Translocation of isoprenoid linked complex sugars across membranes 8 2 OH Polyisoprenol 7 2 OH Dolichol
Feb 01, 2016
Translocation of isoprenoid linked complex sugars
across membranes
8
2 OH Polyisoprenol
7
2
OH Dolichol
Bacterial envelopes
Gram negative (Salmonella)
Gram positive (S.aureus)
Cell wall
membrane
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
Bacterial Cell Wall SynthesisBacterial Cell Wall Synthesis
A AA E KM
G
A AA E KM
G
out
in
A AA E KM
G
A AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
A AA E KM
G
Vancomycin
RamoplaninMersacidin NisinPlectacin
D-cycloserineFosfomycin
Bacitracin
penicillins
Mureidomycin
Moenomycin
Sites of Action of AntibioticsSites of Action of Antibiotics
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism
A AA E KM
G
A AA E KM
G
out
in
A AA E KM
G
A AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
A AA E KM
G
Vancomycin
RamoplaninMersacidin Nisin
D-cycloserineFosfomycin
Bacitracin
penicillins
Mureidomycin
Moenomycin
Sites of Action of AntibioticsSites of Action of Antibiotics
In order to identify proteins involved in the In order to identify proteins involved in the transport process we need:transport process we need:
• an assay that can determine the optimal an assay that can determine the optimal conditions for Lipid II transportconditions for Lipid II transport
• a method that is able to identify the a method that is able to identify the components involved in the transport processcomponents involved in the transport process
Is it a spontaneous process?Is it a spontaneous process?
DAGDAG CerCerCholChol
LPCLPCPCPC gangliosideganglioside
1010-1 -1 s seconds 10 h >10 hs seconds 10 h >10 h
Lipid structure predicts flip rate in model membraneLipid structure predicts flip rate in model membrane
What about polyisoprenoid based lipids?
CHR1NH
(CH2)4
NH2
R2C
O
+
Pi
Pi
G
MK A A
8
2
Dansyl chloride
Pyrene chloride
NBD chloride
FITC
A E
synthesis of NBD-labeled Lipid II
Lipid II
NBD-Lipid II
I2-stain FluorescenceTLC
Lipid II topology in membranesLipid II topology in membranesDithionite assayDithionite assay
•Based on irreversible quenching of fluorescent NBD-label by dithionite
Does Lipid II spontaneous flip/flop across the membrane?
DOPE/DOPG/NBD-Lipid II 75:25:0.2
0 25 50 10075
time (s)
NB
D-f
luo
resc
ence
(a.
u.)
0
1
2
NBD-based lipid flip/flop testNBD-based lipid flip/flop test
Does Lipid II spontaneous flip/flop across the membrane?
DOPE/DOPG/NBD-Lipid II 75:25:0.2
0 25 50 10075
time (s)
NB
D-f
luo
resc
ence
(a.
u.)
0
1
2
dithionite
triton X-100
50%
Lipid II Does not spontaneously flop across the membrane!
DOPE/DOPG/NBD-Lipid II 75:25:0.2
?
Incubation at RT
time (hrs)
% p
rote
ctio
n
75
25
100
00 1 202 3
Protein activity is necessary for efficient transport of Lipid II across the bacterial plasma membrane
Dithionite assay failed in biological membranes……
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism
A AA E KM
G
A AA E KM
G
Pi
Pi
G
MK A A
8
2
A E
out
in
A AA E KM
G
A AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
A AA E KM
G
Vancomycin
RamoplaninMersacidin Nisin
D-cycloserineFosfomycin
Bacitracin
penicillins
Mureidomycin
Moenomycin
Sites of Action of AntibioticsSites of Action of Antibiotics
Options?Options?
Can the specific recognition of Can the specific recognition of Lipid II by vancomycin be used Lipid II by vancomycin be used
to determine the Lipid II to determine the Lipid II topology?topology?
A Lipid II translocation assay based on A Lipid II translocation assay based on FRETFRET
Pi
Pi
G
M
Plasma membrane
Fluorescent lipid II
in
out
Pi
Pi
G
M
Pi
Pi
G
M
Plasma membrane
Fluorescent lipid II
in
out
A Lipid II translocation assay based on FRET
A Lipid II translocation assay based on FRET between Lipid II and vancomycin
Pi
Pi
G
M
Pi
Pi
G
M
Plasma membrane
Vanco
Fluorescent lipid II
in
out
Pi
Pi
G
M
Pi
Pi
G
M
Plasma membrane
Vanco
Fluorescent lipid II
FRET
in
out
A Lipid II translocation assay based on FRET between Lipid II and vancomycin
FRETFRET
Criteria for choosing donor & acceptor fluorophores:
– Sufficient separations in excitation spectra
– Overlap between emission spectra of donor and excitation of acceptor
– Reasonable separation in emission spectra
A FRET-based Lipid II translocation assayA FRET-based Lipid II translocation assay
Make use of:
-NBD-labeled Lipid II
-coumarin or rhodamine labeled vancomycin
Coumarin spectrum NBD spectrum
Which sites to choose on Which sites to choose on vanco?vanco?
Preparation of fluorescent vancomycinPreparation of fluorescent vancomycin
Label vancomycin with: Reacts with:
-7-amino-4-methylcoumarin carboxyl
-Tetramethylrhodamine cadaverine (TMR) carboxyl
-AMCA-X (coumarin) amine
-DACITC (coumarin) amine
-7-methoxycoumarin-3-carboxylic acid amine
-Lissamine Rhodamine B (LRB) amine
Analysis of vanco-TMR and vanco-LRBAnalysis of vanco-TMR and vanco-LRB
Excitation and emission spectra
wavelength (nm)
400 450 500 550 600 650
Abs
/Flu
(A
.U.)
0.0
0.5
1.0
1.5
2.0
2.5
3.0excit. NBD-LII emis. NBD-LII excit. vanco-TMR emis. vanco-TMR excit. vanco-LRB emis. vanco-LRB
Lipid II Vanco
vancomycin
Tetramethyl rhodamine cadaverine
Absorption max ~550
Labeling did not reduce activity
TMR-vancomycinTMR-vancomycin
Pi
Pi
G
M
Are NBD-Lipid II and TMR-vancomycin able Are NBD-Lipid II and TMR-vancomycin able to form a complex and does that result in to form a complex and does that result in
FRET?FRET?
Vanco+ ?=
In buffer containing triton X-100
Fluorescence spectra of NBD-LII and vanco-TMRexcitation 481 nm
wavelength (nm)
500 520 540 560 580 600 620 640
Flu
ore
scen
ce (A
.U.)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
vanco-TMR NBD-LII NBD-LII + vanco-TMR Sum of black and red
Fluorescent spectra of the individual moleculesFluorescent spectra of the individual molecules
Fluorescence spectra of NBD-LII and vanco-TMRexcitation 481 nm
wavelength (nm)
500 520 540 560 580 600 620 6400.0
0.5
1.0
1.5
2.0
2.5
3.0
vanco-TMR NBD-LII NBD-LII + vanco-TMR Sum of black and red
Vanco-TMR recognizes NBD-Lipid II, resulting Vanco-TMR recognizes NBD-Lipid II, resulting in FRETin FRET
FRET…
Flu
ore
scen
ce (A
.U.)
Now what?Now what?
how to proceed from here?how to proceed from here?
E. coli membrane vesicle E. coli membrane vesicle systemssystems
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDP G
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism
A AA E KM
G
A AA E KM
G
ISO RSO
In vitro Lipid II translocationIn vitro Lipid II translocation~FRET assay~~FRET assay~
Getting NBD-UMpp inside the RSOs (Freeze-thawing versus incubation)LI/LII
freeze-thaw or incubation
Wavelength (nm)
500 520 540 560 580 600 620
Flu
ore
sce
nce
(A.U
.)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4LII freeze-thawLII incubationbackground freeze-thawbackground incubationLI freeze-thawLI incubation fr/th incub control
LI LII LI LII LI LII
In vitro Lipid II translocationIn vitro Lipid II translocationFRET assayFRET assay
General procedure
•Right side out (RSO) vesicles E. coli W3899
•NBD-UDP-MurNAc-pentapeptide + UDP-GlcNAc
•Two freeze-thaw cycles
•Lipid II synthesis (on ice)
•Fluorescence spectra in the
presence of vanco-TMR (15 °C)
Freeze-thaw
Synthesis
Pi
Pi
G
M
Membrane
Fluorescent lipid II
in
outPi
Pi
G
M
15 °C
Vanco
FRET
vanco-TMR
A AA E KMUDP
UDP G
A AA E KMUDP
UDP G
RSO
The appearance of FRET demonstrates The appearance of FRET demonstrates that Lipid II translocation occurredthat Lipid II translocation occurred
Lipid II + vanco-TMR
Wavelength (nm)
500 520 540 560 580 600 620
Flu
ore
sce
nc
e (A
.U.)
0.0
0.2
0.4
0.6
0.8
1.0
t=0t=5t=10t=20t=30
Spectra at 0, 5, 10, 20 and 30 min at 15 °C with vanco-TMR
Bert Rutten, Aurin Vos & Sacha Dalhuijsen