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