Tethered bilayer membranes

for protein-membrane
interaction studies
Dr. Gintaras Valinius
Vilnius university
Institute of Biochemistry

7th NanoSchool
2013-05-10
Vilnius

Models of biological membranes

Vecicles

BLM

SSM

tBLM
2

Structure of the talk

Engineering of tethered bilayer

Application of tBLMs for protein/membrane interactio

udies

Surface biofunctionalization: two

concepts
I. Weak interaction, e.g.
electrostatics

II. Strong/weak
interaction:
Chemical anchoring and
Hydrophbic insertion

Molecular anchoring
Hydrophobic segment

Hydrophilic segment

Surface active
group, e.g. thiol
(-SH)
5

The following molecules are currently available :

Dimiristoil , WC14 (EO6)
Dioleoil, HC18 (EO6), DC18 (EO9)
Dipalmitoil, FC16 (EO9)
Palmiloil/oleoil FayC (EO6)

Rapid solvent exchange

Step 1: Monolayer
formation
ethanol

Step 2: Lipid transfer

ethanol

Au

Au

Ng 2008 SURF talk

Formation of anchor layer can be

monitored:
Contact angle variation
140
120 angle, deg
Contact
100
80
60
40
20
0
0
20 40 60

80

100 120

WC14, mol %
McGillivray, D.J.; Valincius, G.; Vanderah, et al. Biointerphases.
2, 21-32 (2007).

Another ex-situ option is

ellipsometry:
4
Ellipsometric thicness, nm

3
2

3.3 nm

1
0

50

100

WC14, mol %
McGillivray, D.J.; Valincius, G.; Vanderah, et al. Biointerphases.
2, 21-32 (2007).

IR spectroscopy provides some

structural information

Diloleoil-

Dimiristoil-

10

Real-time monitoring of anchor layer

formation by SPR

11

How can we detect anchoring of

phospholipid bilayer?
Conductor
(electrolyte)
Dielectric

Si/Au
Electric

Conductor
(metal)

capacitance :

12

Electrochemical impedance
spectroscopy (EIS) is a fast and powerful
tool for monitoring
Anchor SAM
-3.50
-3.00

0.1 Hz

Im C, mF/cm2
-2.50

HC18/DOPC

-2.00
-1.50

Membrane

-1.00
-0.50
0.00
0.00

0.1 Hz
1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Re C, mF/cm2

Decrease of electric capacitance singnals membrane formation

13

Typical periphery devices used for EIS

measurements

14

Neutron reflectometry is another

technique which allows detailed
structural analysis of interface
NR measures ratio
between the intensity
of reflected and (k2)
incident (k1) neutrons

Red incident and reflected n-beam;

Orange refracted n- beam;
Green sctattered n-beam

15

Neutron reflectometry is sensitive

to the layered structure along
normal to a surface
Qz, -1

Log (reflected intensity)

Qz

16

Layers create interference patterns that can be

used for reconstruction of the structure
through modeling

17

MAGIK reflectometer at NIST Center for

Neutron Research, Gaithersburg, MD

18

ISIS Pulsed Neutron Facility, Rutherford-Appleton

Laboratory, Oxfordshire, UK

2012-03-09

19

Tethered phospholipid membranes are suitable for NR.

Light atoms exhibit high scattering and typical
wavelengths are from 2 to 10 .

Contrast H/D is also widely utilized to resolve complex structures.

20

Typical NR reflectivity curves

and modeled material
distribution curves

WC14 (dimiristoyl)/diphytanoyl bilayer

21

NR resolves structure
through a fitting of
the reflectivity
curve

WC14(dimiristoyl)/
Diphytanoyl tBLM

McGillivray et al. Biointerphases. 2 (2007) 21-33

22

Lateral structure can be

interrogated by atomic force
microscopy

WC14/DPhyPC

23

Vesicle interaction with surfaces:

assembly and modification of tethered bilayer
membranes

24

Vesicle fusion to the anchor SAM

600
500

Wash out

400
300

SPR demonstrates
Full coverage of surface
EIS demonstrate intactness
of the bilayer

200
100
0

500 1000 1500 2000 2500 3000 3500

Time/seconds

25

Material transfer from vesicles is

visualized by fluorescence microscopy

LR-DOPE labeled
DOPC vesicles

Chol-NDB labeled
DOPC vesicles
26

Cholesterol transfer can be

monitored by EIS

27

Cholesterol transfer facilitates reconstitution of pore

forming toxins sensitive to cholesterol content
160
Membrane conductance, mS

140
120
100
80
60
40
20
0

28

NR reveals structural details of material

exchange
Fosfolipidin
membrana

D2O

D2O
Aukso
sluoksnis

Aukso
sluoksnis

H2O

H2O

A bilayer from d54DMPC before vesicle exchange, B bilayer after exchange with
hDMPC vesicles. ISIS INTER reflectometer. Oxfordshyre, England, 2012.

Budvytyte et al. Langmuir. 29, (13), 43204327, (2013)

Details of
material
distributi
on
changes

BEFORE

AFTER

Budvytyte et al. Langmuir. 29, (13), 43204327, (2013)

30

Material (cholesterol) can be also extracted

from tBLMs using vesicle exchange

31

Conclusions I

The technology for surface immobilization of bilayer allows assembly of complex

compositions, including those from natural lipid components.

Physical techniques allows verification of structure of tBLMs with the nanometer

(for NR - Angstrom) resolution.

Composition of tBLM could be modified via vesicle exchange process

32

Applications
1. Membrane disrupting enzymes
2. Pore-forming toxins
3. Proteins (peptides) affecting dielectric properties of

membranes

33

Monitoring phospholipase activity with WC14-based tBLMs

PL D
PL C

PLA2
Ca2+ co-factor

Scooting mode

Bilayer capacitane :
34

Single-frequency EIS measurements can

detect and provide real-time detection of
enzymatic activity

+2 PLA2

Valincius, G.; et al. J.Phys. Chem. B.; 110 10213-10216

(2006).

35

NR reveals structural changes in

the course of lipid hydrolysis
nSDL, -2

Outer layer, ~33% D2O

36

Pore- forming -hemolysin from

S.aureus

Song, L., Hobaugh, M., Shustak, C., Cheley, S., Bayley, H., Gouaux, J.E. Structure of
staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science v274
pp.1859-1866 , 1996

37

tBLM allows precise positioning and structure

information on reconstitution of pore-forming protein

Duncan J. McGillivray, Gintaras Valincius, Frank Heinrich, et al. J, 96 (4) 1547 - 1553, (2009).
38

Functionality of reconstituted
protein

39

tBLMs may be used to detect (possibly

identify) cholesterol-dependent bacterial
toxins (e.g. vaginolysin from Gardanela
vaginalis)

40

tBLM may be used to quickly asses production

of toxins in cell culture and antibody efficiency
16

tBLM conductancce uS

16730
M5

12

100
8
10
4

VLYconcentra oninstrains, nM

1000

0
tBLM

16h

24h

48h

Grown me

VLY from G.vaginalis

41

Amyloid oligomers are

associated with Alzheimers
disease

42

Different protocols produce different

A oligomers
Protocol I (1-2 nm)

Protocol II (5-10 nm)

Protocol III (fibrils)

43

Toxicity of -amyloid oligomers

is size-dependent

44

Large (5-10 nm) A1-42 oligomers does

not affect electric properties of tBLMs
-7.5e-7
346_DOPC_101.txt
346_DOPC_1.6uM Ab_big_102.txt

E''

-5.0e-7

-2.5e-7

0
0

2.5e-7

5.0e-7

7.5e-7

E'

45

Small (1-2 nm) A1-42 oligomers affect both the

capacitance and conductance of tBLMs
(dielectric damage hypothesis).

WC14/DOPC
Gintaras Valincius, Frank Heinrich, Rima Budvytyte, et al.
Biophys. J. 95 4845-4861, (2008).
46

Practical applications: new generation

of electrochemical detectors for HPLC
Human
sample

Primary
purification

Separation &
Concentration

Chromatograph
y

Amyloid-
fractions

Detectors
D2

D1

UV

Total amyloid-
Necrotic-active
Apoptotic-active
47
47

Conclusions II

tBLMs provide biologically relevant media for protein reconstitution.

tBLMs allow to position proteins in a uniform manner and conduct

structural measurements with atomic resolution.
tBLM technology provide s experimental and technological platform for
the detection of the activity of membrane-associated proteins (peptides).

48

Acknowledgements
Rima Budvytyt , Mindaugas Mickevicius, Tadas Ragaliauskas , Tadas
Penkauskas, VU BChI
Dr. David Vanderah, NIST Gaithersburg, MD, USA
Dr. Vilmant Borutait, Ramune Morknien, Paulius ias LSMU, NI.
Dr. Aurelija virblien VU BTI
Habl. dr. Gediminas Niaura Center of Physical Sciences and
TechnologyVilnius,
Prof. Mathias Losche and Dr. Frank Heinrich , Carnegie Mellon Univ.
Pittsburgh, PA
Duncan McGillivray , Oakland University, Oakland ,New Zealand.
This research has been funded by RCL (ImfaBITE, AMILOIDE and
MALPALMA projects); also by the University of Maryland, Ohio State
university and Carnegie Mellon University; also EC support for instrument
access (NR, ISIS) through the I3 grant scheme is acknowledged.
49