V11: Folding of Membrane Proteins

1
V11 Folding of Membrane Proteins
Membrane proteins are in general either helical
proteins (see bacteriorhodopsin or beta-proteins
structure, left) (see porin-structure, right)
2
Folding of helical membrane proteins
Paradigm by Engelman Popot 2-step
mechanism (i) ?-helices fold after being inserted
into membrane (ii) folded ?-helices then assemble
to form entire protein Todays
program 1 recent discoveries on
translocon-mediated insertion into lipid
bilayer. 2 apply protein engineering to
helix-connecting loops in bR ? kinetics 3
rupture individual bR proteins out of membrane by
atomic force microscopy
3
Folding of helical membrane proteins (II)
White, FEBS Lett. 555, 116 (2003)
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Hydrophobicity Scales
White, FEBS Lett. 555, 116 (2003)
5
Translocon-assisted folding of TM proteins?
Upper picture (model!) the newly synthesized
polypeptide chain of a membrane protein is
inserted from the ribosome into the membrane via
interaction with a TM complex, the translocon
(EM map shown). lower picture experiment
largely supports the concerted view. What
determines insertion into the membrane ?
White, FEBS Lett. 555, 116 (2003)
6
Integration of H-segments into the microsomal
membrane
Ingenious experiment! Introduce marker that shows
whether helix segment H is inserted into membrane
or not. a, Wild-type Lep has two N-terminal TM
segments (TM1 and TM2) and a large luminal domain
(P2). H-segments were inserted between residues
226 and 253 in the P2-domain. Glycosylation
acceptor sites (G1 and G2) were placed in
positions 9698 and 258260, flanking the
H-segment. For H-segments that integrate into the
membrane, only the G1 site is glycosylated
(left), whereas both the G1 and G2 sites are
glycosylated for H-segments that do not integrate
in the membrane (right).
b, Membrane integration of H-segments with the
Leu/Ala composition 2L/17A, 3L/16A and 4L/15A.
Bands of unglycosylated protein are indicated by
a white dot singly and doubly glycosylated
proteins are indicated by one and two black dots,
respectively.
Hessa et al., Nature 433, 377 (2005)
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Insertion determined by simple physical chemistry
measure fraction of singly glycosylated (f1g) vs.
doubly glycosylated (f2g) Lep molecules
c, ?Gapp values for H-segments with 24 Leu
residues. Individual points for a given n show
?Gapp values obtained when the position of Leu is
changed. d, Mean probability of insertion (p)
for H-segments with n 07 Leu residues.
Hessa et al., Nature 433, 377 (2005)
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Biological and biophysical ?Gaa scales
a, ?Gappaa scale derived from H-segments with the
indicated amino acid placed in the middle of the
19-residue hydrophobic stretch. Only Ile, Leu,
Phe, Val really favor membrane insertion. All
polar and charged ones are very unfavored. b,
Correlation between ?Gappaa values measured in
vivo and in vitro. c, Correlation between the
?Gappaa and the WimleyWhite water/octanol free
energy scale for partitioning of peptides.
Hessa et al., Nature 433, 377 (2005)
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Positional dependencies in ?Gapp
Tyr and Trp are favorable in interface region.
a, Symmetrical H-segment scans with pairs of Leu
(red), Phe (green), Trp (pink) or Tyr (light
blue) residues. The Leu scan is based on
symmetrical 3L/16A H-segments with a Leu-Leu
separation of one residue (sequence shown at the
top the two red Leu residues are moved
symmetrically outwards) up to a separation of 17
residues. For the Phe scan, the composition of
the central 19-residues of the H-segments is
2F/1L/16A, for the Trp scan it is 2W/2L/15A, and
for the Tyr scan it is 2Y/3L/14A. The ?G app
value for the 4L/15A H-segment GGPGAAALAALAAAAALAA
LAAAGPGG is also shown (dark blue). b, Red lines
show ?G app values for symmetrical scans of
2L/17A (triangles), 3L/16A (circles), and 4L/15A
(squares) H-segments. c, Same as b but for a
symmetrical scan with pairs of Ser residues in
H-segments with the composition 2S/4L/13A.
Hessa et al., Nature 433, 377 (2005)
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Folding kinetics of bR
Fluorescence bO ? I1 ? I2 ? IR ? bR bO
denatured bR in SDS (4 TM helices) I1 fastest
kinetic phase after mixing of SDS and DHPC/DMPC
micelles, 4 10 ms, increase in
fluorescence I2 important folding intermediate,
another 1.25 TM helices form (CD)
Allen et al. J Mol Biol 308, 423 (2001)
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What effect do the loops have on folding kinetics
of bR?
Scheme shows which loops were replaced by
structureless linkers of Gly-Gly-Ser repeats.
The loops were replaced in turn by linkers of
the same length as the wild-type loop. Linkers
of two different lengths were used to replace the
BC loop one shorter than the wild-type loop
(BC1) and one the same length as the wild-type
loop (BC3).
Allen et al. J Mol Biol 308, 423 (2001)
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Kinetics of formation of native-like chromophore
for wt and loop mutants
(a) Kinetic spectra for the two time constants
resolved in time-resolved absorption studies
during folding of wild-type ebO to bR, showing
the wavelength-dependence of the amplitude of the
130 seconds and 4180 seconds components. (b)
Changes in 560 nm absorbance during folding of
ebO, AB, CD and EF loop mutants and at 500 nm for
BC1 mutant. (c) Changes in 560 nm absorbance
during folding of ebO and the DE loop mutant and
at 541 nm for the FG mutant.
Allen et al. J Mol Biol 308, 423 (2001)
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Effects of loop mutants on folding kinetics
Mutation of CD or EF loops shows slower
apoprotein folding to I2 mutation of FG loop
shows slower rate of the events
accompanying retinal binding to the protein.
Allen et al. J Mol Biol 308, 423 (2001)
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AFM topography of a purple membrane
Typical high-resolution AFM topograph of the
cytoplasmic surface of a wild-type purple
membrane. BR assembles in trimers that arrange in
a hexagonal lattice. To catch an individual
protein (white circle), we zoomed in by reducing
the frame size and the number of pixels. After
the AFM tip was positioned, it was kept in
contact with the selected protein for about 1 s
while a force of 1 nN was applied to give the
protein the chance to adsorb on the stylus. In
15 of the cases, the protein can then be
extracted.
Oesterhelt, F et al. Science 288, 143 (2000)
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Force profile
The stylus and protein surface were separated at
a velocity of 40 nm/s while the force spectrum
was recorded. The interaction between tip and
surface, which is expressed in the marked
discontinuous changes in the force, indicates a
molecular bridge between tip and sample. This
bridge reaches far out to distances up to 75 nm,
which corresponds to the length of one totally
unfolded protein.
Oesterhelt, F et al. Science 288, 143 (2000)
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Check membrane to see what happened
After the adhesive force peaks were recorded, a
topograph of the same surface was taken to show
structural changes. Note that a single monomer
is missing. Thus, the recorded force spectrum
may be correlated to extraction of an individual
protein from the membrane.
Oesterhelt, F et al. Science 288, 143 (2000)
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Force extraction profiles
Several force spectra taken on wild-type BR are
shown. A typical repeating pattern is visible.
All curves show four peaks located around
10, 30, 50, and 70 nm.
Oesterhelt, F et al. Science 288, 143 (2000)
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What are the regular features?
Thirteen spectra are superposed on the second
peak. This results in an exact cover of the
third and fourth peaks, whereas the first peak
remains scattered. Gray lines are force
extension curves calculated by the worm-like
chain model with a Kuhnlength of 0.8 nm, which is
known to describe the elasticity of an unfolded
poly-amino acid chain.
Oesterhelt, F et al. Science 288, 143 (2000)
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Model to explain force extraction spectra
This model explains the peaks in the force
spectra as the sequential extraction and
unfolding of a single BR. A rupture length of
more than 60 nm can be recorded only if the
COOH-terminus has adsorbed on the tip. If a
force is applied on the COOH-terminus, helices F
and G will be pulled out of the membrane and
unfold. Upon further retraction, the unfolded
chain will be stretched and a force will be
applied on helices D and E until they are
extracted from the membrane. Thus, peak
2 reflects unfolding of helices D and E and peak
3 reflects unfolding of helices B and C. Peak
4 shows extraction of the last remaining helix A.
Oesterhelt, F et al. Science 288, 143 (2000)
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3-dimensional structure of bR
(A) BR is a 248-amino acid membrane protein that
consists of seven transmembrane ?-helices, which
are connected by loops. (B) Three-dimensional
model and top and bottom view show spatial
arrangement of the helices. Helices F and G are
neighboring helices A and B and thus can
stabilize them.
Oesterhelt, F et al. Science 288, 143 (2000)
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How to check correctness of model? Mutations!
Force curves were recorded on BR where the E-F
loop was cleaved enzymatically. (A) Selection of
the longest force curves taken on the cleaved BR.
No recorded spectrum showed a rupture length
beyond 50 nm. Only three main peaks are
visible-around 5, 25, and 45 nm--and the second
is a double peak. (B) Superposition of
17 spectra on the second peak results in an exact
cover of all but the first peak. (C) Because
loop F-G is cut out, force curves with a length
of 45 nm can be recorded only when the free end
of helix E is fixed to the tip. Thus, the first
peak reflects extraction of helices D and E and
the second reflects extraction and unfolding of
helices B and C the last peak shows extraction
of the last remaining helix A. Consequently, the
intermediate peak between peaks 2 and 3 reflects
stepwise unfolding of helices A and B.
Oesterhelt, F et al. Science 288, 143 (2000)
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bR mutant G241C with specific anchoring of
COOH-terminus
(A) Force spectra of G241C where a terminal
cysteine was introduced near the COOH-terminus at
position 241, allowing specific attachment to a
gold evaporated tip. In these experiments, the
percentage of full-length force curves increased
to 80. (B) Thirty-five force curves are
superposed and WLC fits with lengths
corresponding to the model shown in Fig. 2 are
drawn. In contrast to the measurements in which
we used unspecific attachment, we also could
resolve the substructure of the first peak, which
reflects unfolding of helices F and G.
Oesterhelt, F et al. Science 288, 143 (2000)
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Unfolding bR from purple membrane at various
temperatures
(A ) Force curves of individual BR molecules
recorded at 25C. To show common unfolding
patterns among single-molecule events, the force
spectra recorded at different temperatures were
superimposed. (BF) BR unfolded at different
temperatures. Required pulling forces are
smaller are higher temperatures!
Janovjak et al. EMBO J. 22, 5220 (2003)
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Unfolding pathways of bR
Janovjak et al. EMBO J. 22, 5220 (2003)
(AD) Unfolding events of individual secondary
structures. (A) Occasionally the first major
unfolding peak shows side peaks at about 26, 36
and 51 aa. The peak at 26 aa indicates the
unfolding of the cytoplasmic half of helix G up
to the covalently bound retinal, which is
embedded in the hydrophobic membrane core. The
peak at 36 aa indicates the G helix to be
unfolded completely. At 51 aa, helix G and the
loop connecting helices G and F are unfolded and
the force pulls directly on helix F until this
helix unfolds together with loop EF. (B) The side
peaks of the second major peak indicate the
stepwise unfolding of helices E and D and
loop DE. The peak at 88 aa indicates the
unfolding of helix E, that at 94 aa of the loop
DE, and the peak at 105 aa indicates unfolding of
helix D. (C) The side peaks of the third major
peak indicate the stepwise unfolding of helices C
and B and loop BC. The peak at 148 aa indicates
the unfolding of helix C, that at 158 aa of the
loop BC, and the peak at 175 aa indicates
unfolding of helix B. (D) The side peak of the
last major peak indicates the unfolding of
helix A (219 aa) and of the pulling of the
N-terminal end through the purple membrane
(232 aa).
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Unfolding of individual secondary structure
elements
(A) Occasionally the first unfolding peak at 88
aa shows two shoulder peaks, which indicate the
stepwise unfolding of the helical pair. If both
shoulders occur, the peak at 88 aa indicates the
unfolding of helix E, that at 94 aa of loop DE,
and the peak at 105 aa corresponds to the
unfolding of helix D. (B) The shoulder peaks of
the second peak indicate the stepwise unfolding
of helices C and B and loop BC. The peak at 148
aa indicates the unfolding of helix C, that at
158 aa of the loop BC, and the peak at 175 aa
represents unfolding of helix B. The arrows
indicate the observed unfolding pathways. In
certain pathways (black arrows), a pair of two
transmembrane helices and their connecting loop
unfolded in a single step. In other unfolding
pathways (colored arrows), these structural
elements unfolded in several intermediate steps.
Janovjak et al. Structure 12, 871 (2004)
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Unfolding forces of secondary structure elements
depend on temperature
(A) Rupture forces of main peaks, which exhibited
no side peaks. The forces represent the pairwise
unfolding of transmembrane helices E and D
(88 aa), C and B (148 aa) and the unfolding of
helix A (219 aa). (BD) Rupture forces of side
peaks represent unfolding of single ?-helices and
of their connecting loops (see text). The
thermally induced weakening of the unfolding
forces was fitted (dotted lines) using
equation (2).
Janovjak et al. EMBO J. 22, 5220 (2003)
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Probability of unfolding pathways depends on
temperature
Janovjak et al. EMBO J. 22, 5220 (2003)

• The occurrence of main force peaks exhibiting no
  side peaks (solid lines) increased with
  increasing temperature. As a consequence, the
  probability of the main peaks exhibiting side
  peaks (dashed lines) decreased significantly.
• ?-helices of BR unfold preferentially pairwise
  at elevated temperatures.
• The probability of single structural elements,
  such as helices or loops, to unfold in a separate
  event decreases with increasing temperature.

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2-state model to interpret mechanical unfolding
experiments
A simple two-state potential exhibiting a single
sharp potential barrier separating the folded
low-energy state (F) from the unfolded state (U)
can be applied to describe the mechanical
unfolding experiments. Here the unfolding of
single secondary structure elements of the
membrane protein BR is interpreted using this
model. The activation energy for unfolding is
given by ?Gu, while xu (the width of the
potential barrier) is the distance along the
reaction coordinate from the folded state to the
transition state () and the natural (thermal)
transition rate is denoted k0u . DFS experiments
allow determining the width of the potential
barrier and the unfolding rate by monitoring the
unfolding forces as a function of pulling speed.
Janovjak et al. Structure 12, 871 (2004)
29
bR force curves recorded at different pulling
velocities
(A)(D) show superimpositions of around 15 force
versus distance traces each recorded on a single
BR molecule at the pulling speed indicated (10
nm/s A, 87 nm/s B, 654 nm/s C, 1310 nm/s
D, and 5230 nm/s E). As observed from the
superimpositions, the unfolding forces (height of
the peaks) increase with the pulling speed.
Janovjak et al. Structure 12, 871 (2004)
30
Pairwise unfolding pathway of TM helices
The experimental curve to the left shows a
representative unfolding spectrum of a single BR,
while the schematic unfolding pathway is sketched
on the right. The worm-like chain model was
applied to derive the length of the unfolded
elements based on their force-extension pattern
(solid lines). These lengths were then used to
reconstruct the corresponding unfolding pathway.
The first force peaks detected at tip-sample
separations below 15 nm indicate the unfolding of
transmembrane a helices F and G. After unfolding
these elements, 88 aa are tethered between the
tip and the surface (a). Separating the tip
further from the surface stretches the
polypeptide (b), thereby exerting force to helix
E and D. At a certain critical load, the
mechanical stability of helices E and D is
overcome and they unfold together with loop DE.
As the number of amino acids linking the tip and
the surface is now increased to 148, the
cantilever relaxes (c). In a next step, the 148
aa are extended thereby pulling on helix C (d).
After unfolding helices B and C and loop BC in a
single step, the molecular bridge is lengthened
to 219 aa (e). By further separating tip and
purple membrane, helix A unfolds (f) and the
polypeptide is completely extracted from the
membrane (g).
Janovjak et al. Structure 12, 871 (2004)
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Unfolding Forces as a Function of Pulling Speed
For single and groups of secondary structure
elements, the unfolding force increased with the
pulling speed. A logarithmic dependence of the
force on the pulling speed was clearly resolved.
This indicated that a single sharp potential
barrier as shown in Figure 1 was to be crossed to
unfold the structural elements. Force versus
ln(speed) plots for the pairwise unfolding of
helices are shown in (A) and for single secondary
structure elements (i.e., transmembrane a helices
and polypeptide loops) in (B)(F). As unfolding
of helices D, C, and B occurred in two different
unfolding pathways (1 and 2), two data sets were
obtained and analyzed independently. Although in
both pathways these helices unfolded
individually, other helices unfolded together
with extracellular loops, and therefore the
events were analyzed separately.
Janovjak et al. Structure 12, 871 (2004)
32
Unfolding Pathways Depend on Pulling Speed
Janovjak et al. Structure 12, 871 (2004)
Individual bR molecules exhibited distinct
probabilities to follow different unfolding
pathways when unfolded by mechanically pulling on
the C terminus.
Although single helices were sufficiently stable
to unfold in individual steps (dashed lines),
they exhibited a certain probability to unfold
pairwise (solid lines). Changing the pulling
speed affected these unfolding probabilities the
probability of unfolding single secondary
structure elements increased with the pulling
speed. This suggests that in the absence of a
pulling force (smallest pulling speeds) two
transmembrane helices would preferentially show a
pairwise behavior.
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Potential Landscape from Dynamic Force
Spectroscopy
Two possible unfolding routes exist for pairs of
transmembrane helices in BR. From the folded
state (F), the two helices are either unfolded
individually (dashed line) or pairwise (solid
line) to the unfolded state (U ). The shown
approximation of the potential landscape at
native conditions (zero force) was generated by
extrapolating the speed-dependent unfolding
probabilities to zero force. Since the
experimental data showed that between two
possible routes the pairwise unfolding was chosen
more frequently, its potential barrier must be
lower than for unfolding of individual helices.
Janovjak et al. Structure 12, 871 (2004)
34
Summary
2-step mechanism suggested by Engelman
Popot 1) ?-helices fold first after being
inserted into membrane 2) folded ?-helices then
assemble to form entire protein is well
supported by recent experiments. Translocon
complex inserts TM helices into lipid
bilayer. Fluorescence allows to follow folding
events upon denaturation/renaturation. AFM
experiments allow to study cooperativity of
unfolding of secondary structure
elements. Remains integrate these results
combine with simulations.