binding to negatively curved membranes. cell biology with bacteria? 5 µm
DESCRIPTION
Cell biology with bacteria? 5 µmTRANSCRIPT
binding to negatively curved membranes
Cell biology with bacteria?
5 µm
Localization of cell division proteins
Rut Carballido-López
GFP-MinD
How do proteins localize to cell poles ?
(DivIVA as model system)
DivIVA-GFP
(lack of) Information from secondary structure prediction
164 amino acids, mostly helical
coiled coil prediction by LUPAS
secondary structure prediction by PSIPRED
multimerization via coiled coil regions
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
DG = DivIVA-GFP
V = membrane vesicles
Lip = liposomes
D = DivIVA
G = GFP
Binding to another (membrane) protein?
70 %
20 %
30 %
membrane vesicles
Biacore (surface plasmon resonance) with L1-chip
T = min
amphipathic helix of N-terminus (60 aa)
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
Edwards, 2000, EMBO
Cardiolipin Domains in Bacillus subtilis Kawai, 2003, J. Bac.
DivIVA localization in B. subtilis strains lacking certain lipids
wt - PG - CL-PE
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes
Affinity for curvature = induces curvature
‘BAR domains as sensors or membrane curvature’
Peter et al., 2004, Science
Affinity for curvature = induces curvature
‘BAR domains as sensors or membrane curvature’
Peter et al., 2004, Science
Induction of curved membranes ?
liposomes liposomes + DivIVA
DDDDD
DDDD
liposomes
DivIVA
200 nm
Induction of curved membranes ?
200 nm
100 nm
Possible mechanisms:
1) binding to another (cell division) protein
2) binding to a specific lipid species
3) affinity for curved membranes ?
Does curvature really not play a role?
B. subtilis E. coli
E. coli division mutant
MHD63
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
Higher order DivIVA structures
Stahlberg, 2004, Mol. Mic.
( Cryo-negative stain EM )
‘Doggy bones’Ø ~ 25 nm
Ø ~ 100 nm
~ 25 nm
?
?
Conceptual simplification:
1) self interaction (clustering) of subunits
2) subunits should be large (relative to curvature)
3) membrane interaction (weak)
‘Molecular Bridging’
- no other proteins / lipids / or curved proteins necessary -
Monte Carlo simulation
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
- spheres can make 8 contacts
- 2 membrane contacts maximal
- spheres can make 4 contacts
- no limitations in membrane contacts
d = 50 nm d =100 nm
- No restrictions in nr. of interactions
Epp = 2 k bTEpm = 6 k bT
- 4 pp bonds- membrane contact = 1 pp contact
Epp = 2.5 k bTEpm = 5.5 k bT
d = 50 nm d =100 nm
- max 4 pp bonds- membrane contact = 2 pp contact
Epp = 3 k bTEmp = 5.5 k bT
- max 6 pp bonds- membrane contact= 3 pp contacts
Epp = 3.5 k bTEpm = 5.5 k bT
d = 50 nm d =100 nm
-Max 8 pp bonds-membrane contact= 4 pp contacts
Epp = 3.5 k bTEpm = 5.5 k bT
Modelling of doggy bones
