detergent impact on tethered bilayer lipid membranes for protein reconstitution

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