1. Detergent impact on tethered bilayer lipid membranes for protein reconstitution
Matteo Broccio, Haw Zan Goh and Mathias Loesche
Department of Physics,
APS March Meeting 2009 – Pittsburgh, PA

2. Introduction Development of biomimetic membranes
robustness/size/versatility: tethered lipid bilayers
functionality - proteins as in cell membranes
proteins extracted/purified using detergents
direct protein reconstitution → detergents
Detergent impact on membranes
vesicles: binding | saturation | solubilization
vesicles: bulk detergent titration guidance
supported bilayers: [lipid]/[detergent] ?
Heerklotz, Quarterly Reviews of Biophysics, 41, 205 (2008);
Seddon et al., Biochim Biophys Acta, 1666, 105 (2004)
→tethered bilayers: conditions (using detergents) for protein reconstitution?
Materials
lipids: DPhyPC, DOPC, POPE/POPG
nonionic detergents:
Dodecylmaltoside, Decylmaltoside
DPhyPC
dodecylmaltoside

3. Tethered bilayer lipid membrane (tBLM)
lipids s
tether
backfiller
submembrane space
Pros: robust, facile, durable. Cons: asymmetric membranes not possible.
aqueous subspace for protein reconstitution
resilience to multiple buffer exchanges
durability

4. Fluorescence Imaging of tBLM degradation
25 mm
DOPC (0.5% LRPE) tBLM incubation with ~ 85M C12Maltoside

5. Fluorescence Imaging of tBLM recovery
25 mm
DOPC (0.5% LRPE) tBLM buffer rinse after C12Maltoside incubation

6. Impedance Spectroscopy
substrate
interfacial
capacitance
frequency response to V(w):
Z() = V()/I()
tBLM resistance
spreading resistance
tBLM capacitance
stray capacitance
tBLM equivalent circuit model
main parameters:
tBLM Capacitance ~ 10-6 F cm-2
tBLM Resistance ~ cm2

7. Detergent impact on tBLM
DPhyPC tBLM on ~ 30% tethers incubated with 150M C12Maltoside

8. tBLM not dissolved, just leakier…
DPhyPC tBLM incubation with 150M C12Maltoside
C(after rinse) ~ 105%-115% C(initial)
R(after rinse) ~ 40%-60% R(initial)

9. Time course of tBLM electric response
DPhyPC tBLM on ~30% tethers incubated with 150M C12Maltoside
Biphasic conductivity increase: main  ≈ 102s ; secondary t > 103s
Partial insulation recovery upon rinsing with t ≈ 102s

10. Dodecyl- vs decylmaltoside dose-response
detergent-incubated state
vs.
initial state
final state
vs.
initial state
POPE/POPG 3:1 tBLM on ~50% tethers
similar responses to decyl- and dodecylmaltoside, rescaling concentrations to cmc

11. Conclusions
FM and EIS show detergent-induced degradation and partial recovery after buffer rinse
EIS quantifies detergent-induced degradation: resistance and capacitance changes (→ defect formation / dielectric thinning)
below cmc, detergent-incubated tBLMs recover
C12Maltoside and C10Maltoside show same dose-responses
Possible explanation:
upon incubation, detergent induces formation of mixed lipid/detergent micelles retained at the membrane;
upon buffer rinsing, micelles break apart releasing lipid material to membrane
This is a basis for current protein reconstitution efforts
This work is funded by the NIH (1 P01 AG032131) and the AHAF (A ).

13. Detergent impact on DOPC tBLM
DOPC tBLM on ~50% tethers with 150M C12Maltoside