Ion Translocation Across Biological
Membranes
Janos K. Lanyi
University of California, Irvine
Examples of transmembrane ion pumps
Protein
Cofactor, substrate, etc.
MW
Subunits
Mitoch. cytochrome oxidase
hemes, Cu, Fe
130,000
13
Mitoch. cytochrome bc1
hemes, FeS, ubiquinone
225,000
11
Mitoch. ATPase
ATP/ADP
350,000
>20
Mamm. NaK pump
ATP/ADP
280,000
8
Mamm. Ca pump
ATP/ADP
290,000
2
Bacteriorhodopsin
retinal
24,000
1
Retinal based ion pumps
(e.g. bacteriorhodopsin)
1. Energy input is photoisomerization of all-trans retinal
to 13-cis,15-anti
2. Ensuing reaction cycle goes through intermediates
K, L, M1, M2, M2’, N1, N2, O, and back to BR
3. A proton is released to one side in the M2 to M2’ reaction,
and another is taken up from the other side in the N1 to
N2 reaction
4. Net result of a turn-over (~ 10 ms) is transport of a proton
BACTERIORHODOPSIN
a light-driven proton
pump
How does it work?
H
BACTERIORHODOPSIN
a light-driven proton
pump
How does it work?
H
More specifically:
how does
reisomerization of
the 13-cis retinal
drive steps 1 - 5?
BACTERIORHODOPSIN
a light-driven proton
pump
A fundamental problem
in ion pumps is:
how local changes
propagate to other
regions of the protein
to produce functionally
relevant
conformational
changes
Images from
Baudry, Tajkhorshid, Molnar, Phillips, Schulten, J.Phys.Chem.
105, 905-918 (2001)
Frame 1, taken Aug 18-20, 2001
at beamline 5.0.2 (ALS)
Frame 150, taken Aug 18-20,
2001 at beamline 5.0.2 (ALS)
The BR state
A chain of covalent
and hydrogen-bonds
links region of
retinal to Asp-96
Hydrogen-bonded
water at active center
A hydrogen-bonded
network links Asp-85
to Glu-194/Glu-204
retinal
isomerizes
5
retinal
reisomerizes
retinal
relaxes
4
1
3
switch
2
THE BR STRUCTURES
Species
Occupancy Resolution
%
Å
BR (wt)
100
1.47
K (wt), 100K
40
1.43
L (wt), 170K
~60
1.62
M1 (wt), 210K
60
1.43
M1 (wt), 295K
42
1.52
M2 (E204Q), 295K
>93
1.80
M2’ (D96N), 295K
100
2.00
N2 (V49A), 295K
37
1.62
PDB
1M0L
1M0K
1O0A
1M0M
1P8H
1F4Z
1C8S
1P8U
The Active Site
(retinal Schiff base, a water, and the
counter-ion/proton acceptor)
Retinal geometry
in non-illuminated
and illuminated
crystals
Schiff base
(No restraints on angles used in
refinement; starting models both
all-trans and undistorted 13-cis)
Models shown for L from
three independent crystals,
overlapped:
note magnitude of crystalto-crystal variations in
atomic positions
State
C13
angle
Torsion
angles*
C13=C14
C14-C15
C15=NZ
Torsion angles
BR (n=2)
112, 113
-157, -154
179, 177
-163, -172
K (n=4)
145 + 12
-2 + 39
138 + 35
101 + 31
L (n=6)
135 + 10
28 + 9
149 + 21
49 + 18
M1 (n=3)
127 + 7
4 + 26
105 + 42
128 + 28
M2
125
13
-173
-161
M2’
114
-2
-166
-169
Bond angle increases in K and gradually recovers
during the first half of the photocycle
State
Torsion
Torsion
angles
angles
C13
angle
C13=C14
C14-C15
C15=NZ
BR (n=2)
112, 113
-157, -154
179, 177
-163, -172
K (n=4)
145 + 12
-2 + 39
138 + 35
101 + 31
L (n=6)
135 + 10
28 + 9
149 + 21
49 + 18
M1 (n=3)
127 + 7
4 + 26
105 + 42
128 + 28
M2
125
13
-173
-161
M2’
114
-2
-166
-169
Deviations from ideal 13-cis,15-anti (torsion angles of
0o, 180o, and 180o) decrease between K and M2
+
all-trans retinal (BR)
all-trans retinal
-
+
idealized, relaxed 13-cis
expectedretinal
13-cis retinal
-
+
-
13-cis retinal in K with
strain, note increased C13
bond angle
Retinal dynamics
Step-by-step
BR
retinal
Lys-216
wat402
(Luecke et al, 1999; Schobert et al, 2002)
K
photoisomerization
(Schobert et al, 2002)
L
relaxation
(Lanyi & Schobert, 2003)
M1
deprotonation
(Lanyi & Schobert, 2002)
M2
switch to other side
(Luecke et al, 2000)
M2 ’
further relaxation
(Luecke et al, 1999)
Conformational Cascades in
the Protein
(to conduct a proton to the extracellular
surface and deliver a proton from the
cytoplasmic surface )
The hydrogen-bonded network collapses and the
positively charged arg-82 side-chain moves 1.7 Å
down toward glu-194/glu-204
(color code: negative or positive charge)
The hydrogen-bonded network collapses and the
positively charged arg-82 side-chain moves 1.7 Å
down toward glu-194/glu-204
H+
(color code: negative or positive charge)
Formation of a water cluster at Asp-96
2
BR
M2
In N2 omit maps detect new density
peaks modeled as water
BR state
BR plus N2
In N2 a hydrogen-bonded chain of four water molecules
accumulates between the Schiff base and Asp-96
BR
N2
In N’ there is a single-file hydrogen-bonded chain of four
water molecules between the Schiff base and asp-96
H+
BR
N2
Tilt of helix F
Tilt of helices A, B, C, D, and E
“N-like” state
Subramaniam & Henderson,
Nature 406, 653-657 (2000)
“O-like” state
Rouhani et al. J. Mol. Biol. 313,
615-628 (2001)
CONCLUSION
The principle of this ion pump is that the strain in the
distorted photoisomerized retinal gradually relaxes as the
binding site accomodates its changed shape. Inherently, the
relaxation includes deprotonation of the Schiff base. The
cascade of conformational changes that ensues allow
release of a proton at one surface and uptake at the other.
Hypothesis:
half-channels to the two membrane surfaces
exist only to optimize transport
1. Mutations decrease turn-over but do not inactivate pump
2. Replacing the Schiff base counter-ion (Asp-85) with a Thr
or Ser converts proton pump into a chloride pump
3. In eubacterial rhodopsins the extracellular proton release
network is missing, but transport is not affected
1. Mutations decrease turn-over but do not
inactivate pump
Examples:
Cytoplasmic side: D96N (absence of internal proton donor)
prolongs lifetime of the deprotonated Schiff base
Extracellular side: R82Q, E194Q, E204Q (absence of proton
release group) delays proton release to the end of the cycle.
Sequence of proton release and uptake is reversed.
2. Replacing the Schiff base counter-ion (Asp-85)
with a Thr or Ser converts proton pump into a
chloride pump
Rationale: in halorhodopsin (a chloride pump) this residue is
a Thr
wt BR
D85T BR
HR
Asp
Asp
Ala
NH+
Asp
NH+
Thr
NH+
Thr
Cl-
Cl-
H+
3. In eubacterial rhodopsins the extracellular
proton release network is missing, but transport
is not affected
Xanthorhodopsin (from Natronobacter ruber):
Differences between xanthorhodopsin
and bacteriorhodopsin
Particularly evident at
the B-C and F-G
interhelical loops
Cleft at the extracellular
surface
Absence of extracellular network causes
reversed release/uptake sequence
CONCLUSIONS
The principle of this ion pump is that the strain in the
distorted photoisomerized retinal gradually relaxes as the
binding site accomodates its changed shape. Inherently, the
relaxation includes deprotonation of the Schiff base. The
cascade of conformational changes that ensues allow
release of a proton at one surface and uptake at the other.
The “pump" resides in the active site. The
two half-channels that connect it to the membrane surfaces
do not play essential roles. They optimize turn-over.
Publications
• J Sasaki, LS Brown, Y-S Chon, H Kandori, A Maeda, R Needleman & JK Lanyi.
Science 269, 73-75 (1995).
•H Luecke, H-T Richter & JK Lanyi, Science, 280, 1934-1937 (1998).
• H Luecke, B Schobert, H-T Richter, J-P Cartailler & JK Lanyi, J. Mol. Biol. 291, 899-911
(1999).
• H Luecke, B Schobert, H-T Richter, J-P Cartailler & JK Lanyi, Science 286, 255-261
(1999).
• H Luecke, B Schobert, H-T Richter, J-P Cartailler, A Rosengarth, R Needleman & JK
Lanyi, J. Mol. Biol. 300, 1237-1255 (2000).
• B Schobert, J Cupp-Vickery, V Hornak, SO Smith & JK Lanyi, J. Mol. Biol. 321, 715-726
(2002).
• JK Lanyi & B Schobert, J. Mol. Biol. 321, 727-737 (2002).
• JK Lanyi & B Schobert, J. Mol. Biol. 328, 439-450 (2003).
•B Schobert, LS Brown & JK Lanyi, J. Mol. Biol. 330, 553-570 (2003).
•SP Balashov, ES Imasheva, VA Boichenko, J Antón, JM Wang & JK Lanyi, Science 309,
2061-2064 (2005).
•JK Lanyi & B Schobert, J. Mol. Biol. 365, 1379-1392 (2007).
•H Luecke, B Schobert, J Stagno, ES Imasheva, JM Wang, SP Balashov & JK Lanyi,
Proc. Natl. Acad. Sci. U. S. A. 105, 16561-16565 (2008).
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Thanks to:
H.T. Richter
B. Schobert
L. Brown
A. Dioumaev
D. Chen
S.P. Balashov
E. S. Imasheva
JM Wang
Collaborators
R. Needleman, Wayne State
A. Maeda, Kyoto Univ.
H. Luecke, Univ. Cal. Irvine
J. Cupp-Vickery, Univ. Cal. Irvine
S.O. Smith, Stony Brook
(and their co-workers)
Funded by grants from NIH, DOE, and US Army Research
Office
Beamlines at ALS, SSRL, SPring-8 and ESRF