Nano-tech Devices: Towards Protein Control of Surface Activity and Permeability

1. Nano-tech Devices:Towards Protein Control of Surface Activity and Permeability

2. Nano-tech Devices Functional Surfaces: chips, membranes, arrays Signals: getting information in and out Actuation: control of behavior

3. Functional Surfaces

4. Functional Surfaces Signals Actuation Micromachined rough surface Flat surface Rough Surface

5. Functional Surfaces Signals Actuation 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

6. Functional Surfaces Signals Actuation Thr Gly Ser Gly Leu Ile Leu Ala Leu Leu Leu Val SC3 Hydrophobin

7. Functional Surfaces Signals Actuation Changing properties of surfaces Teflon Mica

8. Functional Surfaces Signals Actuation • Lateral force image • light = high lateral force • Topographic Image • light = raised surface - hydrophobin + hydrophobin

9. Functional Surfaces Signals Actuation AFM of the rodlet structure

10. Functional Surfaces Signals Actuation Circular Dichroism ATR-FTIR 2 ) -1 1 dmol 2 (deg cm Intensity 0 -4 Ellipticity x 10 -1 -2 1700 1680 1660 1640 1620 1600 190 200 210 220 230 240 250 -1 Wavenumber (cm ) Wavelength (nm) a b b random coil -helix -sheet -turn Soluble 23 41 16 20 At air-water interface 16 65 9 10 At hydrophobic surface 33 36 17 14

11. Functional Surfaces Signals Actuation Molecules exchange between oligomers TFA/dansyl-SC3+TFA/dabcyl-SC3 TFA/dansyl-SC3/dabcyl-SC3 7 TFA/dansyl-SC3 6 5 Fluorescence intensity (a.u.) 4 3 2 1 0 200 400 600 800 Time (min)

12. Functional Surfaces Signals Actuation Molecules exchange between oligomers TFA/dansyl-SC3+TFA/dabcyl-SC3 7 TFA/dansyl-SC3/dabcyl-SC3 TFA/dansyl-SC3 6 5 Fluorescence intensity (a.u.) 4 3 2 1 0 200 400 600 800 Time (min)

13. Functional Surfaces Signals Actuation Soluble state Teflon -helical state Fluorescence (a.u.) Teflon Fluorescence (a.u.) Wavelength (nm) Wavelength (nm) SC3 associates in solution and dissociates on a hydrophobic surface 1.2 1 TFA/dansyl-SC3 /dabcyl-SC3 + 0.8 Relative fluorescence Teflon 0.6 0.4 TFA/dansyl- SC3/dabcyl-SC3 0.2 0 0 0.2 0.4 0.6 0.8 1 dabcyl-SC3/(dansyl-SC3+dabcyl-SC3)

14. Functional Surfaces Signals Actuation TFA/dansyl-SC3/Teflon TFA/dansyl-SC3/Teflon/65C Add 0.1% Tween80, 15 h 14 Teflon 12 -helical state 10 Fluorescence (a.u.) 8 heating, detergent Or low pH 6 4 2 0 Teflon 400 450 500 550 600 650 Wavelength (nm) -sheet state SC3 in -sheet state clusters on a hydrophobic surface

15. Functional Surfaces Signals Actuation 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

16. Functional Surfaces Signals Actuation soluble -helix form -sheet form very slow medium fast very fast Deuterium Exchange Rates vs Structural State

17. Functional Surfaces Signals Actuation Next Step: Receptor site fusions Mini-hyrdrophobins Alignment

18. Functional Surfaces Signals Actuation Engineering Surface Permeability Goal: control ion permeability through changes in ion selectivity and changes in gating properties

19. Functional Surfaces Signals Actuation Protein Ion Channels 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)

20. Functional Surfaces Signals Actuation In the context of biosensor technology ligand target molecule ‘closed’ ‘open’ 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

21. Functional Surfaces Signals Actuation Ion Channel Selectivity 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

22. Functional Surfaces Signals Actuation R-COO- -OOC-R Ca2+ Ca2+ R-COO- -OOC-R -OC-C-CH2-CH2-COO- I HN I glutamate (E) EEEE locus Example: L-type Ca2+ channels Selectivity filter comprises4 negatively charged glutamates:

23. Functional Surfaces Signals Actuation Model System: Porin (OmpF) of E. Coli Side view Top view

24. Functional Surfaces Signals Actuation 40 Å - + Goal Switch the essentially non-selective porin (OmpF) into a calcium-selective ion channel by mimicing the dielectric environment found in Ca2+ channels

25. Functional Surfaces Signals Actuation D113 D113 E117 E117 Site-directed R132 E132 mutagenesis R82 A82 R42 E42 Wild type EAE mutant Strategy Use site-directed mutagenesis to put in extra glutamates and create an EEEE locus in the selectivity filter of OmpF

27. Functional Surfaces Signals Actuation PLANAR LIPID BILAYER SET UP recordings on a single molecule! Phospholipid bilayer IV-converter ions R f OmpF trimer I f I f - OA V out + V com Voltage clamp: - voltage is set - current is measured I K Trans Cis

28. Functional Surfaces Signals Actuation Zero-current potential or reversal potential = measure of ion selectivity

29. Functional Surfaces Signals Actuation SUMMARY OF RESULTS (1) Ca2+ over Cl- selectivity (PCa/PCl) recorded in 1 : 0.1 M CaCl2 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,seeEAE mutant) further increases the cation selectivity.

30. Functional Surfaces Signals Actuation SUMMARY OF RESULTS (2) Ca2+ over Na+ selectivity (PCa/PNa) recorded in 0.1 M NaCl : 0.1 M CaCl2 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’.

31. Functional Surfaces Signals Actuation ‘Electric stew’ of Nonner et al. with imposed electroneutrality O-1/2 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 Ca2+ Na+ O-1/2 Na+ Na+ Ca2+ O-1/2 O-1/2 O-1/2 O-1/2 ‘GOOD’ ‘TOO CROWDED’ - 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

32. Functional Surfaces Signals Actuation Dynamic Control of Permeability Goal: To put permeability under control of external signals • pH • pressure • temperature • redox potential • electric and magnetic fields • ultrasound • light

33. Model of Mechanosensitive Gating Mechanism of the MscL Protein

36. Functional Surfaces Signals Actuation 2-Bromo-3-(5-imidazolyl)propionic acid pH-Induced Channel Switching 6 5 MscL.BI 4 Abundance (a.u) 3 MscL 2 1 0 15.6 15.8 16.0 16.2 16.4 Molecular Mass (kDa)

37. Functional Surfaces Signals Actuation pH-Responsive Channel Switching N 2-Bromo-3-(5-imidazolyl)propionic acid N N N pK = 5.68 pK = 5.19 pK = 5.97 pK = 6.02 a a a a O N H 2 S N N N N H N 2 pK = 5.4 pK = 6.82 pK = 9.25 pK = 6.62 a a a a

38. Functional Surfaces Signals Actuation pH 7.2 (pA) 100 50 0 N N pH 5.2 100 (pA) 0 + H pH-sensitive channel openings

39. Functional Surfaces Signals Actuation Circulating Liposome Targeted Liposome pH-mediated Drug Release from Proteoliposomes

40. Functional Surfaces Signals Actuation Light-Responsive Channel Proteins UV H O Vis B r S S H O S S B r O O O O 0.6 Open 0.5 Closed 0.4 Absorbance 0.3 0.2 0.1 0.0 200 300 400 500 600 700 Wavelength (nm)

41. 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 Acknowledgments