Molecular Motors: Flagella and Chemotaxis L6-7 (David Stephans)

Question 1

How many flagella do bacteria have?

Answer 1

One or more(3-5)

All types of bacteria have different types of organisation

Question 2

What influences how fast a motor rotates?

Answer 2

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

Question 3

How many discrete steps do flagella rotate in?

Answer 3

26 discrete steps per revolution

14 degrees

Question 4

What proteins is the motor made of?

Answer 4

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

Question 5

What protein is the hook made of?

Answer 5

FlgE (helical conf)
adjacent to motor structure
FlgE has 5.5subunits per turn of the structure
11 fold symmetry

Question 6

What protein is the flagella made of?

Answer 6

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

Question 7

What protein is the stator made of?

Stator forms proton or sodium gradient

Answer 7

MotAB
MotB=sits in periplasmic space-binds to peptidoglycan and anchors stator
MotA=sits in cellular membrane
MotA x4 MotB x2

Question 8

How many stator subunits surround the flagellum?

Answer 8

11-12 per flagellum
symmetry mismatch with motor/MS ring (26 fold symmetry)
Asymmetry used to help act as a break and generate torque

Question 9

What type of stator uses a sodium gradient?

Stators define the specificity of the transported ion (H+ or Na+)

Answer 9

PomA/B in vibrio species (single flagellum)

proton driven flagella exist in E.coli

Question 10

What protein does the stator/MotA interact with to generate torque?

Answer 10

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

Question 11

What residue in MotB is proposed as the H+ acceptor in stator?

Answer 11

Asp32

Question 12

Proposed mechanism for torque generation

Answer 12

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

Question 13

How many H+ are transported per complete rotation?

Answer 13

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

Question 14

ESSAY TITLE

Compare and contrast the differences b/w ATP synthase and molecular motors

Answer 14

_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

Question 15

Facts about stator

Answer 15

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

Question 16

Where is the site of export?

Answer 16

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

Question 17

What is the main protein involved in export?

Answer 17

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

Question 18

What uncoupler inhibits flagella export?

Answer 18

CCCP a proton uncouppler
Blocks PMF and displaces the proton gradient
Export is driven by the PMF

Question 19

ATP hydrolysis in flagella assembly

Answer 19

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

Question 20

What does FlhB do?

IRREVERSIBLE event

Answer 20

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

Question 21

What is FliK?

How long is the hook?

Answer 21

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

Question 22

What direction of flagella movement causes them to bundle together?

Answer 22

ANTI clockwise

bacteria swim smoothly

Question 23

What direction of flagella movement causes the bundles to fly apart?

Answer 23

clockwise

bacteria TUMBLE

Question 24

Define CHEMOTAXIS

Answer 24

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

Question 25

How many methyl sensing chemotaxis proteins (MCPs) do bacteria have?

Answer 25

1-60 different MCPs with different ligand specificity (detect different types of compounds)
E.coli have 4
MCPs cluster together in patches (heterogenous clusters) and allow interplay b/w different signals, or increase sensitivity

Question 26

What is the signalling domain of a MCP?

Answer 26

CheA DIMER
Methyl sites are in the tail
CheW binds b/w CheA and the MCPs (part of the sensory system)
Sensing occurs within the periplasm since small molecules can diffuse through the outer membrane.

Question 27

What is the difference b/w Euks and Proks in terms of the types of amino acid kinases they use in signalling pathways

Answer 27

Euks- involve ser thr and tyr kinases

Proks-usually involve his and asp kinases

Question 28

The Che system

Answer 28

Signal/repellent (want to initiate tumbling) - CheA autophosphorylates on HIS residue
CheA-p phosphorylates CheY on ASP residue
CheY-p binds to FliM of flagella c-ring (switch) causing change in ROTATION direction

C ring = FliG/FliM/FliN

CheZ dephosphorylates CheY (smooth swimming)
CheA is a histidine and aspartate kinase

Question 29

How is the activity of MCPs modulated?

Answer 29

via reversible methylation
CheA-p also phosphorylates CheB to activate it.
CheB-p is a METHYLESTERASE (removes methylation)
This reduces the activity of MCPs as it removes methyl groups from its target proteins
CheR is a METHYLTRANSFERASE (methylates GLUTAMATES on MCPs

Methylated MCPs are more active and more sensitive to molecules
The balance of CheR and CheB-p activity allows MCPs to adapt to the current environment and sense changes in environmental factors
If their activity is down-regulated they will respond more slowly to changes in the environment.

Question 30

What are Euk cilia and flagella built on?

Answer 30

AXONEMES- the proteinaceous core of cilia and flagella
Set of proteins that form core of these structures

Composed primarily of MICROTUBULES

Question 31

What molecules connect microtubules in flagella in Euks?

Answer 31

Nexin
Nexin likes the two MTs together and induces bending rather than sliding of the MTs by the axonemal dyneins.
The coordinated bending action of the motors will drive flagella motility (only if nexin exists)

Question 32

What motors provide force in cilia?

Answer 32

axonemal dyneins
different type compared with those that power cell division and mitosis or those that move around organelles in the cell
These are motors which use ATP to generate force
Linkers stick out from the structure and connect to the MTs

Question 33

What is the architecture/organisation of a axoneme?

Answer 33

9+2 arrangement of MTs
Radial spoke connects the central pair of MTs
Contains inner and outer arms of dynein

Question 34

What are PRIMARY cilia?

Answer 34

Present on most mammalian cells
9+0 structure
NON motile
Roles in environmental sensing and signal transduction
eg detect flow in kidney epithelia/hedgehog signalling pathway (development)