v11: folding of membrane proteins

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)



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



Folding of helical membrane proteins (II)

White, FEBS Lett. 555, 116 (2003)



Hydrophobicity Scales

White, FEBS Lett. 555, 116 (2003)



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 ?



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).



Insertion determined by simple physical chemistry

gg

g

ff

fp

21

1

g

gapp f

fK

2

1


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



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 Wimley–White water/octanol free

energy scale for partitioning of peptides.



Positional dependencies in Gapp


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.



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)



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).




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.




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.



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)



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



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.


Check membrane to see what happened



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.




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.


What are the regular features?



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



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.




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)



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.




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)



(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)



(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



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)



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)



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)



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.





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).



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.


http://www.structure.org/content/article/image?uid=PIIS096921260400098X&imageid=GR4#fig1

http://www.structure.org/content/article/image?uid=PIIS096921260400098X&imageid=GR4#fig1



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.



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.




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.

v11: folding of membrane proteins

Documents

membrane integration

membrane insertion

symmetrical 3l16a hsegments

leuleu separation

glycosylated proteins

helix segment h

red leu residues

leu scan