Journey of bacteriophage M13 major

coat protein

David Stopar

University of Ljubljana

Slovenia

Interactions in macromolecular

assemblies

• Replication cycle of the bacteriophage M13

• Structure of the major coat protein

• Topology of the major coat protein in the lipid bilayer

• Anchoring of the major coat protein in the lipid bilayer

Journey of bacteriophage M13 major coat protein

Schematic representation of bacteriophage M13 replication cycle

M13

E.coli

F-pilus

DNA

gp 5

gp 8

Gene 9 protein

Gene 6 protein

Gene 3 protein

Gene 8 protein

Circular ssDNA

Distal end

Proximal end

Gene 7 protein

Schematic structure of bacteriophage M13 filament

Model of the of bacteriophage M13 protein coat

Nearest-neighbour interactions between protein subunits in the phage particle

k=11 k=11

k=6 k=6

k=0 k=0

• Replication cycle of the bacteriophage M13

• Structure of the major coat protein

• Topology of the major coat protein in the lipid bilayer

• Anchoring of the major coat protein in the lipid bilayer

Journey of bacteriophage M13 major coat protein

Major coat protein structural domains in lipid bilayer

1-5 N-terminus

6-16 amphiphatic helix

17-23 loop

24-46 transmembrane helix

47-50 C-terminus

1

50

Amino acidresidue number

X-ray and NMR limitations for membrane proteins

X-ray: “no” crystals of membrane proteins

NMR: membranes are anisotropic systems

high-resolution NMR limited to micellar systems

solid-state NMR is needed for bilayer structure

13C and 15N-enrichment is needed (difficult and very

expensive!)

2D NMR & DG analysisPapavoine et al. (1998)J. Mol. Biol. 282, 401-419

M13 coat protein in SDS micelles

? ? ? ? ? ? ? ?

Structure not possible in a membrane

Molecular Dynamics (MD) Approach

Starting conformation for

protein in POPC membrane

from SDS (Papavoine et al.,

1998).

Snap Shots of Protein Backbone

0 ns 0.5 ns 1 ns 1.5 ns

41 H-bonds

41 H-bonds

42 H-bonds

43 H-bonds

The effects seen in MD are in the ESR time scale (ns)

Note: one label is measured at a time membrane-water

interface

M13 Coat Protein with Labels Attached

ESR Spin Label Information

Position withstructural restriction

Position with nostructural restriction

N

NOO

O

ESR spin label: 5-maleimido-proxyl

•

Sensitivity of spin labels for different protein sites

1

50

Amino acidresidue number

2Azz

Outer Splitting

Sensitivity of spin labels for different lipids

65

60

55

50

2Azz

(G

)

2422201816141210

amino acid residue

60

55

50

45

40

35

2A

zz (

G)

40302010

amino acid residue

65

60

55

50

2Azz

(G

)

424038363432302826

amino acid residue

PC22

PC14

• Replication cycle of the bacteriophage M13

• Structure of the major coat protein

• Topology of the major coat protein in the lipid bilayer

• Anchoring of the major coat protein in the lipid bilayer

Journey of bacteriophage M13 major coat protein

Spin label relaxation times in DOPC bilayers

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20

Ni2+

(mM)

1/T

1 (se

c x

106 ) A49C

V31C

G38C

1

50

Amino acidresidue number

Relative quenching efficiency by oxygen and

Ni2+

Ni2+

1

50

Amino acidresidue number

0

10

20

30

40

50

60

70

20 30 40 50

residue number

rela

tive

quen

chin

g ef

fici

ency

oxygen

Ni2+

Dependence of spin labeled DOPC acyl

chains on the relaxation enhancement by

oxygen and Ni2+

0

10

20

30

40

50

60

70

-2 8 18 28 38

acyl carbon atom

rela

tive

rela

xatio

n en

hanc

emen

t

Ni2+

oxygen

1

50

Amino acidresidue number

Location of the protein in the lipid bilayer as determined by fluorescence labeling

470

475

480

485

490

495

500

505

510

0 20 40

Amino Acid Residue Number

Em

issi

on m

axim

um (

nm)

1

50

Amino acidresidue number

Trp 26Leu 14

NH3+

Phe 45

Interface

Interface

Hydrocarboncore

20 Å

Lys 40

Topology of the major coat protein in the lipid bilayer

• Replication cycle of the bacteriophage M13

• Structure of the major coat protein

• Topology of the major coat protein in the lipid bilayer

• Anchoring of the major coat protein in the lipid bilayer

Journey of bacteriophage M13 major coat protein

AEDANS max of the A7C protein mutants in lipid

bilayers

M13 major coat protein mutant Emission maximum (nm)

A7C 495

A7C/A10I 492

A7C/F11A 502

A7C/L14A 502

A7C/F11A/L14A 505

AEDANS max in DOPC lipid bilayers

488

489

490

491

492

493

494

495

496

497

T46C T46C/INS T46C/FA

AED

AN

S m

ax (n

m)

1

50

Amino acidresidue number

Tryptophane max in DOPC lipid bilayers

320

322

324

326

328

330

332

334

336

338

Wild type T46C/INS T46C/FA

Try

ptop

han

max

(nm

)

1

50

Amino acidresidue number

Bacteriophage M13 major coat protein anchors

in the lipid bilayer

C-terminus

N-terminus

Leu 14

Phe 11

Lys 8Trp 26

Lys 40Lys 43

Lys 44

Phe 42

Phe 45

Schematic representation of bacteriophage M13 replication cycle

M13

E.coli

F-pilus

DNA

gp 5

gp 8

Model of the major coat protein disassembly and assembly

(1)(3)(2)

N-terminus

C-terminus

Acknowledgements

Wageningen

Marcus Hemminga

Rob Koehorst

Ruud Spruijt

Werner Vos

Cor Wolfs

Göttingen Derek MarshKity A. Jansen

Szeged Tibor Páli

Ljubljana Ivan Mahne

Janez ŠtrancarMilan Schara