nutrient transport in e. coli passive transporters : most nutrients < 600 da - porins ligand...
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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
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
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
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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
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SI
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SNS
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NE S
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SD
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SD R
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VGH
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DK
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1245 367910 81113141516171819202122
L11
L10L9
L8 L7 L6
L5
L4
L3L2
L1
12
F Y
W
W
W
W
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WW
W
W
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F
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Y Y
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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
•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
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)
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
-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
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
- 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)
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
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
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
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
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