nutrient transport in e. coli passive transporters : most nutrients < 600 da - porins ligand...

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Page 1: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients
Page 2: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients
Page 3: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients
Page 4: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Nutrient Transport in E. coli

Passive transporters : Most nutrients < 600 Da - Porins

Ligand Gated Porins - sugars

Active transporters : Larger nutrients such as iron and cobalamin (vitamin B12 : CNCbl) BtuB, FecA, FhuA, and FepA etc.

FhuA

FhuD

FhuB/C

BtuF

BtuC/D

Ferrichrome Vitamin B12

BtuB

Page 5: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Proposed TonB Dependent Transport Cycle

InnerMembrane

H+

Cytoplasmic Space

CNCbl

BtuB

CNCblA B

CNCbl

CNCblTon box

TonB

ExbB

ExbD

C D A

PeriplasmicSpace

Extracellular Space

OuterMembrane

Page 6: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

EPR Spectra for 2 consecutive strands of the barrel

Q D T S P T L VD V A N R F E Q P R S T V L A P TT VV T R Q

LI

A

G

S

T

M

H

K

R

N

Q

V

D

D I D R W Q

TSVNDVLRRL

S

PGVDITQNGGSGQLSSIRGTNASHVLVLID G V R L N L A G V S G S A D L S Q P I A L V Q R

RT

TI I N V V

V E Y I R G P R S A V GSD

AI

G G

D

GP

SI

T

A

E

SNS

DV

N

E

G

GW

QQ

Q

Q

TS

D

R

L

A

G

K

G

P

A

D

VN

E

G

S LGK

T

VT

D

HT

H

L

TL

R L

H

DAST

F

G

G

NR

TN

ET

S

KA

D

GDL

H

GN

R

N

IL

KQL

I

S

S

S

D

Q

Q

T

RL

G

K

P

M

VN

E

GV

T

D T

R

LG

G

R

T

NE S

G K

A

G

D

H

GN

RN

SD

H

ED

LT

SA

SD R

G

VI

VGH

G

I

A

QK

Q

I

QG

T

QQ

Q

TL

V

T

TG

LF

A

G S

IV

E

S

RA

GK

A

EG

T

G

N

RN

S

AF

QK

KIS

KI

AK

N

Q

DT

TD D

QQ

Q

V

A

GA

GKT

Q

AK

LI

P

DL

T

LS AG

T

VA

E

RS V

G

K

Y

TA

I

LH

S TV LD G

VEA L

G E IEG

GS

LF

G

D

DN

TQ

L

S

T

K

N

DV

VAV

G

P

T

TL

P

G

D

TQ

R

V

A

A

A

GS

D

QS

L

V

A

N

E

QP

RS

T

L

A

PT

N

G

G

GL

S

I

RG

T

N

GV

S

GS

A

DL

P

A

E

DD

P

QD

T

PT

D

R

EP

G

Q

LR

G

T

V

L

DG R

L

N

L

G

V

G

S

A

D

L

S

DD

DD

T

D

DT

DK

D

T

T

1245 367910 81113141516171819202122

L11

L10L9

L8 L7 L6

L5

L4

L3L2

L1

12

F Y

W

W

W

W

W

W

WW

W

W

W

F

F

F

F

F

F

F

F

Y

YY

Y

Y

Y

Y

YYYY Y

Y

Y

Y

Y

Y

Y

Y

Y

YYYY

YY

Y

Y

YY

Y Y

Y

Y

Y

Y

Y

YY

Y Y Y YF

YH

135

162

187

214

240

264

285

325

343

304

361

381

403

424

444

465

490

512

534

557

576

F594

T

YY

Fanucci et al. Biochemistry, 41, 11543, 2002

Page 7: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

•Pulsed EPR - direct T1 measurements

Vs

•Continuous Wave Power Saturation - indirect T1 measurements

Enhancements in relaxation due to paramagnet tell you about collisional frequency and hence accessibility

Strategy: Add a paramagnetic species that can collide with spin label~ relative enhancements in relaxation times

Accessibility is measured via Relaxation Measurements

Page 8: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Power Saturation of the EPR Resonance

Ametal

oxygen

membrane

A = I•P1/2 •[1 + (21/ - 1)P/P1/2]-

oxy = P´1/2oxy - P´1/2nit

met = P´1/2met - P´1/2nitaccessibility

0 2 4 6 8 10

Sqrt (P)

nit

oxy

met

P1/2 = 5.6

P1/2 = 10.6

P1/2 = 19.1

-2

0

2

4

-40 -20 0 20 40

Distance from phosphate (A)

Aqueous

Hydrocarbon

Nitroxide Accessibility and Membrane Depth from EPR

= ln (P1/2oxy/P1/2metal)

Page 9: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Calibration Points: bR and DOXYL-PC

PDB ID: 1ap9

5-DOXYL +1.5 8.1 7-DOXYL +2.1 10.510-DOXYL +2.4 14.012-DOXYL +2.8 16.0

bR109R1 +2.2 11.0 bR116R1 +4.1 20.5 bR117R1 +4.2 22.5 bR124R1 +2.0 10.5

Sample Distance

Frazier, A.A. et al. Biochem, 42 (2003), 96-105

Page 10: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

-2

0

2

4

-40 -20 0 20 40

Distance from Phosphate (A)

A B C Dtan h d istan ce -

C2 domain

doxyl-PC

bacteriorhodopsin

Modeling of C2A on the Membrane Surface

174

175173

176

142202

171

201240 170

229239

231

236233

197

197b

199

237

234

Frazier, A.A. et al. Biochem, 42 (2003), 96-105

Page 11: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

OO O

O

H

O

HN

O

O

NO

O

O

O

ONi

H2O

OH2

BtuB as a Ruler From the Bilayer Surface

DOGS-NTA-Ni

Ni(II)

Ni(II)EDDA

ca. 14 Å

Page 12: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

- 2 0 - 1 0 0 1 0 2 0 3 0

- 2

0

2

4

6

a q u e o u s

D is ta n c e f r o m P h o s p h a te s ( Å )

m e m b r a n e

1 2

8

4

0

P1/

2 DO

GS

-Ni-

NT

A

O 2

N i ( I I ) E D D A

a q u e o u s

O 2

O 2D O G S -N i-N T A

m e m b r a n e

BtuB as a Ruler From the Bilayer Surface

Ni(II)

Ni(II)EDDA

ca. 14 Å

= ln (P1/2oxy/P1/2metal)

Page 13: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Conclusions

KcsA

Ni(II)EDDA

DOGS-Ni-NTA

BtuBbR

C2A CPLA2

~ 14Å

O2

SDSL “Rulers”

GM2AP oligomerization

GM2AP

A60

~ 2Å

POPC + BMP

Lipid Composition Affects Binding

Page 14: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Secondary Structure Analysis: Spin Label MappingTransmembrane Water Filled Pores

-helix bundle

membrane

-barrel

membrane

aqueous phase

membraneaqu

accessibility

oxygenmetal complex

oxygenmetal complex

2 4 6 8

2 4 6 8

Page 15: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Power Saturation of EPR Spectra for 2 consecutive putative strands of the -barrel

-0.2

-0.1

0.0

0.1

0.2

178 176 174 172 170 168 166 164 162

0.0

0.4

0.8

1.2

1.6

2.0

-0.2

-0.1

0.0

0.1

0.2

146 148 150 152 154 156 158 160 162 164

0.0

0.4

0.8

1.2

1.6

2.0

Ni(

acac

) 2 ac

cess

ibil

ity

Oxy

gen

acce

ssib

ilit

y

residue

-barrel

membrane

aqueous phase

membraneaqu

26 of 36

Page 16: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Power Saturation of EPR Spectra for 2 consecutive putative strands of the -barrel

-0.2

-0.1

0.0

0.1

0.2

178 176 174 172 170 168 166 164 162

0.0

0.4

0.8

1.2

1.6

2.0

-0.2

-0.1

0.0

0.1

0.2

146 148 150 152 154 156 158 160 162 164

0.0

0.4

0.8

1.2

1.6

2.0

Ni(

acac

) 2 ac

cess

ibil

ity

Oxy

gen

acce

ssib

ilit

y

residue

172 170 168 166 164 162

-2

-1

0

1

2

3

-2

-1

0

1

2

3148 150 152 154 156 158 160

Residue

Residue

aqueous

membrane

Fanucci et al. Biochemistry, 41, 11543, 200227 of 36

Page 17: Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients

Bottom ViewSide View

Extracellular

Periplasmic

Lipid Bilayer

Tonbox(6-12)

S1 (25-31)

extendedtonbox (13-17)

Structure of BtuB

• 66 kD protein• beta barrel of 22 antiparallel strands• globular hatch/core domain

Chimento et al. NSB 2003 (10) 394-4017 of 20