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

• robustness/size/versatility: tethered lipid bilayers• functionality - proteins as in cell membranes• proteins extracted/purified using detergents• direct protein reconstitution → detergents

Development of biomimetic membranes

Materials

Heerklotz, Quarterly Reviews of Biophysics, 41, 205 (2008);Seddon et al., Biochim Biophys Acta, 1666, 105 (2004)

Detergent impact on membranes

lipids: DPhyPC, DOPC, POPE/POPG nonionic detergents: Dodecylmaltoside, Decylmaltoside

• vesicles: binding | saturation | solubilization• vesicles: bulk detergent titration guidance• supported bilayers: [lipid]/[detergent] ?

DPhyPC

dodecylmaltoside

→tethered bilayers: conditions (using detergents) for protein reconstitution?

Tethered bilayer lipid membrane (tBLM)

tether

backfiller

s

submembrane space

lipids

aqueous subspace for protein reconstitution resilience to multiple buffer exchanges durability

Fluorescence Imaging of tBLM degradation

DOPC (0.5% LRPE) tBLM incubation with ~ 85M C12Maltoside

25 m

DOPC (0.5% LRPE) tBLM buffer rinse after C12Maltoside incubation

25 m

Fluorescence Imaging of tBLM recovery

Impedance Spectroscopy

frequency response to V():

Z() = V()/I()tBLM capacitance

tBLM resistancespreading resistance

stray capacitance

substrate interfacial capacitance

main parameters:

tBLM Capacitance ~ 10-6 F cm-2

tBLM Resistance ~ 105-106

cm2

tBLM equivalent circuit model

Detergent impact on tBLM

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

DPhyPC tBLM incubation with 150M C12Maltoside

tBLM not dissolved, just leakier…

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Capacitance (uF/ cm2)

initial incubated rinsed

0

20

40

60

80

100

120

Resistance (kOhm cm2)

initial incubated rinsed

C(after rinse) ~ 105%-115% C(initial)R(after rinse) ~ 40%-60% R(initial)

Time course of tBLM electric response

Biphasic conductivity increase: main ≈ 102s ; secondary > 103s

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

Partial insulation recovery upon rinsing with ≈ 102s

POPE/POPG 3:1 tBLM on ~50% tethers

Dodecyl- vs decylmaltoside dose-response

similar responses to decyl- and dodecylmaltoside, rescaling concentrations to cmc

detergent-incubated statevs. initial state

final statevs. initial state

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

This work is funded by the NIH (1 P01 AG032131) and the AHAF (A2008-307).

This is a basis for current protein reconstitution efforts

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

Detergent impact on DOPC tBLM

DOPC tBLM on ~50% tethers with 150M C12Maltoside