Functional Surfaces

Nano-tech Devices:

Towards Protein Control of Surface Activity

and Permeability

Nano-tech Devices

Functional Surfaces:

chips, membranes, arrays

Signals:

getting information in and out

Actuation:

Actuation

Micromachined rough surface

Hydrophobins

• Proteins excreted by fungi • Function in growth and

development

• About 100 amino acids • 8 conserved cysteine

residues

• Self-assemble at hydrophobic - hydrophilic interfaces

Thr

Leu

Ser Gly Gly

Leu Leu

Leu Val

Ala Leu

Ile

SC3 Hydrophobin

Changing properties of surfaces

Teflon

Mica

Lateral force image

•light = high lateral force

Topographic Image

•light = raised surface

- hydrophobin + hydrophobin

AFM of the rodlet structure

ATR-FTIR

1700 1680 1660 1640 1620 1600

In

te

n

si

ty

Wavenumber (cm-1)

190 200 210 220 230 240 250 -2 -1 0 1 2 E lli pt ic ity x 1 0 -4 (d eg c m 2 dm o l -1 ) Wavelength (nm)

Circular Dichroism

-helix -sheet -turn random coil

Soluble 23 41 16 20

At air-water interface 16 ₆₅ 9 10

At hydrophobic surface ₃₃ 36 17 14

Molecules exchange between oligomers

0 200 400 600 800 1 2 3 4 5 6 7 F lu or es ce nc e in te ns ity ( a. u. ) Time (min) TFA/dansyl-SC3+TFA/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3 TFA/dansyl-SC3

Molecules exchange between oligomers

SC3 associates in solution and

dissociates on a hydrophobic surface

Wavelength (nm) Wavelength (nm)

F lu or es ce n ce ( a. u .) F lu or es ce n ce ( a. u .)

400 4 50 500 5 50 600 6 50 0 1 2 3 4 5 TFA/DX-SC3

TFA/DX -SC3+TFA /DAB-SC3

TFA/DX -SC3+TFA/DAB-SC3+Teflon

400 450 500 550 600 650 0 1 2 3 4 5 TFA/dansyl-SC3 TFA/dansyl-SC 3/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3/Teflon 0 0.2 0.4 0.6 0.8 1 1.2

0 0.2 0.4 0.6 0.8 1

SC3 in



-sheet state clusters

on a hydrophobic surface

Actuation

400 450 500 550 0 2 4 6 8 10 12 14 TFA/dansyl-SC3/Teflon TFA/dansyl-SC3/Teflon/65C Add 0.1% Tween80, 15 h

Wavelength (nm) F lu or es ce n ce ( a. u .) -helical state Teflon

-sheet state

Teflon

heating, detergent

600 650

Surface-induced folding of sulfite treated SC3

• Native SC3

• Reduced and reacted form - stable in solution

• Refolding on hydrophobic surface

• Reformation of disulfides by air oxidation

soluble



-helix form



-sheet form

very fast fast

medium very slow

Deuterium Exchange Rates vs Structural State

Actuation

Engineering Surface Permeability

Goal:

control ion permeability through changes

in ion

selectivity

and changes in

gating

properties

Pore forming molecules mediating ion fluxes

across (biological) membranes

Ion channels are

not

mere

nano tubes

but

are characterized by:

-

ion selectivity

-

gating

(opening and closing)

Protein Ion Channels

In the context of biosensor technology

Ion channels are signal amplifiers:

Channel opening results in a flow of ions as large as 108

per second

Ion channels are signal transducers:

A chemical signal (binding event of the target molecule) can be transduced into an electric current

‘closed’ ‘open’

ligand target molecule

What determines the selectivity of an ion channel?

- size of the permeant ion species

- charge of the permeant ion species

- combination of both

- atomic arrangement in that part of the protein

responsible for the ion selectivity

Ion Channel Selectivity

Example: L-type Ca

channels

Selectivity filter comprises

4 negatively charged glutamates:

-OC-C-CH₂-CH₂-COO

I HN

I

glutamate (E)

R-COO

- -

Ca2+

Ca2+

R-COO

-

-

Model System: Porin (OmpF) of

E. Coli

Side view Top view

40 Å +

-Actuation

Switch the essentially non-selective porin (OmpF) into a calcium-selective ion channel by mimicing the dielectric environment found in Ca2+ channels

Strategy

Use site-directed mutagenesis to put in extra glutamates

and create an EEEE locus in the selectivity filter of OmpF

Site-directed

mutagenesis R132

R82

E42 E132

R42 A82

Wild type EAE mutant

E117 E117

D113 D113

PLANAR LIPID BILAYER SET UP

recordings on a

single

molecule!

OmpF trimer

ions

Trans Cis

OA Rf

Phospholipid bilayer

-+

Vcom

Vout If

If

IK

IV-converter

Voltage clamp: - voltage is set

- current is measured

-100 -50 50 100

-150 -50 50 150

E_Ca

WT

EAE

Current (pA)

Voltage (mV) Cis Trans

1 M CaCl₂ 0.1 M CaCl₂

Ca2+ _Ca2+

Cis Trans _{Cis Trans}

IV-plot EAE: current reverses at equilibrium potential of Ca2+ (ECa),

Zero-current potential or reversal potential = measure of ion selectivity

P

/P

WT

2.8

AAA

25

EAE

>100

Ca2+ over Cl- selectivity (P

Ca/PCl)

recorded in 1 : 0.1 M CaCl₂

SUMMARY OF RESULTS (1)

Conclusions:

- Taking positive charge out of the constriction zone

(= -3, see control mutant AAA) enhances the cation over anion permeability.

- Putting in extra negative charge (= -5, see EAE mutant) further increases the cation selectivity.

P

/P

WT

2.2

AAA

3.7

EAE

4.2

Ca2+ over Na+ selectivity (P

Ca/PNa)

recorded in 0.1 M NaCl : 0.1 M CaCl₂

SUMMARY OF RESULTS (2)

Conclusion:

- Compared to WT, EAE shows just a moderate increase of the Ca2+ over Na+ selectivity.

- To further enhance P_Ca/P_Na may require additional negative charge and/or a change of the ‘dielectric volume’.

O-1/2

Na+

Ca2+

Ca2+

with imposed electroneutrality

- Selectivity filter is a dielectric volume rather than a rigid molecular structure

- ‘Goodness of fit’, selectivity, determined by a proper crowding, it takes twice as much Na+ than Ca2+ to compensate the -4e charge of 8 O’s

‘GOOD’ _{‘TOO CROWDED’} Na+ Na+ Na+ O-1/2 O-1/2 O-1/2

O-1/2 O_-1/2

O-1/2 O-1/2

Dynamic Control of Permeability

Goal: To put permeability under control of external signals

– pH

– pressure

– temperature – redox potential

– electric and magnetic fields – ultrasound

– light

pH-Induced Channel Switching

OH

O Br N

N H

2-Bromo-3-(5-imidazolyl)propionic acid

N N N N

N S N

O

N

NH₂

N NH2

pK a= 5.97

pK a= 5.19 pK a= 5.68 pK a= 6.02

pK a= 5.4 pK a= 6.62 pK a= 6.82 pK a= 9.25

pH-Responsive Channel Switching

(p

A

)

0 50 100

pH 7.2

N

(pA )

0

100 pH 5.2

HN

+

pH-sensitive channel openings

pH-mediated Drug Release from

Proteoliposomes

Circulating Liposome Targeted Liposome

Light-Responsive Channel Proteins

200 300 400 500 600 700

Biomade

• M.de Vocht • X. Wang • R. Friesen • H. Meidema • W. Meijberg • A. Sagiroglu

Rush Medical College

• B. Eisenberg • J. Tang

U. Of Miami School of Medicine

• W. Nonner • D. Gillespie

U. Of Groningen

• B. Poolman • B.Feringa • J.van Esch • H. Wosten • J. Wessels • I. Reviakine

• W. Bergsma-Schutter • A. Brisson

École Polytechnique Fédérale de Lausanne