nano-tech devices: towards protein control of surface activity and permeability

Nano-tech Devices:

Towards Protein Control of Surface Activity and Permeability

BBio

Nano-tech Devices

Functional Surfaces:

chips, membranes, arrays

Signals:

getting information in and out

Actuation:

control of behavior

Functional Surfaces

Functional SurfacesSignalsActuation

Micromachined rough surface

Flat surface 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

• Assemblages - very stable


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

Inte

nsity

Wavenumber (cm-1)

190 200 210 220 230 240 250-2

-1

0

1

2

Elli

ptic

ity x

10-4

(deg

cm2 dm

ol-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 8001

2

3

4

5

6

7

Flu

ores

cenc

e in

tens

ity (

a.u.

)

Time (min)

TFA/dansyl-SC3+TFA/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3 TFA/dansyl-SC3


0 200 400 600 8001

2

3

4

5

6

7

Flu

ores

cenc

e in

tens

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)

Flu

ores

cen

ce (

a.u

.)

Flu

ores

cen

ce (

a.u

.)

400 450 500 550 600 6500

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 6500

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

TFA/dansyl-SC3/dabcyl-SC3

TFA/dansyl-SC3/dabcyl-SC3 +Teflon

Rel

ativ

e fl

uor

esce

nce

dabcyl-SC3/(dansyl-SC3+dabcyl-SC3)

Soluble state

-helical state

Teflon

Teflon


SC3 in -sheet state clusterson a hydrophobic surface


400 450 500 5500

2

4

6

8

10

12

14

TFA/dansyl-SC3/Teflon TFA/dansyl-SC3/Teflon/65C Add 0.1% Tween80, 15 h

Wavelength (nm)

Flu

ores

cen

ce (

a.u

.)

-helical state

Teflon

-sheet state

Teflon

heating, detergent

600 650

Or low pH

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 fastfastmediumvery slow

Deuterium Exchange Rates vs Structural State



Next Step: Receptor site fusionsMini-hyrdrophobinsAlignment

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’

ligandtarget 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 Ca2+ channels

Selectivity filter comprises 4 negatively charged glutamates:

-OC-C-CH2-CH2-COO-

I HN I

glutamate (E)

R-COO- -

OOC-R

Ca2+

Ca2+

R-COO-

-OOC-R

EEEE locus


Model System: Porin (OmpF) of E. Coli

Side view Top view


40 Å+ -


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

Goal

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

R82E42

E132

R42 A82

Wild type EAE mutant

E117 E117

D113D113


PLANAR LIPID BILAYER SET UPrecordings on a single molecule!

OmpF trimer

ions

Trans Cis

OA

Rf

Phospholipid bilayer

-

+

Vcom

Vout

IfIf

IK

IV-converter

Voltage clamp:- voltage is set- current is measured


-100 -50 50 100

-150

-50

50

150

ECa

WT

EAE

Current (pA)

Voltage (mV)

Cis Trans

1 M CaCl2 0.1 M CaCl2

Ca2+

Ca2+

IV-PLOT

Cis Trans Cis Trans

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

indicating the channel can discriminate between Ca2+ and Cl-

Zero-current potentialor reversal potential = measure of ion selectivity


PCa/PCl

WT 2.8AAA 25EAE >100

Ca2+ over Cl- selectivity (PCa/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.


PCa/PNa

WT 2.2AAA 3.7EAE 4.2

Ca2+ over Na+ selectivity (PCa/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 PCa/PNa may require additional negative charge and/or a change of the ‘dielectric volume’.


O-1/2

Na+

Ca2+

Ca2+

‘Electric stew’ of Nonner et al.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

O-1/2

O-1/2

O-1/2

O-1/2

O-1/2

O-1/2O-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


Model of Mechanosensitive Gating Mechanism of the MscL Protein

pH-Induced Channel Switching

15.6 15.8 16.0 16.2 16.40

1

2

3

4

5

6

Abu

ndan

ce (

a.u)

Molecular Mass (kDa)

MscL.BI

MscL

OH

O

BrN

NH

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


OH

O

BrN

NH

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

N N N N

NS

N

O

N

NH2

N NH2

pK a= 5.97pK 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


(pA

)

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

S SHO

O OBr

UV

VisSS

O

HO

O

Br

200 300 400 500 600 7000.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

Wavelength (nm)

Open Closed


Acknowledgments

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

• P. Ulrich• H. Vogel

nano-tech devices: towards protein control of surface activity and permeability

Documents

functional surfaces

sc3 native sc3

permeability slide

sc3 associates

arrays signals

control of behavior

low ph slide

tfadansyl sc3dabcyl