binding to negatively curved membranes

1
binding to negatively curved membranes
2
(No Transcript)
3
Cell biology with bacteria?
5 µm
4
Localization of cell division proteins
5
(No Transcript)
6
Rut Carballido-López
7
(No Transcript)
8
How do proteins localize to cell poles ?(DivIVA
as model system)
DivIVA-GFP
9
(lack of) Information from secondary structure
prediction
164 amino acids, mostly helical
secondary structure prediction by PSIPRED
coiled coil prediction by LUPAS
multimerization via coiled coil regions
10
Possible mechanisms 1) binding to another (cell
division) protein 2) binding to a specific lipid
species 3) affinity for curved membranes
11
Binding to another (membrane) protein?
DG DivIVA-GFP V membrane vesicles Lip
liposomes D DivIVA G GFP
12
Biacore (surface plasmon resonance) with L1-chip
T min
13
Possible mechanisms 1) binding to another (cell
division) protein 2) binding to a specific lipid
species 3) affinity for curved membranes
14
Cardiolipin Domains in Bacillus subtilis Kawai,
2003, J. Bac.
15
DivIVA localization in B. subtilis strains
lacking certain lipids
wt
- PG
- CL
-PE
16
Possible mechanisms 1) binding to another (cell
division) protein 2) binding to a specific lipid
species 3) affinity for curved membranes
17
Affinity for curvature induces curvature
BAR domains as sensors or membrane
curvature Peter et al., 2004, Science
18
Affinity for curvature induces curvature
BAR domains as sensors or membrane
curvature Peter et al., 2004, Science
19
Induction of curved membranes ?
liposomes DivIVA
liposomes
200 nm
20
Induction of curved membranes ?
200 nm
21
100 nm
22
Possible mechanisms 1) binding to another (cell
division) protein 2) binding to a specific lipid
species 3) affinity for curved membranes
?
23
Does curvature really not play a role?
B. subtilis
E. coli
24
E. coli division mutant
25
Possible mechanisms 1) binding to another (cell
division) protein 2) binding to a specific lipid
species 3) affinity for curved membranes.., but
not as we know it
26
Higher order DivIVA structures
27
Conceptual simplification
28
Molecular Bridging
1) self interaction (clustering) of subunits 2)
subunits should be large (relative to
curvature) 3) membrane interaction (weak)
- no other proteins / lipids / or curved proteins
necessary -
29
Monte Carlo simulation
30
Monte Carlo simulation

• Rules
• - cylinder 1 x 4 µm
• - DivIVA oligomers (green) spheres of 25 nm
  diameter
• - curvature of membranes at transition from
  lateral wall to sides diameter of 100 nm
• - spheres can make max 8 contacts (doggy bone
  contains at least 8 DivIVA molecules)
• 2 membrane contacts maximal (based on our EM
  data)
• Epp and Epm in the range 1.5-6 k bT
• (equivalent to 1-4 kcal/mol) in range of typical
  weak protein-protein attractions

31
- spheres can make 8 contacts - 2 membrane
contacts maximal
32
d 50 nm
d 100 nm
- No restrictions in nr. of interactions Epp
2 k bT Epm 6 k bT
33
d 50 nm
d 100 nm

• - max 4 pp bonds
• - membrane contact
• 2 pp contact
• Epp 3 k bT
• Emp 5.5 k bT

• max 6 pp bonds
• membrane contact
• 3 pp contacts
• Epp 3.5 k bT
• Epm 5.5 k bT

34

• Max 8 pp bonds
• membrane contact
• 4 pp contacts
• Epp 3.5 k bT
• Epm 5.5 k bT

d 50 nm
d 100 nm
35
Modelling of doggy bones
36
CBCB - Newcastle University Rok Lenarcic Ling
Wu Jeff Errington Sven Halbedel University of
Oxford Wouter de Jong Loek Visser Michael Shaw
University of Edinburgh Davide Marenduzzo