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8. 2. Translocation of isoprenoid linked complex sugars across membranes. Polyisoprenol. OH. 2. Dolichol. OH. 7. Gram positive ( S.aureus ). Gram negative ( Salmonella ). Cell wall. Bacterial envelopes. membrane. G. M. G. G. A. A. A. A. A. A. E. E. E. E. E. E. K. K.

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translocation of isoprenoid linked complex sugars across membranes

8

2

Translocation of isoprenoid linked complex sugars across membranes

Polyisoprenol

OH

2

Dolichol

OH

7

bacterial envelopes

Gram positive (S.aureus)

Gram negative (Salmonella)

Cell wall

Bacterial envelopes

membrane

bacterial cell wall synthesis

G

M

G

G

A

A

A

A

A

A

E

E

E

E

E

E

K

K

K

K

K

K

A

A

A

A

A

A

A

A

A

A

A

A

Lipid I

M

M

MraY

MurG

Lipid II

M

M

G

G

UDP

M

UDP

Bacterial Cell Wall Synthesis

Lipid II

out

in

sites of action of antibiotics

G

M

G

G

A

A

A

A

A

A

E

E

E

E

E

E

K

K

K

K

K

K

A

A

A

A

A

A

A

A

A

A

A

A

Lipid I

M

M

MraY

MurG

Lipid II

M

M

G

G

UDP

M

UDP

Sites of Action of Antibiotics

penicillins

Ramoplanin

Mersacidin

Nisin

Plectacin

Vancomycin

Bacitracin

out

Moenomycin

in

D-cycloserine

Fosfomycin

Mureidomycin

transport of peptidoglycan precursors occurs via an unknown mechanism

G

M

G

G

A

A

A

A

A

A

E

E

E

E

E

E

K

K

K

K

K

K

A

A

A

A

A

A

A

A

A

A

A

A

Lipid I

M

M

MraY

MurG

Lipid II

M

M

G

G

UDP

M

UDP

Transport of peptidoglycan precursors occurs via an unknown mechanism

Lipid II

out

in

sites of action of antibiotics1

penicillins

Ramoplanin

Mersacidin

Nisin

G

Vancomycin

M

Bacitracin

Moenomycin

G

G

A

A

A

A

A

A

E

E

E

E

E

E

K

K

K

K

K

K

A

A

A

A

A

A

A

A

A

A

A

A

Lipid I

M

M

MraY

MurG

Lipid II

M

M

G

G

UDP

M

UDP

D-cycloserine

Fosfomycin

Mureidomycin

Sites of Action of Antibiotics

out

in

in order to identify proteins involved in the transport process we need
an assay that can determine the optimal conditions for Lipid II transport

a method that is able to identify the components involved in the transport process

In order to identify proteins involved in the transport process we need:
slide9

Lipid structure predicts flip rate in model membrane

What about polyisoprenoid based lipids?

10-1 s seconds 10 h >10 h

DAG

Cer

Chol

PC

LPC

ganglioside

slide10

Dansyl chloride

G

M

A

E

A

A

K

Pi

Pyrene chloride

Pi

O

NBD chloride

+

R2

R1NH

CH

C

8

(CH2)4

NH2

2

FITC

slide11

synthesis of NBD-labeled Lipid II

I2-stain

Fluorescence

TLC

NBD-Lipid II

Lipid II

slide12

Lipid II topology in membranesDithionite assay

  • Based on irreversible quenching of fluorescent NBD-label by dithionite
nbd based lipid flip flop test
NBD-based lipid flip/flop test

Does Lipid II spontaneous flip/flop across the membrane?

2

NBD-fluorescence (a.u.)

1

0

0

25

50

100

75

time (s)

DOPE/DOPG/NBD-Lipid II 75:25:0.2

slide14

Does Lipid II spontaneous flip/flop across the membrane?

2

dithionite

triton X-100

NBD-fluorescence (a.u.)

50%

1

0

0

25

50

100

75

time (s)

DOPE/DOPG/NBD-Lipid II 75:25:0.2

slide15

Lipid II Does not spontaneously flop across the membrane!

Incubation at RT

100

75

% protection

?

25

0

0

1

2

3

20

time (hrs)

DOPE/DOPG/NBD-Lipid II 75:25:0.2

slide16

Protein activity is necessary for efficient transport of Lipid II across the bacterial plasma membrane

Dithionite assay failed in biological membranes……

options

G

M

A

E

A

A

K

Pi

Pi

8

2

Options?
can the specific recognition of lipid ii by vancomycin be used to determine the lipid ii topology

Can the specific recognition of Lipid II by vancomycin be used to determine the Lipid II topology?

a lipid ii translocation assay based on fret
A Lipid II translocation assay based on FRET

out

in

Pi

Plasma membrane

Pi

M

Fluorescent lipid II

G

slide20

A Lipid II translocation assay based on FRET

G

M

out

Pi

Pi

in

Pi

Plasma membrane

Pi

M

Fluorescent lipid II

G

slide21

Vanco

A Lipid II translocation assay based on FRET between Lipid II and vancomycin

G

M

out

Pi

Pi

in

Pi

Plasma membrane

Pi

M

Fluorescent lipid II

G

slide22

Vanco

A Lipid II translocation assay based on FRET between Lipid II and vancomycin

FRET

G

M

out

Pi

Pi

in

Pi

Plasma membrane

Pi

M

Fluorescent lipid II

G

slide23
FRET

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 assay
A FRET-based Lipid II translocation assay
  • Make use of:
  • NBD-labeledLipid II
  • coumarinorrhodaminelabeled vancomycin

Coumarin spectrumNBD spectrum

preparation of fluorescent vancomycin
Preparation 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
tmr vancomycin
TMR-vancomycin

vancomycin

Tetramethyl rhodamine cadaverine

Absorption max ~550

Labeling did not reduce activity

are nbd lipid ii and tmr vancomycin able to form a complex and does that result in fret

Vanco

Are NBD-Lipid II and TMR-vancomycin able to form a complex and does that result in FRET?

G

M

?

+

Pi

=

Pi

In buffer containing triton X-100

fluorescent spectra of the individual molecules
Fluorescent spectra of the individual molecules

Fluorescence spectra of NBD-LII and vanco-TMR

excitation 481 nm

3.0

vanco-TMR

NBD-LII

NBD-LII + vanco-TMR

2.5

Sum of black and red

2.0

1.5

Fluorescence (A.U.)

1.0

0.5

0.0

500

520

540

560

580

600

620

640

wavelength (nm)

vanco tmr recognizes nbd lipid ii resulting in fret
Vanco-TMR recognizes NBD-Lipid II, resulting in FRET

Fluorescence spectra of NBD-LII and vanco-TMR

excitation 481 nm

3.0

vanco-TMR

NBD-LII

NBD-LII + vanco-TMR

2.5

Sum of black and red

FRET…

2.0

1.5

Fluorescence (A.U.)

1.0

0.5

0.0

500

520

540

560

580

600

620

640

wavelength (nm)

now what

Now what?

how to proceed from here?

slide34

In vitro Lipid II translocation~FRET assay~

Getting NBD-UMpp inside the RSOs (Freeze-thawing versus incubation)

fr/thincubcontrol

LI LII LI LII LI LII

slide35

FRET

A

A

E

E

K

K

A

A

A

A

15 °C

G

Vanco

M

vanco-TMR

Pi

Pi

G

G

UDP

UDP

M

M

UDP

UDP

In vitro Lipid II translocationFRET assay

RSO

  • 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

out

in

Membrane

Pi

Pi

M

Fluorescent lipid II

G

slide36

The appearance of FRET demonstrates that Lipid II translocation occurred

Spectra at 0, 5, 10, 20 and 30 min at 15 °C with vanco-TMR

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