Chemotaxis Flashcards
(20 cards)
Protofilament configuration
B-hairpin abrupt change in orientation - causes change in repeat distances of flagellin monomers
RH configuration is 1.5x shorter than LH
Flagella rotation and isoform
ACW rotation –> Normal, longer waveform: 9L-2R –> flagella can bundle –> Run
CW rotation –> Curly isoform –> 8L-3R or 7L-4R –> breaks apart the peritrichious flagella bundle –> Tumble
Tar MCP
Extreme negative co-operativity
Maltose binds to MBP, which can bind to periplasmic domain of Tar and have an additive effect
Conformational changes upon ligand binding to MCP
Piston, Twist, Crank
Attractants loosen the MCP and make it more dynamic
Extreme negative co-operativity - the other binding site greatly decreases in affinity
Clustering of MCP
Mixed trimers of homodimers - a ‘receptor squad’
Receptor squads can recruit CheA, and share a common CheA dimer to form a signalling team
Signalling teams can inter-link using CheA/CheW to form clusters at poles of the cell
Transmembrane signalling complex
MCP dimer
2 CheW monomers - Adaptor Protein
CheA dimer - Kinase
CheY
Response regulator - phosphorylated on His by CheA
Short half-life - spontaneously dephosphorylates
Accelerated by CheZ oligomer - by 100-fold
Addition of CheY in isolated cell envelopes w flagella increased CW flagellar rotation
CheR
Constitutively active methyltransferase - methylates Glu residues on MCP
CheB
Activated by phosphorylation on Asp residue by CheA
Methylesterase - removes Me groups on Glu of MCP
Amidase activity - convert Gln side-chains to Glu on MCP
CheA domains
Modular domains:
Transmitter domain of CheA binds ATP
g-phosphate group is transferred to His residue in P1 domain of CheA (trans-autophosphorylation)
CheY binds to P2 domain of CheA
P1 domain transfers this phosphate group to Asp residue on CheY
CheY mutageesis
Screen for phosphorylation-independent activation:
Clustering of mutations in 3 regions:
CheA-binding
FliM-binding
CheZ-binding
Switching factor
Strong co-operative relationship between CheY phosphorylation and CW bias
NOT due to co-operative binding of CheY to FliM (shown by FRET) - so is likely due to a switching factor
Fumurate
Stator complex
Heterohexamer:
MotA tetramer - Interacts with charged residues of FliG
MotB dimer - anchors the complex to the peptidoglycan layer
Force-generating unit
Rotor complex
Multiple FliFG copies, interacts with MotA and FliM
Charged residues of FliG interacts with MotA
C-ring
FliM/N In the cytoplasm - FliM interacts with FliFG (rotor complex)
Force-generating unit
Force-generating unit corresponds to Stator complex
(lac promoter + IPTG of MotA increases flagellar rotation in a stepwise manner that corresponds to number of assembled stator complexes)
Symmetry
Rotor complex has rotational symmetry of 26-units
Flagellar motor steps with a periodicity of 26
C-ring has symmetry of 34 subunits:
26 units of C-ring interact stiochiometrically with 26 subunits of FliFG rotor complex
8 FliM subunits contact a different domain of FliG
CheY binding to FliM
Tethered-bait binding strategy:
CheY-P binds to High-affinity binding site at FliMn of C-ring
Brings CheY-P to close proximity to weaker binding site of FliMm
Displaces FliG from the rotor complex - conformation change in FliG affects MotA component of the stator complex, leads to CW rotation
2 Models of H+ translocation
H+ can bind to the rotor component at some stage
Or H+ may not bind to the rotor component
Second model supported since mutagenesis did not find a critical H+-binding residue
How is H+ translocation coupled to mechanical rotation?
2 models
Proton turbine model - H+ interact with stator and rotor
Conformational change model - key conformational change is required for passage of H+
