio cholera: PomA and PomB in Na+-driven motors
  • flexible hook is attached to filament
Lösung ausblenden
TESTE DEIN WISSEN

How do cone cells see different colors?


Lösung anzeigen
TESTE DEIN WISSEN
  • cones allow for focused and color daylight vision
  • different types of cones (for different colors – blue, green, red)
  • can be activated by different wavelength --> can mix later together for final color
  • have all same chromophore (11-cis retinal) but spectral sensitivity:
    1. S-cones (short wavelength receptor) ~ 12% - blue
    2. M-cones (medium wavelength receptor) variable – green
    3. L-cones (long wavelength receptor) variable – red
  • different light absorption peaks due to different AA groups in opsin part
Lösung ausblenden
TESTE DEIN WISSEN

Which two general types of helicases exist and what is their mechanism of directed DNA translocation?


Lösung anzeigen
TESTE DEIN WISSEN
  • Helicases convert chemical energy of nucleoside triphosphate (NTP) hydrolysis to the mechanical energy necessary to transiently separate the strands of duplex nucleic acids
  • SF1 and 2 consists of two fused RecA-like domains --> inchworm mechanism  
    1. inchworm’ mechanisms that require coordinated alternate binding of nucleic acid at two different sites within the functional unit of the helicase
    2. can function as mono- or dimers --> monomer containing two binding sites or by a dimer with a single binding site per subunit
  • SF3-6 consist of hexameric 6 RecA-like domains --> hexameric helicases 
    1. ssDNA strand tethered through the central tunnel of the hexameric ring
    2. actual unwinding of the DNA duplex is achieved by sterically excluding the non-translocating DNA strand from entering the central tunnel
Lösung ausblenden
TESTE DEIN WISSEN

Explain two methods to determine the diffusion constant of lipids (and proteins) in a lipid bilayer?

Lösung anzeigen
TESTE DEIN WISSEN

Fluorescence after photobleaching

  • target molecule labelled with fluorescent tag - - >measure initial intensity of fluorescence 
  • destroy fluorophore with high intensity laser pulse - - >irreversible non-fluorescent mode of molecules
  • slow diffusion of environment  --> alteration of fluorescence intensity over time due to the diffusion of unbleached molecules into region of interest
How lipids fluore. Tagged? How constant determined from that

Optical tweezers

  1. Highly focused laser beam to provide attractive or repulsive force
  2. Hold with biomolecule coated beads in place - - > measure applied force
Lösung ausblenden
TESTE DEIN WISSEN

What kind of carriers exist?

Lösung anzeigen
TESTE DEIN WISSEN
  • Carriers provide passive pathways for solutes, move down conc. Gradient; Conformational change translocates limited number of solutes
  • Uniporters: facilitate diffusion down conc. Gradient
    • glucose, amino acids
  • Symporters: co-transport of multiple substrates
    • sugar-sodium-symporter, sodium-chloride
  • Antiporters: substrate against conc. Gradient in exchange for ion/molecule down conc. Gradient
    • sodium-hydrogen, sodium-calcium
Lösung ausblenden
TESTE DEIN WISSEN

What energy source can be used for pumps and what can be pumped across membranes?

Lösung anzeigen
TESTE DEIN WISSEN
  • energy used mostly from ATP or light 
  • ions and larger, unpolar substrates  (solutes) pumped across 
  • bacteriorhodopsin (light driven proton pump)
  • rotary motors (F-/ V-ATPases)
  • P-type cation pump (primary ion gradient) --> ABC transporter, sodium/potassium pump
Lösung ausblenden
TESTE DEIN WISSEN

Name three different functions of channels and how their opening is controlled.

Lösung anzeigen
TESTE DEIN WISSEN

Functions:

  1. transport of water, ions across membrane
  2. regulate membrane potentials
  3. signaling (Ca2+)
  4. sensing, important targets for toxins, medicine

Gating:

  • voltage-gated (action potentials, muscle contraction, protein kinase)
  • ligand-gated (ATP, peptide, intracellular ligands (calmodulin, nucleotides), extracelllar (acetylcholin, serotonin))
  • mechanically gated (hearing, balance, touch)
Lösung ausblenden
TESTE DEIN WISSEN

What are the most important proteins (+ their functions) in a prokaryotic replication fork?

Lösung anzeigen
TESTE DEIN WISSEN
  • Helicases: catalyze unwinding of double stranded DNA to ss DNA (breaking H-binds between nucleotides) under ATP hydrolysis
  • Single strand binding protein: prevents rewinding near replication fork
  • Topoisomerase: binds at region ahead from replication fork, prevents supercoiling (overwinding)
  • RNA primase: synthesizes 5 nucleotide-long primers (doesn’t require free 3 Oh-group)
  • DNA polymerase III: adds nucleotides one by one to growing DNA strand, consumption of ATP 
  • DNA pol I removes RNA primers and fills gaps
  • DNA ligase: seals gaps between DNA fragments
Lösung ausblenden
TESTE DEIN WISSEN

What energy source can be used for pumps and what can be pumped across membranes?

Lösung anzeigen
TESTE DEIN WISSEN
  • energy used mostly from ATP or light 
  • ions and larger, unpolar substrates  (solutes) pumped across 
  • bacteriorhodopsin (light driven proton pump)
  • rotary motors (F-/ V-ATPases)
  • P-type cation pump (primary ion gradient) --> ABC transporter, sodium/potassium pump
Lösung ausblenden
TESTE DEIN WISSEN

How can helicase activity be monitored? Sketch and explain two single-molecule assays to study helicase unwinding activity?

Lösung anzeigen
TESTE DEIN WISSEN

optical tweezers and confocal microscopy

  • Both ssDNA strands attached to beads influenced by optical (or magnetic) tweezers
  • Measure destructive force due to unwinding (DNA gets longer) due to helicase activity
  • Pulling force at beads increases the helicase activity

Single-molecule FRET

  • Two fluorophores on DNA (acceptor and donor) --> energy resonance transfer
  • One ssDNA stand immobilized to surface
  • Measure fluorescence of both molecules
  • Proximity: acceptor fluorescence
  • Increasing distance due to helicase activity fluorescence of donor increases (less energy resonance transfer)
  • Large distance due to completed helicase activity à no fluorescence
Lösung ausblenden
TESTE DEIN WISSEN

Why is a primer on the lagging strand made out of RNA?


Lösung anzeigen
TESTE DEIN WISSEN
  • on lagging strand 3'->5' many primers necessary for rapid replication 
  • primase synthesizes RNA quickly but with a higher error rate 
  • RNase H removes RNA primer (which could contain errors) --> targets only DNA/RNA heteroduplex
  • POL I precisely inserts the matching DNA nucleotides 


Lösung ausblenden
TESTE DEIN WISSEN

Explain three different artificial membrane systems!

Lösung anzeigen
TESTE DEIN WISSEN

Black Lipid Bilayer 

  • free standing bilayer in a hole of teflon partitions --> hydrophobic support
  • seperation of two compartments by bilayer
  • synthesized by paintig 1-2% Phospholipidsolution over aperture or by folding bilayer through lowering and raising fluid level in one compartment 
  • investigation of ion channel, engineered single pore sensors 
  • poor stability  

Solid supported lipid bilayer (SLB)

  • bilayer formed near surface 
  • Hydrophilic, smooth solid support: glass, mica, silica
  • More robust & stable than black lipid membranes
  • Aqueous solution only above
  • synthesis through vesicle fusion or Langmuir-Blodgett technique (pulling a hydrophilic substate through a lipid monolayer & subsequently pushing it through another lipid monolayer)
  • study lipid domain formation, lipid and membran protein diffusion, pore formation, intermembrane interactions (vSNARE), membrane processes (protein absorption, -function, &self-assembly)

Liposomes

  • spherical vesicles that consist of an aqueous compartment enclosed by one or several concentric phospholipid bilayer 
  • Hydration of dry lipid film (evaporation - addition of aqueous solution - heat - freeze thaw - filter) 
  • Reverse phase evaporation (higher scalable) 
  • Nano container (reaction chamber), drug delivery system 
Lösung ausblenden
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Q:

Explain the structure and functional principles of the bacterial flagellar motor!


A:
  • flagella are driven by rotary electric motor
    1. 40-100 protons per rotation
    2. 2 protons per step
    3. transfer of one proton has same energy as hydrolysis of ATP
    4. rotates with up to 170 Hz; typically a few 100 Hz with attached flagellum
    5. can switch direction in quarter turn; clutch
      • counterclockwise (CCW): filaments bundle --> swimming
      • clockwise (CW): flagella de-bundle --> tumbling 
      • reversal frequency depends on CheY concentration and phosphorylation state influenced by methylation of the receptor (adaptation) (normal reverssl rate approx. 1s)
  • = self-assembled protein complex around 50 nm diameter and molecular mass of around 11 kDa
  • rotor rotates relative to cell
    1. MS ring: around 26 copies of the FliF proteins --> stepwise rotation
    2. C ring: site of torque generation; inside is export machinery for flagellar assembly
  • stator is anchored in cell wall
    1. stator complex in E.coli: MotA and MotB in H+-driven motors
    2. stator complex in Vibrio cholera: PomA and PomB in Na+-driven motors
  • flexible hook is attached to filament
Q:

How do cone cells see different colors?


A:
  • cones allow for focused and color daylight vision
  • different types of cones (for different colors – blue, green, red)
  • can be activated by different wavelength --> can mix later together for final color
  • have all same chromophore (11-cis retinal) but spectral sensitivity:
    1. S-cones (short wavelength receptor) ~ 12% - blue
    2. M-cones (medium wavelength receptor) variable – green
    3. L-cones (long wavelength receptor) variable – red
  • different light absorption peaks due to different AA groups in opsin part
Q:

Which two general types of helicases exist and what is their mechanism of directed DNA translocation?


A:
  • Helicases convert chemical energy of nucleoside triphosphate (NTP) hydrolysis to the mechanical energy necessary to transiently separate the strands of duplex nucleic acids
  • SF1 and 2 consists of two fused RecA-like domains --> inchworm mechanism  
    1. inchworm’ mechanisms that require coordinated alternate binding of nucleic acid at two different sites within the functional unit of the helicase
    2. can function as mono- or dimers --> monomer containing two binding sites or by a dimer with a single binding site per subunit
  • SF3-6 consist of hexameric 6 RecA-like domains --> hexameric helicases 
    1. ssDNA strand tethered through the central tunnel of the hexameric ring
    2. actual unwinding of the DNA duplex is achieved by sterically excluding the non-translocating DNA strand from entering the central tunnel
Q:

Explain two methods to determine the diffusion constant of lipids (and proteins) in a lipid bilayer?

A:

Fluorescence after photobleaching

  • target molecule labelled with fluorescent tag - - >measure initial intensity of fluorescence 
  • destroy fluorophore with high intensity laser pulse - - >irreversible non-fluorescent mode of molecules
  • slow diffusion of environment  --> alteration of fluorescence intensity over time due to the diffusion of unbleached molecules into region of interest
How lipids fluore. Tagged? How constant determined from that

Optical tweezers

  1. Highly focused laser beam to provide attractive or repulsive force
  2. Hold with biomolecule coated beads in place - - > measure applied force
Q:

What kind of carriers exist?

A:
  • Carriers provide passive pathways for solutes, move down conc. Gradient; Conformational change translocates limited number of solutes
  • Uniporters: facilitate diffusion down conc. Gradient
    • glucose, amino acids
  • Symporters: co-transport of multiple substrates
    • sugar-sodium-symporter, sodium-chloride
  • Antiporters: substrate against conc. Gradient in exchange for ion/molecule down conc. Gradient
    • sodium-hydrogen, sodium-calcium
Mehr Karteikarten anzeigen
Q:

What energy source can be used for pumps and what can be pumped across membranes?

A:
  • energy used mostly from ATP or light 
  • ions and larger, unpolar substrates  (solutes) pumped across 
  • bacteriorhodopsin (light driven proton pump)
  • rotary motors (F-/ V-ATPases)
  • P-type cation pump (primary ion gradient) --> ABC transporter, sodium/potassium pump
Q:

Name three different functions of channels and how their opening is controlled.

A:

Functions:

  1. transport of water, ions across membrane
  2. regulate membrane potentials
  3. signaling (Ca2+)
  4. sensing, important targets for toxins, medicine

Gating:

  • voltage-gated (action potentials, muscle contraction, protein kinase)
  • ligand-gated (ATP, peptide, intracellular ligands (calmodulin, nucleotides), extracelllar (acetylcholin, serotonin))
  • mechanically gated (hearing, balance, touch)
Q:

What are the most important proteins (+ their functions) in a prokaryotic replication fork?

A:
  • Helicases: catalyze unwinding of double stranded DNA to ss DNA (breaking H-binds between nucleotides) under ATP hydrolysis
  • Single strand binding protein: prevents rewinding near replication fork
  • Topoisomerase: binds at region ahead from replication fork, prevents supercoiling (overwinding)
  • RNA primase: synthesizes 5 nucleotide-long primers (doesn’t require free 3 Oh-group)
  • DNA polymerase III: adds nucleotides one by one to growing DNA strand, consumption of ATP 
  • DNA pol I removes RNA primers and fills gaps
  • DNA ligase: seals gaps between DNA fragments
Q:

What energy source can be used for pumps and what can be pumped across membranes?

A:
  • energy used mostly from ATP or light 
  • ions and larger, unpolar substrates  (solutes) pumped across 
  • bacteriorhodopsin (light driven proton pump)
  • rotary motors (F-/ V-ATPases)
  • P-type cation pump (primary ion gradient) --> ABC transporter, sodium/potassium pump
Q:

How can helicase activity be monitored? Sketch and explain two single-molecule assays to study helicase unwinding activity?

A:

optical tweezers and confocal microscopy

  • Both ssDNA strands attached to beads influenced by optical (or magnetic) tweezers
  • Measure destructive force due to unwinding (DNA gets longer) due to helicase activity
  • Pulling force at beads increases the helicase activity

Single-molecule FRET

  • Two fluorophores on DNA (acceptor and donor) --> energy resonance transfer
  • One ssDNA stand immobilized to surface
  • Measure fluorescence of both molecules
  • Proximity: acceptor fluorescence
  • Increasing distance due to helicase activity fluorescence of donor increases (less energy resonance transfer)
  • Large distance due to completed helicase activity à no fluorescence
Q:

Why is a primer on the lagging strand made out of RNA?


A:
  • on lagging strand 3'->5' many primers necessary for rapid replication 
  • primase synthesizes RNA quickly but with a higher error rate 
  • RNase H removes RNA primer (which could contain errors) --> targets only DNA/RNA heteroduplex
  • POL I precisely inserts the matching DNA nucleotides 


Q:

Explain three different artificial membrane systems!

A:

Black Lipid Bilayer 

  • free standing bilayer in a hole of teflon partitions --> hydrophobic support
  • seperation of two compartments by bilayer
  • synthesized by paintig 1-2% Phospholipidsolution over aperture or by folding bilayer through lowering and raising fluid level in one compartment 
  • investigation of ion channel, engineered single pore sensors 
  • poor stability  

Solid supported lipid bilayer (SLB)

  • bilayer formed near surface 
  • Hydrophilic, smooth solid support: glass, mica, silica
  • More robust & stable than black lipid membranes
  • Aqueous solution only above
  • synthesis through vesicle fusion or Langmuir-Blodgett technique (pulling a hydrophilic substate through a lipid monolayer & subsequently pushing it through another lipid monolayer)
  • study lipid domain formation, lipid and membran protein diffusion, pore formation, intermembrane interactions (vSNARE), membrane processes (protein absorption, -function, &self-assembly)

Liposomes

  • spherical vesicles that consist of an aqueous compartment enclosed by one or several concentric phospholipid bilayer 
  • Hydration of dry lipid film (evaporation - addition of aqueous solution - heat - freeze thaw - filter) 
  • Reverse phase evaporation (higher scalable) 
  • Nano container (reaction chamber), drug delivery system 
Cellular Machines

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Chapter 3: Cellular Level

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