molecular mechanism of proton transport in membrane …colloq/talk8/presentation8.pdfmolecular...
TRANSCRIPT
![Page 1: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/1.jpg)
Molecular Mechanism ofProton Transport
in Membrane Proteins
Régis Pomès
Structural Biology & Biochemistry, Hospital for Sick ChildrenDepartment of Biochemistry, University of Toronto
![Page 2: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/2.jpg)
Why study mechanisms of ion transport?
Biological importance
– Ionic gradients are essential to proper biological function nervous system, metabolism, ATP synthesis…
– Membranes block ion flow
– Specific transport is mediated by membrane proteins= channels, transporters, pumps
ion permeation (potassium, chloride, sodium…)ion exclusion (aquaporins = water specific)ion pumps
![Page 3: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/3.jpg)
Malfunction of channels is linked to disease:
– Cystic fibrosis Chloride channel
– Aquaporins cataract, diabetes, …
– But there’s no life without proton pumping!
Medical relevance
![Page 4: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/4.jpg)
cytosol
H+ transport across biomembranes
IM space
matrix
O2 + e- H2O
H+
H+H+ H+
H+ H+ H+H+
cytochromec oxidase
ATP
H+
+ + +
- - -
ADP + Pi
ATP synthase
Chemiosmotic coupling
H+
H+ pumping is required for ATP synthesis
![Page 5: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/5.jpg)
cytosol
H+ transport across biomembranes
Passive H+ transport destroys proton-motive force
H+
H+H+ H+ H+ H+ H+
H+
H++ + +
- - -
H+
(leakage)
gramicidin aquaporins
channels transporters
H+
104 s-1107 s-1
![Page 6: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/6.jpg)
Channels = narrow, water-filled pores
Selective to the passage of certain ions and/or small molecules
Allow ions to cross the dielectric barrier of the membrane
Physical basis of permeation
![Page 7: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/7.jpg)
1. Relay of H+ translocation in gramicidin
2. Exclusion of protons from aquaporins
3. Uptake of protons in cytohrome c oxidase
Proton transport and blockage
![Page 8: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/8.jpg)
Opposed by the dielectric barrier
H+ reactivity special transport properties
The Grotthuss mechanism
Biological Proton Translocation
![Page 9: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/9.jpg)
The Grotthuss mechanism owes its name to a paper published in 1806
Theodor Grotthuss
(1785-1822)
![Page 10: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/10.jpg)
Pomès & Roux, Biophys. J. 1996, 2002
1. Proton relay in the gramicidin channel
![Page 11: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/11.jpg)
0.24 nm
0.24 nm
O
H
H
O
H
H
H+
0.28 nm
O
H
H
O
H
H
H+
• H+ is very reactive it doesn’t exist by itself in biological systems
• The hydrated proton exists primarily in two forms:
Zundel ion hydronium
• These two species differ in the length of the hydrogen bonds
Proton solvation and hydrogen bonds
![Page 12: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/12.jpg)
• The exchange between the two forms of hydrated H+ drives transport:
• This relay process hinges on fluctuations of small amplitude (~1 Angstrom)- on ps timescale
translocation of H+ across large distances (10’s of Angstroms) - in 10-9 second or even faster.
The Grotthuss mechanism: proton exchange
O
H
H
O
H
H
O
H
H
O
H
H
H
H+
+
O
H
H
O
H
H
H+
![Page 13: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/13.jpg)
The exchange between OH3+ and O2H5
+
is the elementary step of proton relay
![Page 14: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/14.jpg)
• Both forms of the hydrated proton are stabilised by hydrogen-bonding donation to 3 neighbors
• In water, hydrogen bonds are constantly formed and broken• In gramicidin, the channel backbone provides ideal coordination
What is the role of the channel in the mechanism?
Proton solvation and hydrogen-bonded networks
O
O
O
O
O
O
O
O
O
O
![Page 15: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/15.jpg)
Mechanism of proton transport in water
![Page 16: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/16.jpg)
Gramicidin offers a local environmentwell suited to rapid proton transport(solvation and mobility)
![Page 17: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/17.jpg)
Role of membrane channels inionic solvation and mobility
• “ideal” channel:– Chemical potential ~ that in water
+ -
–Low barriers
• Multiple “binding sites”
![Page 18: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/18.jpg)
Origin of theattenuation ofH+ conductanceby methanol?
Diffusion ofmethanol ingramicidin
![Page 19: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/19.jpg)
Attenuation of proton permeation by methanol
![Page 20: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/20.jpg)
• Methanol fits in the pore of gramicidin
• It blocks proton relay because it never forms a continuous chain
![Page 21: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/21.jpg)
MeOH2+ does NOT form a continuous chain
+ does NOT tumble when inside the channel
Conclusion: blockage of H+ relay when MeOH is in
Pomès & Yu, Front. Biosc. 2003
![Page 22: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/22.jpg)
2. Proton exclusion from aquaporins
Water-selective channels
109 H2O s-1
Water diffusion is coupledto their reorientation
(DeGroot & Grubmuller, Science 2001)(Tajkhorshid et al., Science 2002)
Exclude ions
Hydrogen-bonded water chain
Physical basisof proton blockage?
![Page 23: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/23.jpg)
![Page 24: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/24.jpg)
Proton exclusion from water channels
Some observations:
Arg206 could prevent approach of +charge-charge repulsion
Adverse polarisation of water molecules incompatible with intrusion of proton
Local interactions with N68, N203incompatible with solvation of hydrated proton
+
O
![Page 25: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/25.jpg)
Free energy profile for waterreorientation
Strong preference for bipolar organizationopposes the turn stepof structural diffusion
![Page 26: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/26.jpg)
Free energy profile forH+ transfer
![Page 27: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/27.jpg)
Ionic solvation: charge-dipole interactions
• Peptide bonds also have a dipole
• The dipole moment of peptide bonds and α helices stabilisesions
δ+
δ-O
CN
H
μpep
δ+
δ-
• O is electronegative, H is electropositive.
• The dipole moment of water molecules (charge separation) stabilises ions:
O
H
Hμwat
δ+
δ+
δ-
+-
+
- -- -
+
![Page 28: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/28.jpg)
Adverse charge-dipole interactions give rise to proton exclusion
![Page 29: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/29.jpg)
ε = 80
ε = 4
ε = 4
δ+
δ-
+
-+
+
+
+-
-
-
-
-
+
δ+
δ+
δ+
δ-
δ-δ-
δ-
+
Continuum electrostatic calculations
Total electrostatic energy =
reaction field dielectric boundaries
+
static field charge distribution
![Page 30: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/30.jpg)
Hop PMFFull ESP = charge + dielectricNo membrane dielectric
Chakrabarti et al., Structure 2004
Electrostatic origin of the free energy barrieropposing proton translocation
Size selectivity at R206
Static field = effect of charge distribution of the channel
![Page 31: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/31.jpg)
PMF vs PB in aquaporin channel variants
H+ PMF is essentially determined by the distribution of charged and polar groups
![Page 32: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/32.jpg)
Structural determinants of H+ blockage: charge-dipole interactions
wild type
M3, M7 helicesoff
M3, M7 dipoles off
![Page 33: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/33.jpg)
H-O-H
+
Mechanism of proton exclusion in aquaporins
+
Favored
Forbidden
??+
-
![Page 34: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/34.jpg)
Compromising proton impermeability of aquaporins?
Single point mutation introducing a negative charge at the NPA motif:
Asn68 Asp
Predicted to leak protons…
Doesn’t express!
-
Chakrabarti et al. J Mol Biol 2004
![Page 35: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/35.jpg)
O2 + 4e- +4H+ = 2H2O
3. Proton pumping by cytochrome c oxidase
![Page 36: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/36.jpg)
+
+
+
+
E286
D132 N139
A proton wire in the D channel
![Page 37: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/37.jpg)
Water-mediated proton uptake
time (ps)
z (Å
)
![Page 38: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/38.jpg)
Kinetics of proton uptake in oxidase
The D channel is a proton sink: strongly non-equilibrium
The kinetics of proton uptake is modulated by dynamic fluctuations of the water chain
The RLS corresponds to a bottleneck at residue 139
What is the origin of decoupling in N139D and N207D mutants?
N139D
N207
![Page 39: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/39.jpg)
Conclusions
Detailed balance of microscopic forces physical basis of proton transport and
blockage in membrane proteins
Atomistic simulations events hard to observe experimentally generate/refine testable mechanistic
hypotheses
Next: mechanism of redox-coupled H+ pumping in cytochrome c oxidase
![Page 40: Molecular Mechanism of Proton Transport in Membrane …colloq/Talk8/Presentation8.pdfMolecular Mechanism of Proton Transport in Membrane Proteins Régis Pomès Structural Biology &](https://reader034.vdocuments.net/reader034/viewer/2022052016/602e64286dfbd54e7e421e53/html5/thumbnails/40.jpg)
Acknowledgments
TorontoDr. Stéphanie BaudDr. Nilu Chakrabarti
Dr. Elisa FaddaMartin Kurylowicz
Chris MadillSarah MansourTom Rodinger
Dr. Tony MittermaierDr. Ching-Hsing Yu
CISS-3Paul Lu
Martin Karplus
CollaboratorsBenoît Roux
Mark SchumakerEmad Tajkhorshid
Funding HSC, NIH, CIHR, CRC/CFI