Post on 16-Jan-2016
description
Unraveling Helix 2 of Colicin E1
Background
Zakharov et al., BBA. 2004
family of antimicrobial proteins
secreted upon environmentalstress (regulated by SOS system)
three modes of action:1. depolarization through ion channels2. inhibition of protein and peptidoglycan synthesis3. degradation of nucleic acids
1994 – crystal structures of polypeptide fragments of Col E11997 – complete crystal structure of channel-forming P190 fragment
Cleavage sites: Zhang and Cramer, 1992
Palmer and Merrill, JBC, 1994
Parallax method of depth dependent fluorescence quenching of trp
Y367 classed as moderately buried
Tory and Merrill, JBC, 1999
Fluorescence and FRET study
bim
olec
ular
que
nch
cons
tant
Δ λ
em
issi
on
ma
x (W
)lif
etim
e
red
blue
reduced accessibility
Y367 immersed or interfacial
Tory and Merrill, BBA, 2002
Red-edge excitation shift analysis
no REESexposed or embedded
moderate to large REESinterfacial
no REESburied
Y367 interfacial and sequestered from aqueous solvent
Musse et al., sometime soon!
Helix one displayed an alpha-helical nature in both soluble and membrane-bound states.
No elongation or blending of helices 1 and 2.
These results support the toroidal pore model.
Models
Zakharov and Cramer, BBA, 2002 Zahkarov et al., Biophys. J., 2004
Model for colicin import
Zakharov and Cramer, BBA, 2002
Colicin membrane insertion
Zakharov and Cramer, BBA, 2002Zakhorov et al., BBA, 2004
Shai, BBA, 1999
Models for membrane insertion
My Job
Helix 2 of Colicin E1
E365
K366
Y367
S368
K369
M370
A371
Q372
E373
L374
A 375
D376
K377
S378
K379
G380
hydrophobicpolaracidicbasic
Mutagenesis, Expression, Purification and Labelling
S378
G380
P190H
Mutant Yield
Y367C 4.6
S368C 2.5
K369C 25
M370C 3.0
A371C 13
A371C-C 47
Q372C 7.0
E373C 4.4
L374C 1.4
A375C 2.5
D376C 8.0
K377C 2.6
K377C-C 34
S378C 11.4
K379C 23
G380C 28
P190H 20
C505A 50
mBBr
well characterized
relatively small (about the size of a tryptophan)
uncharged, non-perturbing (structure or binding)
essentially non fluorescent until conjugated
fluorescence quenched by near-by W and, to a lesser extent, Y
Mutant % labelling
Y367C 110
S368C 100
K369C 85
M370C 56
A371C 9
A371C-C 9
Q372C 82
E373C 90
L374C 95
A375C 97
D376C 84
K377C 200
K377C-C 83
S378C 60
K379C 73
G380C 81
Bimane Labelling Efficiency
M370C - C505 7.23 Å
A371C – C505 10.98 Å
E373C - C505 6.95 Å
L374C - C505 7.21 Å
K377C - C505 8.32 Å
Analysis
Intrinsic Trp Fluorescence
indication of folded integrity of mutant protein compared with WT using 295 nm excitation avoids fluorescence from the 9 Y residues the three trp residues are in rigid environments and exhibit limited flexibility
Trp λ emission maximum
Mutant Native Labelled
Y367C 333 332
S368C 326 326
K369C 326 326
M370C 329 325
A371C 325 324
A371C-C 324 324
Q372C 325 327
E373C 327 327
L374C 332 331
A375C 326 324
D376C 326 325
K377C 328 NA
K377C-C 324 325
S378C 326 326
K379C 323 324
G380C 325 325
P190H 324 NA
C505A 324 NA
SPQ in vitro Channel Assay
test of the pore-forming ability of the mutant protein
P190H
0
5
10
15
20
25
0 2 3 5 7 8 10
time (min)
au
protein TX-100
SPQ
Mutant % P190H Rate
Y367C 9
S368C 102
K369C 74
M370C 82
A371C-C 46
Q372C 57
E373C 63
L374C 79
A375C 162
D376C 126
K377C-C 67
S378C 105
K379C 92
G380C 68
C505A 67366 368 370 372 374 376 378 380 382
0
2
4
6
8
10
12
14
Rat
e
Residue Number
P190H C505A
0
20
40
60
80
100
120
140
160
180
P19
0H
C50
5A
Y36
7
S36
8
K36
9
M37
0
A37
1
A37
1-C
Q37
2
E37
3
L374
A37
5
D37
6
K37
7-C
S37
8
K37
9
G38
0
% P
19
0H
ra
te
Bimane Fluorescence
reports on accessibility of bimane probe which relates to “location” within the tertiary structure of the protein
Musse and Merrill
standard apparent polarity scale curvebimane-N-acetyl-Cysdioxane-water solvent system
Bimane λ emission maximum
Mutant Soluble LUV
Y367C 470 469
S368C 474 471
K369C 480 470
M370C 469 466
A371C NA NA
A371C-C 455 457
Q372C 472 472
E373C 473 471
L374C 468 470
A375C 464 464
D376C 473 471
K377C-C 482 469
S378C 471 460
K379C 481 476
G380C 479 474
Surface Area Solvent Accessibility
GETAREA 1.1Solvent Accessible Surface Areas, Atomic Solvation Energies, and Their Gradients for Macromolecules
Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555
Area Per residue
Job identifier: get_a_13306
Probe radius : 1.400
Residue Total Apolar Backbone Sidechain Ratio(%) In/Out
ILE 345 121.48 109.19 13.62 107.86 73.2 o
LYS 346 151.45 27.52 15.63 135.82 82.6 o
ASP 347 102.95 34.60 0.13 102.82 91.0 o
ALA 348 6.96 6.68 3.62 3.34 5.1 i
VAL 349 74.04 74.04 0.00 74.04 60.5 o
ASP 350 89.67 30.09 8.52 81.15 71.8 o
ALA 351 36.79 35.19 7.92 28.87 44.5
THR 352 0.04 0.04 0.00 0.04 0.0 i
VAL 353 89.36 89.33 0.13 89.23 73.0 o
SER 354 68.44 41.93 7.49 60.95 78.7 o
SASA and bimane λem max
Mutant Total Area λem max
Y367C 1.35 470
S368C 2.99 474
K369C 126.82 480
M370C 6.14 469
A371C-C 0.76 455
Q372C 60.79 472
E373C 80.86 473
L374C 0.98 468
A375C 0.00 464
D376C 70.03 473
K377C-C 95.47 482
S378C 0.00 471
K379C 87.14 481
G380C 67.90 479366 368 370 372 374 376 378 380 382
-20
0
20
40
60
80
100
120
140
160
180
200
220
366 368 370 372 374 376 378 380 382-20
0
20
40
60
80
100
120
140
Anisotropy
predicts local environment within tertiary structure by measuring rotational property of bimane moiety on cysteine residue
low value = free movinghigh value = restricted movement
Probe mobility (1/r)
Mutant Soluble LUV
Y367C 5.21 4.65
S368C 5.92 5.85
K369C 9.43 7.41
M370C 10.53 4.93
A371C 9.26 6.21
A371C-C 7.41 5.15
Q372C 5.21 5.15
E373C 7.87 6.54
L374C 6.21 5.49
A375C 5.78 5.41
D376C 8.70 6.85
K377C-C 10.64 6.85
S378C 6.37 4.83
K379C 9.71 8.20
G380C 10.53 7.87366 368 370 372 374 376 378 380 382
4
6
8
10
12
Pro
be
Mo
bili
ty (
1/r)
Residue Number
Soluble LUV
>30%
10-25%
<5%
Quantum Yield
= number of photons emitted/the number of photons absorbed = fraction of fluorophore that decays through emission
low value = accessiblehigh value = not accessible
Quantum Yield
Mutant ΔQF relative QF
Y367C 0.154 1.312
S368C 0.149 1.882
K369C -0.010 0.979
M370C -0.007 0.973
A371C NA NA
A371C-C 0.039 1.438
Q372C 0.035 1.156
E373C 0.023 1.057
L374C 0.002 1.010
A375C 0.278 2.390
D376C 0.005 1.011
K377C-C 0.147 1.241
S378C 0.346 1.779
K379C 0.015 1.039
G380C 0.074 1.147
366 368 370 372 374 376 378 380 382-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
366 368 370 372 374 376 378 380 382-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Qua
ntum
Yie
ld L
UV
-sol
uble
Residue Number
Qu
antu
m Y
ield
LU
V-S
olu
ble
Residue Number
366 368 370 372 374 376 378 380 3820.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
366 368 370 372 374 376 378 380 3820.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
Re
lati
ve Q
ua
ntu
m Y
ield
Residue Number
Rel
ativ
e Q
uan
tum
Yie
ld
Residue Number>30%
10-25%
<5%
Dual Quenching Assay Erwin London
depth study quenching with KI (shallow) and 10-DN (deep) quenching is insensitive to variation in lipid content 10-DN retains “free energy” over range of bilayer depth could do study with each quencher alone, but ratio between shallow and deep increases sensitivity and eliminates non-depth related quenching effects (excited state lifetime of bimane) ratio is linearly dependent on depth of trp residue
low ratio value = accessible high ratio value = buried
DQA
Mutant (Fo/F10-DN-1)/(Fo/FKI-1)
Y367C 0.10
S368C 0.07
K369C 0.03
M370C 0.51
A371C-C 0.48
Q372C 0.05
E373C 0.18
L374C 0.19
A375C 0.70
D376C 0.10
K377C-C 0.04
S378C 0.34
K379C 0.40
G380C 0.34
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Y36
7C
S36
8C
K36
9C
M37
0C
A37
1C-C
Q37
2C
E37
3C
L374
C
A37
5C
D37
6C
K37
7C-C
S37
8C
K37
9C
G38
0C
Residue Number
F 0/F
-1 (a
u)
Shallow
Deep
Deep/Shallow
>0.4
0.11-0.4
<0.1
366 368 370 372 374 376 378 380 382-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
DQ
A
Residue Number
366 368 370 372 374 376 378 380 382-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
SummaryBimane lambda emission max (membrane-bound vs soluble):
Blue shifted (455-460 nm): A371, S378Intermediate (461-470 nm): Y367, K369, M370, L374, A375, K377Red shifted (>471 nm): S368, Q372, E373, D376, K379, G380
Soluble anisotropy:Accessible (0.095-0.135): K369, M370, A371, E373, D376, K377, K379, G380Inaccessible (0.157-0.192): Y367, S368, Q372, L374, A375, S378
These residues are considered buried, but mBBr may be mobile within “pocket” of protein structure.These residues are considered exposed, but probe may be immobilized by tertiary structure contacts.
Membrane bound anisotropy: Little change (<5%): Y367, S368, Q372, A375Moderate change (10-25%): K369, E373, L374, D376, S378, K379, G380Significant change (>30%): M370, A371, K377
Quantum Yield: Little change (<5%): K369, M370, E373, L374, D376, K379Moderate change (10-25%): Y367, Q372, K377, G380Significant change (>30%): S368, A371, A375, S378
S368 and A371 had rather low soluble QF values (<0.18, the observed value for Cys-bimane standard)which may have been caused by quenching of the bimane signal by Y367.
Dual Quenching Analysis:Accessible (<0.10): Y367, S368, K369, Q372, D376, K377In-between (0.11-0.4): E373, L374, S378, K379, G380Buried (>0.4): M370, A371, A375
Sobko et al, FEBS Lett, 2004