Proteins for Bio-Electronics ? - PowerPoint Presentation

1. Proteins for Bio-Electronics ?Electron Transport across Proteins & Peptides SupportMinerva Foundation, MunichIsrael Science FoundationIsrael Council of Higher EducationPecht-fest 6-2017 withMordechai Sheves & Israel Pechtthese pictures are cartoons!

2. 2Proteins in “Solid-State” electronics?Single bio-molecule ElectronicsElectron transfer protein cytochrome b562 bound between two electrode surfaces.Della Pia et.al.ACS Nano, 2012, 6, 355Nanoscale, 2012,4, 7106Artés et. al. Nano Lett. 2012, 12, 2684Artés et. al. ACS Nano 2011, 5, 2060–2066Transistor-like Behavior of Single Metalloprotein JunctionsBand Gap Tuning of Holoferritin by Metal Core ReconstitutionRakshit et. al. Langmuir, 2011, 27, 9681Rakshit et. al. Langmuir, 2013, 29, 12511

3. Proteins are Soft Materials “soft bioelectronics”1st challenge to study such materials is to contact them electrically, reproduciblyaffecting them minimally.It is even harder to make contacts for reliable current transport measurements(i.e., beyond applying bias)

4. padLift-off float-on (LOFO) e.g.: Au, PEDOT-PSS Non-destructive electronic contacts to soft matter: Hg or In-Ga, ’ready-made’ metallic pad, evaporated PbSiPb~1nmsubstrate / contactlinker layerAmetalIdealized cartoonHg dropHgSubstrateBackcontactGaOx!high pHg!~0.2 mm 2 ~ 106 – 107 (formally 1010) proteins/contact

5. Dielectrophoretic assembly of nanowires suspended nanowire techniqueNoy, Ophir, Selzer, 2010, Angew Chem.Sepunaru et al., (2015) JACS Y. Selzer (TAU)> ~10% junctions “work”Formally ~ ≥ 1000 (small) proteins / contact; ~ 0.05 μm27TRP+orPeptide or protein junction

6. Yoram Selzer &Rani Arielly(TAU)Molecular junction

7. IdealizedCartoons!We can work with nanoscale contacts; here is a closer look at such experiments:Nanoscopic A10 nmMetallic substrate 2 μm~10 – ~ 20 ? proteins/contact ~ 400 nm2

8. …..…..…..…..…..contact…..intimate 10-20 µm2 contact to a 10-20 nm2 / protein monolayer? like contacting each grass leaf (~5 cm2) on 70×100 m2 soccer field[Akkerman]But also large contact area experiments have problemsIs also a Cartoon!!still, higher over-all currents  large measuring ability gain;aren’t we measuring pinholes between these large molecules? Unlikely: comparable results with different roughness top & bottom contacts.0.2 mm2~ 106 – 107 (formally 1010) proteins/contact

9. Conductive substrateSi (p++)Linker layer Substrate - smooth !!!! Metal or Semiconductor Linker layer - self-assembled monolayer short molecule with functional terminal group (“+” or “-” charge) Protein layer – should be dense, free of pinholes Top contact – deposition and composition compatible with ‘soft’ biological material9SiO2Preparation of “Solid-State” Protein JunctionsElectrical top contact- AFM; TEM  Cryo-ED; XRR Ellipsometry; UV-Visible Abs.; Fluorescence- FT-IR; Surface PotentialMonolayer CharacterizationCan proteins act as active material outside Of natural environment?

10. Proteins (& peptides) are Soft Materials “soft bioelectronics”After learning how to contact them electronically, reproducibly, reliably and affecting them minimallywe find that (as monolayers) they are amazing(solid state-compatible) electronic materials

11. of protein monolayersolid-state junctionsConductive substrate(CH2)3 LINKER layerElectrical top contactidealizedcartoonJin et al., PNAS, 2006 …..... Ron et al, JACS. 2010Current-Voltage characteristicsm~5.5 nm~2.5 nm~2.5~4.5

12. Can the proteins “survive” (partial) dehydration ?bR568M412PhotochemicalThermalbR PhotocycleBacteriorhodopsin (bR)Ron et al, JACS 2010UV-Vis. AbsorptionDifference SpectrumPhotocycle of monolayer LOV protein YtvA Mukhopadhyay et al., PCCP, 2016

13. Photo-conductance across Bacteriorhodopsin (bR) monolayer Green irradiation – Formation of metastable M-state Jin et. al. PNAS 2006 , 103, 8603Photo-effect originates from M-state accumulation (conformation change) Idealized cartoons

14. * L. Sepunaru et al., (2011) JACS ** X. Yu et al., (2015) ACS Nano For Azurin :@ denaturation temperature irreversible decrease in conductance *and in situ vibrational spectroscopy shows Amide 1 & 2 of proteins in the junction**Proteins can “survive” (partial) dehydration (II) !

15. at least some proteinsconduct electrons “too efficiently”show tunneling-like behaviour over “too long” distancesNow we can look at some results:

16. Amdursky et al., Adv. Mater. 9-2014Protein vs. conjugated & saturated molecule conduction this is funnynano-scale@RT@ 100 mVLog(current) @ 0.1 V vs. length plot of nano-scale measurement dataWhile long-range Electron Transport involving several proteins is known (respiration, photosynthesis), but across one protein,.. that’s different

17. Protein vs. conjugated & saturated molecule conduction Log(current) @ 0.1 V vs. length plot of macro-scale measurement dataphRPS1 (Groningen)PS1 (WIS)PS1 ( MIT)Ferritin(NUS)bRAmdursky et al. Adv. Mater. 9-2014, updated 8-2016Globular Proteinsmacro-scale @RTthis is weirdidealizedcartoon@ 100 mV@ RT

18. at least some proteinsconduct electrons “too efficiently”show tunneling-like behaviour over “too long” distancesNow we can look at some results:

19. with (holo), and without (apo) Cu ion300 meVSepunaru et al., J. Am. Chem. Soc. 2011Temperature dependence of “solid state” Electron Transport, ETp, of Azurin~2.5 nmConductive substrateLinker layerElectrical top contactidealizedcartoon@ 50 mV@ RT

20. BacterioruberinRetinalHalorhodopsin (phR), a Cl- pumpMukhopadhyay et. al. J. Am. Chem. Soc. 20156-6.5 nm

21. Temperature-dependent electron transport, ETp, of bacteriorhodopsin (bR) and Halorhodopsin (phR)HalorhodopsinBacteriorhodopsinMukhopadhyay et al. J. Am. Chem. Soc. 2015Sepunaru et al. J. Am. Chem. Soc., 20126-6.5 nm5.5-6 nm@ 50 mV

22. Temperature (in)dependent ETp of PS I (~ 7-9 nm !)Sepunaru et al., 2012 unpublished;samples from Carmeli & Carmeli, TAU- 50 mV+ 50 mV @RTcf. Castaneda Ocampo …...Herrmann, Chiechi JACS 2015(@ 500 mV )

23. MPS = sodium 3-mercapto-1-propanesulfonate2ME = 2-mercaptoethanolTemperature (in)dependent ETp of PS I (~ 7 nm !)Castañeda Ocampo, O. E. et al.. J. Am. Chem. Soc.. (2015)@ 500 mV

24. @ 1 VTemperature (in)dependent ETp of Ferritin (~ 10 nm)Kumar …. Nijhuis, Adv. Mater. (2016)

25. But , …..nihil novi sub solemאין חדש תחת השמשEcclesiastes קהלת 1.9

26. Methods: For solution electron transfer, ET: e.g., Flash-quench, Electrochemistry For solid-state electron transport, ETp: e.g., STM, CP-AFM, macro-junction

27. e-hνin solutionin solid state!ETpETSpectroscopyElectrochemistrye-N. Amdursky et. al. (PCCP 2014)Biomolecular electronic junctione-+-+How does Electron transfer differ from Electron transport ?

28. STMCP-AFMMacroscopicSpectroscopicElectrochem.J = kET/ constantAmdursky et al.,Adv. Mater. -2014@RTHow do Electron transfer results compare toElectron transport ones ?

29. RT Conduction across protein monolayers is, length-normalized, comparable to that across conjugated molecule monolayers and , for membrane proteins, can be even better. Several proteins show temperature – independent current transport, even across 6-10 nm monolayers So, what did we learn till now?How can electrons cross proteins?

30. Flickering resonance Hopping  Redox sites in chain move in/out of resonance, Beratan & SkourtisIncoherent transport Localized charge hops between consecutive sites Possible Electron Transfer (ET) and ETp mechanisms Superexchange-mediatedTunnelingThermal fluctuations degenerate D & A levels Tunnel along bridge cf. e.g., Gray & Winkler ~~ temp. independentfrom J. Blumberger, Chem. Rev., 2015

31. Charge Transfer in Dynamical Biosystems, or The Treachery of (Static) ImagesBeratan et al., Acc. Chem. Res., 2015Proteins (and peptides) are Soft Materials; ergo: structural dynamics  energetic disorderQuantum path for ET in ProteinBlumberger, Chem. Rev., 2015these are not tunnelling paths, but CARTOONS !Possible Electron Transfer (ET) and ETp mechanisms

32. 32Tunneling bySuperexchangeFlickering resonance Hopping Thermal fluctuations degenerate D & A levels Tunnel along bridge Gray et al.  Redox sites in chain move in/out of resonance Beratan et al. Incoherent transport Localized charge hops between consecutive sites from Blumberger et al. Chem. Rev., 2015Possible transport mechanisms

33.  kBTThermally fluctuating energy states Potential landscape(Dynamic)Thermally activated hoppingFlickering resonance tunnelingMolecular junctionDonorResiduesTunneling by superexchange SequentialtunnelingAcceptorC. Bostick, S. Mukhopadhyay, I. Pecht, M. Sheves,D. Cahen, D.LedermanSubmitted.http://arxiv.org/abs/1702.05028

34. What controls Electronic Transport in and across proteins ?H-bondspeptide backbonecofactorscontacts✔soonCunlan Guo Lior Sepunaruwith Leeor Kronik (DFT) Koby Levy (MD)See poster P11-9 Thu p.m.(✔)

35. Factors affecting peptide ET (  ETp )e-See poster P11-9 Thu p.m.

36. 7TRP6TRP5TRP4TRPβ = 0.58 ± 0.06 Å-1RT7KLength effect on ETp via HOMO-peptide (oligo-Tryptophan)Sepunaru et al., JACS 2015Completely temperature –independentpossible mechanism: tunneling

37. Electronic structure of oligo-Tryptophan with length

38. Strong residue effect on electron transportGas phase computations (CH3)Sepunaru et al., JACS 2015

39. Jaklevic RC et. al. PRL 1966, Galperin M et. al. Science 2008Inelastic electron tunneling spectroscopy (IETS)Are there molecules in the junction ?Some tunneling electrons can lose (or gain) energy by exciting (excited) vibrations of the molecules between junctions. The inelastic tunneling event is related with molecular vibrations: Inelastic tunneling channel opens when It can be compared with IR and Raman spectroscopy. IETSeV = ℏωvib

40. Differential Conductance ∂I/∂VCurrent -Voltage(∂2I/∂V2)/(∂I/∂V)νas(NH3+) νas(NH3+) ν(NH)ν(NH)+H3NSepunaru et al., JACS 2015; PhD thesis 2014Yes, there are molecules in the junction !PEPTIDE7-Lys, protonated@ ~ 10 K

41. and also proteins:IETS of Azurin on suspended nanowire C-HC-HAu-SAu-S?C-S C-S?Au-S and S-C stretchings are clearly seen. Electron injected into protein through Au-S-C.41X. Yu et al., (2015) ACS Nano @ ~ 10 K

42. Amide IAmide IICH2 Wag**IETS of Az – Peak PositionsX. Yu et al., (2015) ACS Nano @ ~ 10 K

43. Adding Secondary Structure to the PeptidesSepunaru et al., JACS 201520-AlaHepta-Ala20-LysHepta-Lys

44. How do electrons get in, and out of proteins?Some questions that describe what we do not yet knowHow do electrons cross proteins? See poster P11-9 Thu p.m.

45. 45Temperature dependence is all about Electrode-Protein COUPLING ( Electrostatics)Top electrodeSubstrateTop electrodeSubstrateMukhopadhyay et al. J. Am. Chem. Soc. 2015Sepunaru et al. J. Am. Chem. Soc., 2011?CouplingAu-S or S-S?CouplingCoupling?Once the electron is IN, there seems to be no barrier till it EXITS

46. Effect of coupling to electrodese-Role of individual Trp residue (indole) in ETp of hetero-peptidesCouplingCoupling

47. 21±2 Å20±2 Å26±2 ÅEllipsometrythicknessA. MPA - 1W 6A W-1B. MPA- 3A 1W 3A W-4C. MPA -6A 1W W-76 Å14 Å3 ÅAll peptides are ~3 nm long, from MD simulation W = Trp, trytophan A = Ala, alanineMPA = 3-Mercaptopropionic acid MD simulation by Yulian Gavrilov, Koby Levy (WIS)Guo, Yu et al., PNAS, 2016Study coupling to electrodes with HETERO-Peptides

48. W-7 > W-1 > W-4I-V characteristicsMPA-7W & MPA-7A I-V curves from Sepunaru et al., JACS, 2015W-7W-1W-47W7A7W ≈ W-7 > W-1 > W-4 >> 7ADominant mechanism: TunnelingGuo, Yu et al., PNAS, 2016

49. Inelastic electron tunneling spectroscopy (IETS) of HETERO-peptidesIndole the side chain of Trp, is involved in ETp for all the hetero-peptidesCH2 vibration(cm-1)C=C vibration from indole (cm-1)CH2 wagging(cm-1)~2940~1580~14007A7WW-1W-4W-7C.Guo, X. Yu et al., PNAS, in press

50. Compare (DFT) theory of peptide electronic structure (gas phase comput.) HOMO of peptide with Trp is higher than of 7A;HOMO of peptide with Trp is localized on the indole ring.W-1W-1W-4W-77A7W~-5.1W-7W-1W-47AConductance (I @ 0.3 V): W-containing peptides >> 7A 7WGuo, Yu et al., PNAS, 2016with experimental currents

51. Energy barrier height and peptide-electrodes coupling in Au-peptide-Au junctionsConsider peptide ETp fitting single-level transport modelEFε0ΓLandauer Modelε0: Energy barrier height Γ: Peptide-electrode coupling Cf. also Baldea, PRB, 2012, 85, 035442From Landauer formula: Fitting I-V curve, , V)  

52. Compare (DFT) theory of peptide electronic structure (gas phase comput.) HOMO of peptide with Trp is higher than of 7AW-1W-4W-77A7WW-7W-1W-47AConductance (I @ 0.3 V): W-containing peptides >> 7A 7WGuo, Yu et al., PNAS, 2016with experimental currents

53. Other theory – experiment comparison withUV Photoelectron Spectroscopy, UPS, of peptide monolayers on Au The trend of energy barrier for peptide monolayer is similar to the energy barrier of peptide in gas phase. The decrease of energy barrier comes from Au-S binding. 7AW-1W-4W-77Wof monolayerGuo, Yu et al., PNAS, 2016

54. Energy barrier height and peptide-electrodes coupling in Au-peptide-Au junctionsConsider peptide ETp fitting single-level transport modelEFε0ΓLandauer Modelε0: Energy barrier height Γ: Peptide-electrode coupling Cf. also Baldea, PRB, 2012, 85, 035442From Landauer formula: Fitting I-V curve, , V)  

55. Some possible transport modelsPossible mechanism I) Tunneling, mediated by super-exchange Possible mechanism II) Tunneling, mediated by single-level transport, , V)  Guo, Yu et al., PNAS, 2016

56. Energy barriers for ETp via peptides from different methods & couplingW-1W-4W-77AW-7N ≈ 100 Trends for peptide-electrode coupling, Γ, and ETp current are similar, also for UPS, except W-7; additional coupling between 2nd contact and peptide promotes peptide ETp.Guo, Yu et al., PNAS, 2016 

57. How do electrons get in, and out of proteins?Some questions that define what we don’t knowHow do electrons cross proteins?

58.

59. ConclusionsETp efficiency depends on amino acid side chain. Dominant transport mechanism – (elastic) tunneling Secondary structure– lowers barrier for ETpCoupling to electrodes ≠ chemical bonding Some reviews: Acc. Chem. Res. 2010, 43, 945 Adv. Mater. 2014, 42, 7142 (Rep. Progr. Phys.) 2017 abs/1702.05028 (ArXiv) Electron transport through a protein can be fully coherent “IN  OUT hypothesis” : once electron enters, it can reach, without relaxation or scattering, the other electrode, i.e., coherentlyIF coupling is T- (in)dependent  transport is T- (in)dependent…. implications for (bio)physics of Electron Transfer in & out of proteins, for its control, and for bioelectronics

60. Thanks to + Lior Sepunaru (UCSB)Nadav Amdursky (Technion)Izhar Ron (Isr. Biol. Inst.)---------------------------Kronik group:Sivan Refaely-Abramson (LBL/UCB) Piyush AgrawalDavid Egger (U. Regensburg)---------------------------Levy group:Yulian GavrilovSachi Mukhopadhyay JerryFereiro Xi Yu (Tianjin U.)CunlanGuo

61. Conductance across protein monolayersis “too efficient”Is tunneling-like, also over “too long” distances If, indeed electrode-protein coupling controls electron transport , ETp, across proteins, let’s speculate: redox entry & exit of e--s control ET across proteins, while main ET(p) mechanism in protein is coherent tunneling, ... and redox group protects protein from electron reducing powerSummary of our protein electronics adventures (till now) Future *  monolayer solid-state gating (explore / define energetics) * Study protein-protein ETp  bacterial nanowires  …  devices ?