Molecular Motors: Flagella and Chemotaxis L6-7 (David Stephans) Flashcards
How many flagella do bacteria have?
One or more(3-5)
All types of bacteria have different types of organisation
What influences how fast a motor rotates?
The more protons there are, the faster it rotates (linear relationship)
The proton pump fills the periplasmic space with protons which rush back down concentration gradient through the motor-generates force
Rotation speed varies directly with the ion concentration
How many discrete steps do flagella rotate in?
26 discrete steps per revolution
14 degrees
What proteins is the motor made of?
FliF (single protein)-M and S rings
M=membrane S=supRAmembane (above the memb)
motor is made up of multiple copies of the single protein in FliF
FliF has 26 fold symmetry
Can rotate either clockwise or anticlockwise
What protein is the hook made of?
FlgE (helical conf)
adjacent to motor structure
FlgE has 5.5subunits per turn of the structure
11 fold symmetry
What protein is the flagella made of?
Flagellin = protein FliC=gene name
Central core/rod surrounded by proteinaceous coat
5.5 subunits per trun
11 fold symmetry
Symmetrical to hook
Hook and flagella directly couple to drive the rotation
What protein is the stator made of?
Stator forms proton or sodium gradient
MotAB
MotB=sits in periplasmic space-binds to peptidoglycan and anchors stator
MotA=sits in cellular membrane
MotA x4 MotB x2
How many stator subunits surround the flagellum?
11-12 per flagellum
symmetry mismatch with motor/MS ring (26 fold symmetry)
Asymmetry used to help act as a break and generate torque
What type of stator uses a sodium gradient?
Stators define the specificity of the transported ion (H+ or Na+)
PomA/B in vibrio species (single flagellum)
proton driven flagella exist in E.coli
What protein does the stator/MotA interact with to generate torque?
FliG (C-ring on rotor)
When you add an ion down this system you disrupt this contact - interferes with electrostatic contact
When the stator is released from the FliG rotor the motor can rotate
What residue in MotB is proposed as the H+ acceptor in stator?
Asp32
Proposed mechanism for torque generation
Protonation of Asp32 in MotB causes conf change in MotA which moves off the rotor
Causes cycles of torque against FliG probably involving charge interactions
How many H+ are transported per complete rotation?
1200
No numerical correlation b/w H+ and step size
LOTS OF PROTONS continuous stream of H+ go through the motor to move past the stator
ESSAY TITLE
Compare and contrast the differences b/w ATP synthase and molecular motors
_Structures
_Mechanism
Both have stator
Motors are functionally but not structurally or mechanistically analogous to stator in ATP sythase (OSPC + b-subunit)
FliI ATPase is structurally similar to alpha and beta subunits of the ATP synthase
Both use proton gradient to drive rotation
Motors use 1200 protons whereas ATP synthase transports 8-15 protons per rotation depending on number of C subunits
Facts about stator
Some bacteria have 2 or more types of stator with different ion specificity
Stator subunits are dynamic-can swap out flagella complex
Mot and Pom stators can be effective in both H+ or Na+ environments/competitive advantage-can survive in 2 environments
Some species can have HYBRID motors
Where is the site of export?
export apparatus is in the centre of MS/C rings
bacteria synthesise flagellum proteins and need to export them out of the membrane through the hollow core of the complex during assembly
What is the main protein involved in export?
FliI ATPase is a central component of flagella export
FliI brings in ATP and assembles with a few more proteins (FliH/FliJ)
FliI(6) assembles into a hexameric ring which drives the export
What uncoupler inhibits flagella export?
CCCP a proton uncouppler
Blocks PMF and displaces the proton gradient
Export is driven by the PMF
ATP hydrolysis in flagella assembly
ATP hydrolysis is not the primary energy source for export. It is used as a timer for CONTROLLING the assembly of the complex
ATP hydrolysis in FliI releases FliI/J/H complex after substrate delivery
FliI ATPase disassembles the complex so ATP hydrolysis STOPS export/PMF drives export
What does FlhB do?
IRREVERSIBLE event
Acts as an export SWITCH for flagella assembly
Sits like a “plug” within the system
In tact FlhB will export rod/hook proteins. It then undergoes intrinsic cleavage which slows down this export-breaks covalent bond of FlhB. A conformational change takes place using FliK which switches the system to export filamentous protein to make the flagella
What is FliK?
How long is the hook?
Molecular tape measure
The hook is consistently 55nm long
Export specifically must switch from hook proteins to filamentous protons when hook reaches this length.
The protein FliK acts as a switch, it is being stretched and eventually connects to the hook cap (on the top of the complex) with the base of the motor - dist=55nm
This causes a conf change to trigger transition in FliB to switch from hook to filamentous proteins
What direction of flagella movement causes them to bundle together?
ANTI clockwise
bacteria swim smoothly
What direction of flagella movement causes the bundles to fly apart?
clockwise
bacteria TUMBLE
Define CHEMOTAXIS
the process of moving in specific directions in response to environmental cues
Bacteria can sense their environment but are too small to detect a GRADIENT in space
They detect a gradient in TIME
environmental cue biases the system
entirely chemically driven
