Detergent impact on tethered bilayer lipid membranes for protein reconstitution

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 • nonionicdetergents: • Dodecylmaltoside, Decylmaltoside DPhyPC dodecylmaltoside

3. Tethered bilayer lipid membrane (tBLM) lipids s tether backfiller submembrane space • 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 C12Maltosideincubation

6. Impedance Spectroscopy substrate interfacial capacitance tBLM resistance frequency response to V(w): Z() = V()/I() spreading resistance tBLM capacitance stray capacitance tBLM equivalent circuit model main parameters: tBLM Capacitance~ 10-6 F cm-2 tBLM Resistance~ 105-106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 (A2008-307).

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