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11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 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
Today‘s 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
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 3
Folding of helical membrane proteins (II)
White, FEBS Lett. 555, 116 (2003)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 4
Hydrophobicity Scales
White, FEBS Lett. 555, 116 (2003)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 5
Translocon-assisted folding of TM proteins?
White, FEBS Lett. 555, 116 (2003)
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 ?
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 6
Integration of H-segments into the microsomal membrane
Hessa et al., Nature 433, 377 (2005)
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.
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 96–98 and 258–260, 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).
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 7
Insertion determined by simple physical chemistry
gg
g
ff
fp
21
1
g
gapp f
fK
2
1
Hessa et al., Nature 433, 377 (2005)
c, Gapp values for H-segments with 2–4 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 = 0–7 Leu residues.
measure fraction of singly glycosylated (f1g) vs. doubly glycosylated (f2g) Lep molecules
appapp KRTG ln
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 8
Biological and biophysical Gaa scales
Hessa et al., Nature 433, 377 (2005)
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 Wimley–White water/octanol free
energy scale for partitioning of peptides.
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 9
Positional dependencies in Gapp
Hessa et al., Nature 433, 377 (2005)
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 GGPGAAALAALAAAAALAALAAAGPGG 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.
Tyr and Trp are favorable in interface region.
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 10
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 11
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 12
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 13
Effects of loop mutants on folding kinetics
Allen et al. J Mol Biol 308, 423 (2001)
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.
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 14
AFM topography of a purple membraneTypical 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 15
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)
Force profile
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 16
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)
Check membrane to see what happened
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 17
Force extraction profilesSeveral 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 18
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)
What are the regular features?
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 19
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)
Model to explain force extraction spectra
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 20
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 21
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 22
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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 23
Unfolding bR from purple membrane at various temperatures(A ) Force curves of individual BR
molecules recorded at 25°C. To show
common unfolding patterns among single-
molecule events, the force spectra
recorded at different temperatures were
superimposed.
(B–F) BR unfolded at different
temperatures.
Required pulling forces are smaller are
higher temperatures!
Janovjak et al. EMBO J. 22, 5220 (2003)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 24
(A–D) 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).
Unfolding pathways of bRJanovjak et al. EMBO J. 22, 5220 (2003)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 25
(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)
Unfolding of individual secondary structure elements
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 26
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).
(B–D) 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 27
Probability of unfolding pathways depends on temperature
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.
Janovjak et al.
EMBO J. 22, 5220 (2003)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 28
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
ΔG‡u, 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 30
Janovjak et al. Structure 12, 871 (2004)
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 α 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).
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 31
Unfolding Forces as a Function of Pulling SpeedFor 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 α 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 32
Unfolding Pathways Depend on Pulling Speed
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.
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.
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 33
Potential Landscape from Dynamic Force SpectroscopyTwo 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)
11. Lecture SS 20005
Optimization, Energy Landscapes, Protein Folding 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.