Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia...
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![Page 1: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/1.jpg)
Journey of bacteriophage M13 major
coat protein
David Stopar
University of Ljubljana
Slovenia
Interactions in macromolecular
assemblies
![Page 2: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/2.jpg)
• 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
![Page 3: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/3.jpg)
Schematic representation of bacteriophage M13 replication cycle
M13
E.coli
F-pilus
DNA
gp 5
gp 8
![Page 4: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/4.jpg)
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
![Page 5: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/5.jpg)
Model of the of bacteriophage M13 protein coat
![Page 6: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/6.jpg)
Nearest-neighbour interactions between protein subunits in the phage particle
k=11 k=11
k=6 k=6
k=0 k=0
![Page 7: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/7.jpg)
• 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
![Page 8: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/8.jpg)
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
![Page 9: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/9.jpg)
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!)
![Page 10: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/10.jpg)
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
![Page 11: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/11.jpg)
Molecular Dynamics (MD) Approach
Starting conformation for
protein in POPC membrane
from SDS (Papavoine et al.,
1998).
![Page 12: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/12.jpg)
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)
![Page 13: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/13.jpg)
Note: one label is measured at a time membrane-water
interface
M13 Coat Protein with Labels Attached
![Page 14: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/14.jpg)
ESR Spin Label Information
Position withstructural restriction
Position with nostructural restriction
N
NOO
O
ESR spin label: 5-maleimido-proxyl
•
![Page 15: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/15.jpg)
Sensitivity of spin labels for different protein sites
1
50
Amino acidresidue number
2Azz
Outer Splitting
![Page 16: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/16.jpg)
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
![Page 17: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/17.jpg)
• 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
![Page 18: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/18.jpg)
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
![Page 19: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/19.jpg)
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+
![Page 20: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/20.jpg)
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
![Page 21: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/21.jpg)
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
![Page 22: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/22.jpg)
Trp 26Leu 14
NH3+
Phe 45
Interface
Interface
Hydrocarboncore
20 Å
Lys 40
Topology of the major coat protein in the lipid bilayer
![Page 23: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/23.jpg)
• 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
![Page 24: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/24.jpg)
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
![Page 25: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/25.jpg)
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
![Page 26: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/26.jpg)
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
![Page 27: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/27.jpg)
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
![Page 28: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/28.jpg)
Schematic representation of bacteriophage M13 replication cycle
M13
E.coli
F-pilus
DNA
gp 5
gp 8
![Page 29: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/29.jpg)
Model of the major coat protein disassembly and assembly
(1)(3)(2)
N-terminus
C-terminus
![Page 30: Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ef95503460f94c0ad8e/html5/thumbnails/30.jpg)
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