Single-molecule covalent chemistry in a protein nanoreactor

Transcription

1 Single-molecule covalent chemistry in a protein nanoreactor Summer School on Biosensing Jacobs University Bremen 28th June 2009 Laboratory of Hagan Bayley University of Oxford

2 We can do lots of things with nanopores: stochastic sensing engineered pores adapters polymer fishing lines polymer analysis counting/ identification sequencing [?] relatively little work on covalent chemistry

3 Utility in sensing There are lots of reactive analytes: chemical warfare agents pesticides anticancer drugs onions

4 Chemistry at the single-molecule level: using a transmembrane protein pore as a nanoreactor α-hemolysin pore as a nanoreactor favorable dimensions

5 Current measurements are made by single channel electrical recording in planar bilayers Perturbations of the ionic current tell us what is happening inside the pore, we see individual interactions or reaction steps for single molecules cis V V = 40 mv 700 ps = 30 pa 1 M NaCl trans In this talk: various V, salt, filtering, timescales etc.

6 Protein nanopore detection provides kinetic information τ on = 1/k on [A] τ off = 1/k off K d = k off / k on The signal contains kinetic information with ~10 µsec resolution under favorable circumstances

7 Single molecule covalent chemistry inside the pore irreversible reactions reversible covalent chemistry irreversible chemistry with turnover limits and future prospects

8 Advantages: short-lived intermediates in any step minor pathways fast reactions to ~10 µsec no tags required, e.g. fluorophores high reagent concentrations possible good control over temperature, ph, salt

9 Single molecule covalent chemistry inside the pore irreversible reactions reversible covalent chemistry irreversible chemistry with turnover

10 Chemistry of caged proteins Loudwig, S. and Bayley, H. Light-activated proteins: an overview, pp , in Dynamic studies in biology: phototriggers, photoswitches and caged biomolecules, M. Goeldner and R. Givens, eds., Wiley-VCH Verlag, Weinheim, (2005)

11 hν

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14 lifetime of carbamate fast step, after slow step pk a = 6.4

15 Chemistry of caged proteins slow! Good for cell biology Too slow for e.g. protein folding

16 Single molecule covalent chemistry inside the pore irreversible reactions reversible covalent chemistry irreversible chemistry with turnover

17 Azobenzene isomerization at the single-molecule level Loudwig, S. and Bayley, H. J. Am. Chem. Soc. 128, (2006)

18 Reactive toxic molecules - examples, chemical warfare agents Lewisite VX nerve gas mustard gas

19 Arsenic(III) as a poison environment (skin disease, cancer) therapeutic agents (Ehrlich, trypanosomiasis, leukemia) chemical weapons (Lewisite) rat poison, murder (As 2 O 3 - tasteless)

20 Arsenic(III) as a poison environment (skin disease, cancer) therapeutic agents (Ehrlich, trypanosomiasis, leukemia) chemical weapons (Lewisite) rat poison, murder (As 2 O 3 - tasteless)

21 Organoarsenic(III) chemistry

22 Organoarsenic(III) chemistry

23 αhl pore containing a single cysteine projecting into the channel lumen P SH

24 4-sulfophenylarsine oxide reacts reversibly with P SH

25 Chiral arsenic adducts 250 P SH count B A (G137C) 1 (WT) Conductance (pa) rare transition, overrepresented here

26 kaon = (14+1) x 103 M-1s-1 kbon = ( ) x103 M-1s-1 kaoff = ( ) s-1 kboff = ( ) s-1 kab = ( ) s-1 kba = ( )s-1 kaon kab kboff=( )x103 M-1s-3 kbon kba kaoff=( )x103 M-1s-3

27 Single molecule covalent chemistry inside the pore irreversible reactions reversible covalent chemistry irreversible chemistry with turnover

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29 fast step, after slow step

30 rate of cyclization step

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32 Polymerization monitored step by step cf.

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34 How far can this technology be pushed? reaction network (stereochemistry) isotope effect

35 Stereochemistry of nucleophilic reaction at As P 1 SH 5 A S As OH R 1 2 B OH S As D S As R 2 S R C S As S R 1 R 1 R 2

36 P 1 SH 5 A S As OH R 1 2 B OH S As D S As R 2 S R C S As S R 1 R 1 R 2

37 1 P SH 17 D A S As S As OH R2 S R 1 OH S As S As 8 R 6 1 R 2 S E 2 14 S As S R 3 R F B 5 16 C R 3 S S As 10 R 1 R 1 R 1

38 Complex reaction network

39 .. allowed and forbidden transitions... P HO As HO SO 3 SH R 1 = SO 3 A S As OH R 1 B OH S As H 2 N R 2 = O OH D S As O OH R 2 S R 1 S As S C R 2 R 1 R 1 R 3 = O HO H 2 N O O NH NH E S As S R 3 R 1 S R 3 S As F R 1

40 ... set limits on the stereochemistry... inversion, retention, racemization...

41 Measuring an isotope effect

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45 single molecule- lifetime of intermediate: τ* D /τ* H = (k -1,H + k 2,H )/(k -1,D + k 2,D ) bulk: k obs-h /k obs-d = (k 1H k 2H /k 1D k 2D )(k -1,D + k 2,D )/(k -1,H + k 2,H ) for k 2 relatively large (fast second step) τ* D /τ* H = (k 2,H )/(k 2,D )... see full isotope effect k obs-h /k obs-d ~ 1 because = k 1H /k 1D ~ k -1,D /k -1,H ~ 1 (and are rate limiting)

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47 Next level issues/problems: Is water in the pore like bulk water? 400 molecules in the barrel Protein surface effects Are reactants driven into the pore? charged reactants neutral reactants- electroosmosis Can large flexible molecules get in? note that for a sensor all this does not matter

48 Is water in the pore like bulk water?- probably radii constriction

49 X-ray structures of mutant αhl βcd complexes M. Montoya & E. Gouaux

50 Are charged reactants driven into the pore? - yes, but effect can be small at low applied potentials K d ( V) = K d (0)e-z F δ V/RT

51 Are neutral reactants driven into the pore by electrosmosis? Yes- can reduce current and therefore EOF by manipulating applied potential and ion selectivity

52 Can large flexible molecules get in?- They pay a price (i) POLYMER'-SH + N S S N POLYMER'- S S N (ii) POLYMER'- S S N + HS-ALPHA HL POLYMER'-S- S-ALPHA HL Π = [PEG] pore /[PEG] bath = e -N(a/D) 5/3 for an infinitely long tube (de Gennes) ln k = -N(a/D) 5/3 + ln k[peg] bath k, measured rate constant D, diameter pore a, persistence length of polymer N, number of repeats in polymer

53 Expanding the applicability of nanopore technology to covalent chemistry temperature ph solvents chemistry other than SH range of accessible rate constants (noise) more precise measurements

54 Protein pores at high temperatures α-hemolysin, leukocidin and OmpG are stable at high temperatures and show no sharp transitions in conductance

55 (M113F/K147N) 7, βcd, adamantane-1-carboxylate

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57 next: reactive side chains

58 QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. visit our web site QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Former single-molecule chemists: Seong-Ho Shin Tudor Luchian Sandra Loudwig Mackay Steffenson Haichen Wu Oxford group: Arijit Banerjee Marc Boudreau Stephen Cheley Timmy Choi Anne Hammerstein Leon Harrington Marsi Henricus Andrew Heron Deanpen Japrung Jochen Klingelhoefer Lingbing Kong Wen-wu Li Siran Lu Giovanni Maglia Rethi Madathil Ellina Mikhailova Kunal Mehta Sarah Rogers Pinky Raychaudhuri James Reeve Dvir Rotem Maria Salichou Tanuj Sapra Samantha Shanley David Stoddart Ruhma Syeda Emma Wallace

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