抗生素课件(英文)-Diffusion-in-nanostructures-Role-of.ppt

1、Diffusion in nanostructures:Role of interaction potentials Sergey M.BezrukovLaboratory of Physical and Structural Biology,NICHDNational Institutes of Health together with:V.Adrian Parsegian,Alexander M.Berezhkovskii,Philip A.Gurnev,Ekaterina M.Nestorovich,Amos B.Oppenheim,and Mathias WinterhalterRol

2、and Benz:Asymmetry?Do you mean voltage-induced asymmetry?Alf Honigmann:Translocation or not?(A particle can leave the channel from the side it enters).Boris Shklovskii:It is all about linear vs.non-linear response.Sunday,August 26,morning,between 9:00 and 10:00Channel asymmetry,Maxwells demons,non-l

3、inear response,and all that-1.0-0.50.00.51.01.502x10-14x10-16x10-18x10-11x100Which one is more effective?A teaser:Plan of the talk:Consider the simplest binding-site model Show experiments on single l-phage docking to maltoporin Introduce and explain the benefits of the diffusion model(e.g.resolve t

4、he teaser)The simplest“binding site”model of ion channel()loffk1()()lroccoffoffkk()loffk()roffkThe simplest“binding site”model of ion channel()loffk1()()lrfreeonlonrkCkC()lonk()ronkThe simplest“binding site”model of ion channel:The flux()()()()()(),lrlronlfreetransfreefreefreeoccroffrtransrloffoffJk

5、C P PPkPkkConsider equilibrium:,lrlrrlCCJJ()()()()lrononlroffoffkkkkTranslocations vs.returns:()loffk()()()()()()()rroffrontransrlrloffoffononkkPkkkkChannel blocked from one side()1*()()()1llronoccoffoffoccronkkkk()lonk()loffkPlan of the talk:Consider the simplest binding-site model Show experiments

6、 on single l l-phage docking to maltoporin Introduce and explain the benefits of the diffusion model(e.g.resolve the teaser)AKClKClKClKClA60 Dia.HoleLipid MonolayerElectrodeWater TubeTeflon Chamber15 Teflon FilmConstructing a lipid bilayer:Native Maltoporin is a homotrimerEach subunit is a 421 a.a.1

7、8 stranded b b-barrelLoops L4,L5 and,L6 form a complex near the extracellular channel vestibule(amino acid residues relevant for phage binding are shown in red)Maltoporin:trimeric b b-barrelEffect of phage on the membrane containing Maltoporin trimers Current corresponding to 10 Maltoporin trimers i

8、s reduced by phage at the cis-side addition Phage addition to the trans-side of the membrane did not produce any significant changes in the current10 pS50 ms150 mV0 mVIon conductance is stable in the absence of maltohexaose 10 pAIon current and maltohexaose transport in single Maltoporin trimer Malt

9、ohexaose molecules bind to a specific site inside the pore and transiently block ion current150 mV0 mVThe 3 channels are identicaland independentSpectral analysis:Reaction=two-state Markovian processkonkoff10 pA50 ms0 M 10M 30 M t=0.2 msThe 3 channels are identicaland independentSpectral analysis:Re

10、action=two-state Markovian processkonkoffThe 3 channels are identicaland independentSpectral analysis:Reaction=two-state Markovian processkonkoffkonkoff10 pA50 ms0 M 10M 30 M t=0.2 msSingle blockDouble blockTriple blockUnblockcis-trans-/+150 mV/0 mV/In the absence of maltohexaose and l phage Maltopo

11、rin trimer is permanently openMonitoring the functional properties of the single Maltoporin trimer embedded into bilayercis-trans-/+150 mV/0 mV/Transmembrane current gets transiently blocked when maltohexaose is applied to the cis-side of the membranePhage was added simultaneously to the same(cis)si

12、de of the membraneMonitoring the functional properties of the single Maltoporin trimer embedded into bilayercis-trans-/+150 mV/0 mV/Docking of phage decreases ion conductance and simultaneously inhibits maltohexaose penetration in all the three pores of MaltoporinMonitoring the functional properties

13、 of the single Maltoporin trimer embedded into bilayercis-trans-/+150 mV/0 mV/Subsequent application of maltohexaose to the trans-side produces current fluctuationsProbing the phage-bound state of Maltoporin for independence of pore blockage:Thermodynamics33!(1)!(3)!kkkPppkkBinomial distribution pro

14、bes independence of blocking events Pk:probability of k(out of 3)blocked poresk:#of blocked poresp:probability of finding a single pore blockedp is determined by equilibrium constant K eq and sugar(maltohexaose)concentration(C)obtained from the same current traces Blocking events are independentG2G3

15、G1cis-trans-G1G2G3Gatrans-l l-phage G2G3G1cis-Phage docking introduces hierarchy in Maltoporin conductance Phage-free Maltoporin trimer 1=2=3 Phage-bound Maltoporin trimer 1 2 3 3 2 1 3 2 1maltohexaose trans-side G2G3G1cis-trans-G1G2G3Gatrans-l l-phage G2G3G1cis-Phage introduces a new common pathway

16、 for ion fluxes in Maltoporin Phage-free MaltoporinPhage-bound MaltoporinPhage introduces a formation of one common entryway,connecting the extracellular face of Maltoporin with the outer membrane solution500 pS-200-10001002000.00.51.01.5Time constant,msTransmembrane voltage,mV phage-bound channel f

17、ree channel Maltohexaose residence time increases upon phage binding Maltohexaose residence time in Maltoporin pore(at the trans-side sugar application)increases upon l l phage binding This finding suggests that the phage binding site does not overlap with the zone of sugar binding()*()1lonoccoccron

18、kk*occoccPhage introduces a new common pathway for ion fluxes in Maltoporin Plan of the talk:Consider the simplest binding-site model Show experiments on single l-phage docking to maltoporin Introduce and explain the benefits of the diffusion model(e.g.resolve the teaser)Particle and Channel?Particl

19、e and Channel?Particle and ChannelParticle and Channel?Analytical Approach L 0 Bulk Channel Bulk U(x)x l U 0 Particle Potential in the Channel:L2aComputer simulationsComputer simulationsParticle and Channel?(b)(a)Diffusion with stickingFree diffusion()0142()trLU xbchpDdxeaDxbL2aChannel()loffJ()()rro

20、nonrJk C()roffJ()()llononlJk C0exp8bRLUDbInstead of treating a channel as a featureless“binding site”we considered single-particle dynamics inside its pore0142exptrbchPD LUD Rb*Rectangular potential well of depth U0 occupying the entire channel*024681012141601000200030004000c1=30 Mc1=50 MFlux,Transl

21、ocations per SecondDepth of Potential Well,kBTc1=10 M200221exp1exp2blrlrbchD R CCJR L CCD LUUD RbbFlux:*Rectangular potential well of depth U0 occupying the entire channel01x1062x1063x1064x1065x106Flux,s-1bUbFl/2-3-1135-4-2024Dependence of the flux on the depth(b bU)and tilt(b bFl/2)of the potential

22、 well:-1.0-0.50.00.51.01.502x10-14x10-16x10-18x10-11x100Which one is more effective?Introducing entropic potential 22ln,()expentBentBUxk TRconstUxc xk Tc xR-1.0-0.50.00.51.01.502x10-64x10-66x10-6JlSimulations:dadxl-1.0-0.50.00.51.01.502x10-64x10-66x10-6JlSimulations vs.Theory:Entropic PotentialdadxlReferences:The phage story:J.Molecular Biol.,2006,359:1447-1455.Theory:Biophys.J.,2005,88:L7-L19;Phys.Rev.Lett.,2006,97:020601;J.of Physics:Condensed Matter,2007,19:065148J.Chem.Phys.,2007 in press.