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

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

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

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

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

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 ~ 105-106 cm2

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

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)

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

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

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).

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