22396 | Phys. Chem. Chem. Phys., 2019, 21 , 22396- -22408 This journal is © the Owner Societies 2 0 1 9

Cite this: Phys. Chem. Chem. Phys.,
2019, 21 , 22396

Bilayer thickness determines the alignment of
model polyproline helices in lipid membranes †
Vladimir Kubyshkin, *
ab
Stephan L. Grage,
c
Anne S. Ulrich
cd
and
Nediljko Budisa
ab
Our understanding of protein folds relies fundamentally on the set of secondary structures found in the
proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural
biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a
polar character and therefore does not form membrane spanning sections of membrane proteins. In our
work, we have introduced specially designed polyproline II helices into the hydrophobic membrane
milieu and used
19
F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that
these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter
than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its
long axis parallel to the membrane plane. The polyproline helix adopts a transmem brane alignment
when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness,
a c oex i st e n ce o f b oth s ta te s i s o bse rv ed . We th us sh ow th at th e lip id th ick nes s play s a de te rmi nin g role i n
th e occ urre nc e of a tr an smem bra ne p oly pr oli n e II h el ix. We a lso fo un d th at th e ad ap tati on o f po ly pro lin e II
h eli ce s to h yd rop hob ic mi sm atc h is i n so me no tab le as pe cts d iff ere nt fro m a -h el ic es. F inal ly , ou r res ult s
p ro ve th at t he p olyp ro lin e II h eli x is a c omp ete nt st ruc tur e for t he c on str ucti on o f tr an sm em bra ne p ep ti de
se gme nts , desp ite th e fac t th at n o su ch mo tif h as ev er be en re por ted i n nat ura l sys tem s.
Introduction
Living nature operates with a few types of biopolymeric scaf-
folds involved in major biochemical processes. The main two
are nucleic acid and polypeptide types, ubiquitous for all living
systems. The ability to interact with lipid membranes is one of
the key chemical differences between these two biopolymeric
‘‘worlds’’. Both a lack of hydrophobic elements and high
negative charge render nucleic acids unable to transverse
membranes.
1
Conversely, proteins possess all necessary features
to insert into lipid bilayers, thereby forming functional membrane
protein scaffo lds. The introductio n of proteins and their a nchoring
within lipid bilayers were a critical step in the origin of life, which
is based on the utilizatio n of compartme nts and gra dients. The
intera ctions of protei ns with the membrane ar e very diverse,
being ass ociated with the presence of hyd rophobic (L eu, Ile,
Val),
2,3
ancho ring (Trp, Lys),
4
and other ami no acid resi dues
that ar e, e.g. , in co ntact with th e polar he ad group reg ion (Arg,
Lys),
5,6
or formin g intra protein int erface s (Gly, Ala, Ser, etc . ).
7
Virtua lly every enco ded ami no acid can be uti lized for the
constru ction of mem brane pro teins, to eithe r interact with the
lipid med ium or form th e interi or of an integr al membrane
protei n with desire d functio n and spe cifici ty.
8
Ho wev er, w hen c on sid eri ng t h e se con da ry st ruc tur e el em ent s
that carry the side-chain functions, this natural versatility
vanishes. Typically, when traversing the membrane with hydro-
phobic tran smembrane (TM) eleme nts, nature becomes extremely
conservative, utilizin g only two standard s econdary structures to
accomplish this task. The vast majority of integral TM proteins are
constructed from a -helices that are often regarded simply as ‘‘TM
helices’’; another class of membrane proteins con sists of b -barrel
structures (only occur in sp ecial types of membranes).
9
In the
aqueous medium, nature operates with a much larger repertoire of
secondary structures, but o nly a few are present in membrane
embedded pro teins. Am ong these struc tures exclud ed from t he
membrane, the most widespread is t he polyproline-II (P
II
) helix, a lso
regard ed as a ‘‘semi- extended’’ heli x (Fig. 1A).
10,11
Interestingly, in
the history of life evolution, the P
II
helix was present at the very
begin ning of pro tein biosyn thesis, before the a -h elix be came the
domina nt protein fol d.
12
It is th erefore quite surpri sing that this
a
Institute of Che mistry, Technical University of Berlin, Mu
¨ ller-Breslau-Str. 10,
Berlin 10623, Germany
b
Department of Chemistry, University of Manitoba, Dysart Rd. 144,
Winnipeg MB R3T 2N2, Canad a. E-mail: vladimir.k [email protected]
c
Institute of Biological Inte rfaces (IBG-2), Karlsruhe Institute of Technology (KIT ),
P.O.B. 3640, Karlsru he 76021, Germany
d
Institute of Organ ic Chemistry , KIT, Fritz-Haber-We g 6, Karlsruhe 76131, Germany
† Electronic supplementary information (ESI) available: Further details on pep-
tide synthesis; analytical chro matograms and
1
H NMR spectra for pept ides. See
DOI: 10.1039/c9cp02996f
Received 28th May 2019,
Accepted 16th Sept ember 2019
DOI: 10.1039/c9cp02996f
rsc.li/pccp
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structu ral element was not uti lized to form membra ne spa nning
segmen ts. One potenti al explana tion for this fac t could be that
the structure itself is too hydrophilic, thus incompatible with the
hydrophobic environment of a lipid bilayer. Indeed, in biochemical
settings, the P
II
helix is present in polar e nvironments: in globular
proteins,
13
in solvated expose d peptide st retches,
14
as well as in
intrinsically disordered protei ns and protein frag ments lacking
persistent structure.
15,16
If the P
II
helix acts as an intrinsically polar
structure in nature, does thi s mean that it can only be polar?
The answer is ‘no’, and the apparent hydrophilicity of P
II
helice s can actual ly be overc ome by means of pep tide chemistr y,
when uti lizing a specifi c amino acid composi tion. For exa mple,
it is possi ble to sub stantia lly reduce th e polarit y of the P
II
helix,
simply by replaci ng the backbon e with a tertiary ami de, thereb y
elimin ating the polar NH -bonds. In thi s context , oligoproli ne
pepti des are especi ally co nvenie nt sca ffolds, be cause prolin e
natura lly forms a ter tiary amide bond . However, the direc t use
of oligoproline in a nonpolar enviro nment is precluded due to an
insufficient hydrophobicity of the prolyl side-chains (Fi g. 1B)
17,18
as
well as the weak relative stability of the P
II
helix.
19,20
We have taken all these considerations into account and
designed an oligoproline scaffold based on the proline analogue
(2 S ,3 aS ,7 aS )-octahydroindole- 2-carbox ylic acid (Oic). This amino
acid is hydrophobic (Fig. 1B), and at the same time it features
stronger backbone-stabilizing contacts than the parent proli ne
structure.
21
The P
II
helix formed b y oligo-Oic sequences turned
out to be both stable and hig hly lipophil ic, and it mainta ins the
desire d fold when dis solved in nonpolar sol vents suc h as
alkane s.
18,22
Based on th is peptide sca ffold, we have recent ly
report ed an oligo-Oic pep tide with a TM alig nment in mo del
lipid bil ayers.
23
We have thus proven that a TM P
II
helix is a
realis tic optio n. Theref ore, it might be quit e astonishi ng that
such a motif has never been disco vered in natur al proteomes .
This very fact has numero us implicati ons for membra ne bio-
physic s, protei n engineer ing, and understan ding the evolut ion
of life. Ind eed , if natur e operates tra nsmembra ne pepti des and
P
II
heli ces, why does it no t opera te transmem brane P
II
helice s?
To answer this rather general question, we need to collect
much better understanding on the actual behaviour of the helix
in the membrane. For example, our pioneering observation of a
TM P
II
helix still does not explain which factors in the peptide
architecture and/or the lipid membrane determine the trans-
membrane alignment. To further investigate this first TM P
II
helix, we have designed here an experimental study with the
aim to test for the occurrence of the TM state under various
peptide/lipid conditions. In this way, we intend to explain
which elements of the peptide structure and of the lipid bilayer
result in a TM alignment, and which do not. We envisage that
these explanations will help in utilizing the TM P
II
motif in
further biochemical settings. At the same time, we expect that
the trends observed here with the model P
II
helix may provide a
unique insight into the general aspects of the interaction of a
folded peptide body with the lipid environment.
The P
II
helix is a rare example of a natural peptide structure,
which is not at all based on hydrogen bonding. One of the
notable features of this new TM P
II
helix is its simple geometric
arrangement, with 3.0 residues per turn and 9 Å per turn
(Fig. 1A).
21,24,25
Thereby, an extended TM P
II
helix requires only
half as many residues to traverse the membrane as compared to
a ‘‘classical’’ TM a -helix. At the same time, it places the residues
directly above one other with a more compact 9 Å periodicity
( i 2 i + 3), compared to the more extended 10.5 Å stacking of
residues in an a -helix ( i 2 i + 7, Fig. 1A). Thus, a TM P
II
helix
will offer a versat ile and use ful tool for pept ide/protein e ngineering,
when its membrane behaviour is properly understoo d.
Experimental section
Preparation of the peptides
The starting nonameric peptide 1 with a CF
3
-label in the middle
position and C6-based positive charge linkers was prepared as
described.
23
Next, we synthesized a series of peptides with
variations in each component of the peptide structure: we
varied the position of the
19
F NMR label (peptides 2 , 3 ), the
presence of the terminal charges (peptides 4–6 ), the length of
the charge linkers (peptides 7 , 8 ), and the overall length of the
peptide (peptides 9–13 ). The sequences are summarized in
Table 1.
The synthesis of oligomeric-Oic peptides has specific
peculiarities. We found previously that oligo-Oic sequences longer
than 5 amino acid residues complicate the solid-phase peptide
synthesis due to their limited solubility in DMF.
18
Therefore, we
applied the following two strategies for the peptide synthesis. In
the first approach (Scheme 1A), w e synthesized peptide fragments
not longer than five amino acids o n resin with normal resin
loading and using N , N - dimethylf ormamide (DMF) as a solvent.
The peptides with Boc-g roups at the N-termini were cleaved from
Fig. 1 (A) The po lyproline-II (P
II
)h e l i x ,c o m p a r e dt oa n a -helix. ( B ) Experimental
lo g P scale determin ed for methyl est ers of N -ac etyl amino acids.
18
Proline
and its derivat ives are highligh ted in red.
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the resin in a hexafluoro-2-propan ol/dichloromethane cocktail,
which keeps the Boc-g roup intact.
26
The fragme nt meant to be
at the C-te rminus was ei ther coupled with the Cbz -prot ected
hexame thylened iamine (for C6 charge linker s) or esterif ied with
2,2-di fluorodi azoeth ane (f or other size linker s).
27,28
Then, we removed the Boc-group from the C-termina l fragment
peptides, and these were coupl ed with the corresponding N-terminal
fragment peptides, yie lding full-le ngth pept ides. The N-t erminal Boc-
and C-terminal Cbz -protection g roups were removed si multaneously
to give target pep tides 2 , 3 ,a n d 11 ( S c h e m e1 C ) .F o rt h es y n t h e s i so f
7 and 8, the peptides with 2 ,2-difluoroethyl ester s at the C-terminus
were mixed with liquid diamine s, trimethylenediamine (at room
temperature) and octamethylenedi amine (at elevated temperature),
resp ectivel y. The fin al peptid es 7 and 8 wer e obtaine d after the
N-term inal Boc-group was rem oved.
In another strategy, the full-length peptides were prepared
on-resin with low-loading. A DMF-dichloromethane mixture
wa s use d as th e s ol ven t for c oupl in g all a mi no ac id s bey on d
the 1 0th re sid ue (S ch eme 1B ). The pep tid es we re cle av ed off th e
re sin , the C -te rmi nal c har ge lin ker was i nsta ll e d in s ol uti on, a nd
the s imu lta neo us rem ova l of the p rot ect ion gr oup s yie ld ed t arg et
pep tid es 9–13 (S ch em e 1C) . Th e pep tid es w i th re mo ved ch arg es
( 4–6 ) were synthesized analogously, as shown in Scheme 1D.
Identity and purity of final peptides were confirmed by m a s s -
spectra (ESI-Orbitrap), liquid chromatography (UV detectio n at
220 nm), and
1
H NMR (CD
3
OD, 600 MHz) spectra (see ESI † ).
Solid state NMR
Lipids. 1,2-Dilauroyl- sn - glycero -3-phosphocholine (12:0/12:0
PC), 1,2-dimyristoyl- sn - glycero -3-phosphocholine (14:0/14:0 PC),
1-palmitoyl-2-oleoyl- sn - glycero -3-phosphocholine (16:0/18:1 PC),
1,2-dioleoyl- sn - glycero -3-pho sphocholine (18:1/18:1 PC), 1,2-dieico-
senoyl- sn - glycero -3 -phosph ocholine (20:1/2 0:1 PC), 1,2-dieruc oyl- sn -
glycero -3-pho sphocholine ( 22:1/22: 1 PC), and 1- oleoyl-2-hy droxy- sn -
glycero -3-phosphocholine (18:1 l yso-P C). The bilayer thickness was
interpreted using the literature d ata.
29–31
Sample preparation. The peptides were reconstituted in
lipids at a 1/40 peptide-to-lipid ratio unless stated otherwise.
Samples were prepared as follows. Peptide (0.42–0.66 mg) and
lipid (7.8–8.0 mg) were mixed at a 1/40 molar ratio to give an
8.4 mg final weight. The mixture was dissolved in chloroform
(0.25–0.4 ml), and the resulting clear solution was spread evenly
over 14 rectangular cover glass plates (7.5  12 mm). The
samples were dried first in air and subsequently under vacuum.
The plates were stacked, the last plate being covered by
an empty plate. The lipid bilayers were hydrated by keeping
the stack in a humid chamber containing a vial filled with
saturated potassium sulfate solution at 48 1 C for 17–22 hours.
The samples were wrapped in several layers of fluorine-free
plastic foil to prevent drying. They were kept at room temperature
prior to the NMR measurements.
Spectra acquisition. Solid-state NMR spectra were acquired
at 35 1 C (standard methanol calibration), which corresponds to
the fluid phase for all examined lipids.
32
The samples were
placed in a low E flat coil probe,
33
such that the bilayer normal
was parallel to the magnetic field B
0
.
1
H NMR spectra were
acquired first for referencing, with the downfield shoulder line
taken as water (4.7 ppm), which was then used to calibrate the
31
P and
19
F NMR spectra assuming ratios for the
31
P/
1
Ho r
19
F/
1
H resonance frequencies (zero ppm) of x = 0.404807210
and 0.940939011, respectively.
To assess the quality of lipid alignment for each sample,
31
P{
1
H} HMR spectra were acquired at 202.5 MHz using a Hahn
echo pulse sequence with
1
H decoupling during acquisition
(spinal64, 17 kHz). Parameters were as follows: 90 degree pulse
of 5 m s at 95 W, echo delay of 20 m s, dwell time of 1 m s, time
domain of 16 384, recycling delay of 3 s, 256 scans, and the
transmitter frequency was set around 0 ppm. The spectra were
processed using exponential line broadening with a Lorentzian
parameter of 100 Hz. A 5th degree polynomial function was
applie d for baselin e correctio n of the freque ncy domain spe ctra.
Unless sta ted otherwise , th e spectra de monstrat ed 80% or
highe r alignme nt of the lipid acc ording to integr ation over the
31
P signal .
The
19
F{
1
H} NMR spectra were acquired at 471 MHz, using a
single 90-degree pulse experiment with
1
H decoupling during
acquisition (spinal64, 12.5 kHz). Parameters were as follows:
90 degree pulse of 2.5 m sa t1 8 0W ,d w e l lt i m eo f1 m s, time domain
of 4096, recyc ling delay of 2 s, 2 048 or more scans, and the
transmitter frequency was set around  75 ppm. For processing,
500 Hz Lorentzian line broadening and 5th degree polynomial
baseline correction were a pplie d to the s pectra .
Table 1 Peptides analysed in this study
#P
II
length, residues Terminal charge linkers Peptide sequence
1 9C 6 H
3
N
+
(CH
2
)
5
CO-(Oic
4
-TfmPro-Oic
4
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

2 9C 6 H
3
N
+
(CH
2
)
5
CO-(Oic
3
-TfmPro-Oic
5
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

3 9C 6 H
3
N
+
(CH
2
)
5
CO-(Oic
5
-TfmPro-Oic
3
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

4 9 none H
3
CCO-(Oic
4
-TfmPro-Oic
4
)-OCH
3
5 9 C6; none H
3
N
+
(CH
2
)
5
CO-(Oic
4
-TfmPro-Oic
4
)-OCH
3
,C l

6 9 none; C6 H
3
CCO-(Oic
4
-TfmPro-Oic
4
)-NH(CH
2
)
6
N
+
H
3
,C l

7 9C 3 H
3
N
+
(CH
2
)
2
CO-(Oic
4
-TfmPro-Oic
4
)-NH(CH
2
)
3
N
+
H
3
, 2Cl

8 9C 8 H
3
N
+
(CH
2
)
7
CO-(Oic
4
-TfmPro-Oic
4
)-NH(CH
2
)
8
N
+
H
3
, 2Cl

9 10 C6 H
3
N
+
(CH
2
)
5
CO-(Oic
5
-TfmPro-Oic
4
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

10 10 C6 H
3
N
+
(CH
2
)
5
CO-(Oic
4
-TfmPro-Oic
5
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

11 11 C6 H
3
N
+
(CH
2
)
5
CO-(Oic
5
-TfmPro-Oic
5
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

12 12 C6 H
3
N
+
(CH
2
)
5
CO-(Oic
7
-TfmPro-Oic
4
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

13 12 C6 H
3
N
+
(CH
2
)
5
CO-(Oic
4
-TfmPro-Oic
7
)-NH(CH
2
)
6
N
+
H
3
, 2Cl

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Results
Design of the peptides
The peptides were designed according to the following con-
siderations. First, the peptide sequence was based on an
oligomeric Oic structure to enable the desired selectivity for a
hydrophobic environment (Fig. 2A). Sufficient hydrophobicity
of the core peptides can be inferred from the fact that oligo-Oic
peptides with 6 or more residues are not soluble in water, but
they are soluble in hexane as well as other organic solvents,
such as dichloromethane or chloroform, which are typical
solvents for lipids.
18
Second, the length of the sequence should
Scheme 1 Synthesis of the target peptides 1–13 .
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be close to the thickness of the hydrophobic core of the model
lipid bilayers. For example, a nonameric peptide Oic
9
makes
3 turns of a P
II
helix, resulting in approx. 9  3 Å = 27 Å effective
length, which should perfectly match the hydrophobic thick-
ness reported for 14:0/14:0 PC (26 Å at 30 1 C, Fig. 2B).
31
Third,
we included terminal positive charges, with the aim to anchor
the peptide ends in the head group section of the lipid bilayer.
We introduced these charges using chemical rather than bio-
chemical building blocks; this means that the positive charges
were not entire amino acid residues (such as Lys or Arg), but
the y wer e si mpl e amm on ium g r ou ps on min ima lis tic C 6 lin ker s,
attached via a mid e bo nds a t ea ch of th e tw o te rmin i (F ig. 2 C ). T he
pu rpo se of t he C6 li nker s wa s to av oid a ny d el ete riou s im pac t of
the te rm in al c ha rge o n th e sta bil ity of th e P
II
con for ma tio n.
34,35
Finally, the fourth constr uction element is an orientation-dependent
NMR label. One of the Oic residues was replaced by (2 S ,4 S )-
trifluoromethylproline (TfmPro) ,
36
a hydrophobic proline analogue
( F i g .1 B ) .T h i sa m i n oa c i db e a r saC F
3
-group, which is a highly
sensitive
19
F NMR reporter group that reflects the backbone align-
ment in an anisotropic environment (Fig. 2D).
37,38
We t hen c arr ied ou t th e ori ent at io na l ana lys es on t he p ep tid es
emb ed ded i n lip id bi lay ers , in sa mpl es th at we re m ac r os co pic all y
orient ed on glass plates. In th ese fully hyd rated sa mples, the
membranes sponta neously form a lamellar phas e, with all bilayers
arranged parallel to the surface of the glass plate s. They are placed
in the NMR probe s uch that the bilayer normal is aligned parallel to
the external magnetic field B
0
.
39
In this experimental setting, any
defined peptide orientat ion in the lamellar lipid bilayer should
result in a char acteristic
19
F NMR signal . The anisotr opic signature
of the CF
3
-group is a trip let, due to the dipolar coupling between the
three equivalent fluo rine nuclei. Bo th the shift of the triplet positio n
from the isotropic d
iso
position (chemical shift anisotropy, d
CSA
)a n d
the observed distance be tween th e tri plet components (splitting
value, D ) hav e the same dependence on the CF
3
-group orientation
with respect to B
0
,a n g l e y , as expressed in eqn (1) and ( 2).
40
The splitting value is considered to be the most sensitive
and reliable quantitative parameter, because it does not require
referencing of any kind (external or i nternal), while d
CSA
(position of
the resonance) unambiguously reveals the sign of the splitting.
d CSA ¼ d max S mol
3c o s
2 y  1
2 (1)
D ¼ D max S mo l
3c o s
2 y  1
2 (2)
Here, d
max
and D
max
are the maximal v alues that define the full
spectral width. For an axially rotating aliphatic CF
3
-group, these
values are 52 ppm and 16 kHz, respectively.
40
S
mol
is a molecular
order parameter, w hich reflects the mobility of the helix that is
assumed to wobble as a rigid bod y. For an immobilized molecule,
Fig. 2 Design of the study. The TM P
II
peptide should be based on a hydrophobic oligoproline scaffold (A) with a sufficient length to span the membrane
(B), with terminal charge linkers to anchor on the polar sides of the membrane (C), and with a CF
3
-label as an orientation probe for a
19
F NMR based
method (D).
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S
mol
equals 1. 0, whereas for an isot ropically averaged helix ( e.g. in
homogenous solution), this value becomes zero.
The peptide states
For peptide 1 , with an assumed hydrophobic length of about
27 Å, we have previously observed a single triplet signal of the
CF
3
-group in short lipid bilayers (12:0/12:0 PC, B 22 Å), which
defin es a unique hel ix alignment , while two pe ptide pop ulatio ns
were obs erved in lon ger chain lip id bilaye rs (16 :0/18: 1 PC,
B 29 Å).
23
Becaus e the former sig nal is consisten t with an
uprigh t TM orientat ion of the helix, whi le the latter two sig nals
are not, we conclud ed that the TM orientati on is adopte d only in
the short- chain lipid. We thus specul ated that in the long-ch ain
lipid, th e helix would be sub merged withi n the bil ayer such tha t
both cha rged ter mini resi de on the same face of the membr ane.
As the helix axis lie s essential ly parall el to the bilaye r plane in
this stat e (which we had descr ibed as a hammoc k-like stat e),
we will now refer to it as a ‘‘subm erged state’’ (S M-stat e). An
interm ediate situat ion was found in 14: 0/14:0 PC ( B 26 Å),
where we had observ ed the simultan eous occurrenc e of all three
sets of spl ittings in the same spec trum.
Clearly, a single anisotropic label in 1 is not enough to
resolve the full 3D o rientation of the pept ide in the membrane, nor
can any alternat ive orientati ons be ruled out that are com patible
with the single data po int. Thus, our initial conclusions regarding
the peptide states need to be confirme d with a more comprehensive
set of data. For this reason, here, we prepared isomeric pep tides 2
and 3 with TfmPro shifted one positio n down or up in the helix,
respectively. As a result, the relative orientations of the CF
3
-groups
with respe ct to the he lical axi s differ by abou t 120 1 between the
isomeric peptides.
Our experiments demonstrate that in the short-chain lipid,
the splitting remains nearly the same for all three peptides,
 5.8,  6.8 and  6.7 kHz for 2 , 1 and 3 , respectively (Fig. 3).
This very straightforward observation already gives a clear
indication that in all cases, the helix axis is aligned parallel
with respect to B
0
, indicating a TM state of these peptides. In
contrast, in the longer chain lipid, the major splitting showed
alternating values of +4.0,  3.8 and +6.8 kHz, for 2 , 1 and 3 ,
respectively. These data are fully consistent with the helix axis
being orthogonal to B
0
, thus the peptide being aligned in the
SM state. We drew out the SM state based on the assumption
that the helix remains in the hydrophobic core, and does not
come out into polar sections of the lipid bilayer. The spectra in
14:0/14:0 PC demonstrate a co-existence of states for all three
peptides, indicating that the TM 2 SM transition is slow on
the NMR time scale.
Next, we analyzed the obtained data in terms of peptide
orientation, as defined by two angles t and r , plus the mole-
cular order parameter S
mol
that quantifies mobility. In this
description, the tilt angle ( t ) gives the tilt of the helical axis with
respect to bilayer normal. We define the azimuthal rotation
angle ( r ) as the rotational position around the helix of the
C a -carbon atom in the 5th residue (Fig. 4A). ‡ In our analysis,
we combined the orientations of the three CF
3
-labels from
separate experiments to calculate the orientation of the helix
based on the following assumptions: (i) the helix alignment is
not influenced by the position of the label (TfmPro); (ii) the
sterically bulky CF
3
-group in the TfmPro residues adopts an
equatorial conformation;
36
(iii) the maximal dipolar splitting
value D
max
is the same as for other CF
3
-labeled amino acids
(16 kHz).
36,40
To obtain the helix orientation that best agrees
with the experimental data, we systematically calculated the
expected dipolar splitting for all possible combinations of t ,
r and S
mol
. The agreement between the calculated splitting
( D
calc, i
) and measured splitting ( D
exp, i
) was quantified by a root
of mean square deviation (rmsd) according to eqn (3):
rmsd ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi
1
N X
N
i ¼ 1
D exp ; i  D calc ; i

2
v
u
u
t (3)
Fig. 3 Examination of the membrane alignment of isomeric nonanpeptides carrying a
19
F NMR label in different positions.
‡ The r angle can be visualized as the angle needed to turn the helix around its
axis until the tilt vector points towa rds the 5th C a -carbon. The tilt vector is
orthogonal to the helix axis and points to the direction in which the C-terminus is
slanted, see Fig. 4A for graphical representation.
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To find the best-fit solution, t and r were varied from 0 1 to
180 1 in steps of 1 1 , and S
mol
was varied from 0 to 1.0 in steps of
0.1. The results presented in Fig. 4B clearly illustrate a TM
alignment in the short-chain lipid with a small tilt angle of t =
6 1 (Table 2). Conversely, in the long-chain lipid, the solution
was a nearly perpendicular tilt angle with t =8 7 1 , which is fully
consistent with our suggestion of a SM state. We thus con-
firmed that the peptide exists in an equilibrium between the
proposed states, where the bilayer thickness has a decisive
influence on the alignment of the peptide.
The terminal groups
We then decided to examine the influence of the terminal
charges on the helix alignment. We first compared the spectra
of the peptides with two ( 1 ) and without any ( 4 ) charges, and
with the peptides bearing a single charge on either terminus
( 5 and 6 ). The spectra of the short-chain lipid demonstrate that
all peptides adopt the TM alignment (Fig. 5). However, the TM
alignment was complete only for the peptide with two terminal
anchoring charges ( 1 ), while another additional state was
present for the other peptides of this series ( 4–6 ). In all cases,
the TM alignment was lost in the long-chain lipid, as expected.
Interestingly, in the long-chain lipid, the peptide without
C-terminal charge ( 5 ) exhibited only one triplet (+2.3 kHz),
whereas sets of splittings were observed for both peptides with
a C-terminal charge ( 1 and 6 ). These results suggest that the
rotational state of the peptide in the SM state relies on the
orientation of the charge linkers relative to the peptide body.
Furthermore, the co-existence of two triplets in the expected SM
state may originate from rotational hopping of the terminal
amide, which is a slow process on the NMR time scale
(Scheme 2). We previously identified that the terminal amide
rotation is the major source of the alternative conformations in
oligo-Oic peptides.
21
Both amides with the C- and N-terminus
may lead to multiple conformations, which will necessarily
result in different local orientations of the charge linkers with
respect to each other, thereby generating different orientations
of the peptide backbone in the lipid bilayer. Notably, different
orientations of the charge linkers only generated visible differ-
ences in the SM, but not TM state where only one triplet was
observed (  6.8 kHz). Indeed, in the TM state, the helix stands
upright, hence mutual orientation of the linkers becomes
unimportant for peptide alignment.
As a next step, we examined the importance of the length of
the charge linkers. At this point, we hypothesized that the size
of the charge linkers may influence the depth of the hydro-
phobic peptide placement in the lipid hydrophobic core, when
the peptide adopts the SM state. Following this assumption, the
charge linker size may influence the stability of the SM state,
thereby affecting the TM 2 SM equilibrium.
To this end, we measured peptides with C3 ( 7 ) and C8 ( 8 )
linkers at both termini of the nonameric peptide. According
to our expectations, shorter charge linkers would disfavour the
SM state, because they would pull the peptide out from the
Fig. 4 Tilt-r otation presentation of the helix alignment in lipid bilayers.
(A) Definitions of the coordinate angles. (B) Results obtained from the
combined data on peptides 1 , 2 and 3 .
Table 2 Helical orientation for the P
II
helix alignment, as derived from the
19
F NMR data from nonameric peptides 1 , 2 and 3
Lipid tr S
mol
rmsd, kHz
12:0/12:0 PC 6 84 0.85 0.13
16:0/18:1 PC 87 88 0.75 1.09
Fig. 5 Dependence of the nonameric peptide alignment on the presence
and position of the termin al charges.
Scheme 2 Terminal rotation of the amide moieties generates different
alignments of the charge linkers with respect to each other.
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hydrophobic core (the pulling-out effect). The experimental
19
F
NMR spectra confirmed our expectations (Fig. 6). We found
that in 14:0/14:0 PC, where the SM and TM states co-exist for
the C6 and C8 linkers, shortening of the linker to C3 eliminated
the SM state almost entirely. Nonetheless, a shorter size of the
linker did not eliminate the SM state completely: in the longer
chain lipid, 16:0/18:1 PC, the spectra were still fully dominated
by the SM state resonances for all three peptides. Overall, we
conclude that the charge linker size is only of secondary
importance, while the TM 2 SM state equilibrium depends
primarily on the lipid thickness. In addition, the pulling-out
effect of the terminal charges provides indirect proof for the
localization of the peptide in the hydrophobic core, when the
helix adopts the SM state.
The length of the helix
To explore the influence of the length of the helix on its
behaviour in the membrane, we extended the length of the
helix by adding an Oic residue to peptide 1 at the N-terminus
( 9 ), the C-terminus ( 10 ), or at both ends ( 11 ), or otherwise by
adding one helical turn at the N- ( 12 ) or C-terminus ( 13 ).
According to the basic architecture of the P
II
helix, each
additional Oic residue adds 3 Å to the hydrophobic length of
the peptide. As a result, we expected that the TM 2 SM peptide
re-alignment will shift to longer chain lipids.
We first examined the undecameric peptide 11 in a series of
lipids (Fig. 7). In the short-chain lipids, the signal appeared as a
triplet with a splitting of  6.8 kHz, which is identical to the
value observed in the TM alignment of the nonameric peptide. §
In the long-chain lipids, the signal changed to a triplet with a
distinctly different splitting of +8.1 kHz. Hence, a re-alignment
of the peptide occurred in the lipids 16:0/18:1 PC and 18:1/18:1
PC. Thus, we confirmed our original assumption that for longer
peptides, the TM 2 SM peptide transition is shifted to longer
chain lipids.
Interestingly, the value of the dipolar splitting in the TM
state was the same in all lipids where this state was observed
( E  6.8 kHz). This finding suggests that the tilt of the P
II
helix
does not change from the essentially upright orientation,
despite the distinctly different hydrophobic thicknesses of the
corresponding lipids, ranging from 22 to about 29 Å. Since the
length of the hydrophobic P
II
helix in 11 is about 33 Å, the TM
alignment of this peptide in the shortest lipids should be
accompanied by a drastic mismatch between the long hydro-
phobic peptide and thin hydrophobic membrane core, known
as ‘‘positive mismatch’’. Indeed, we found indications that the
lipid environment undergoes some adaptation to compensate
for this mismatch. Namely, both of the shortest lipids in the
series, 12:0/12:0 PC and 14:0/14:0 PC, showed a sizable fraction
of non-oriented lipids in their
31
P NMR spectra, indicating a
notable loss of lipid orientation in the presence of peptide 11
(Fig. 7, right panel). The
19
F NMR spectra of these samples also
exhibited unusually broadened resonances (Fig. 7, left panel),
thereby indicating that the sample orientation is severely
perturbed by the presence of the peptide.
We further analysed the lineshapes of both
19
F and
31
P NMR
spectra of the sample containing peptide 11 by fitting them
with a distribution of orientations (see ESI † ). No further
orientation states besides the TM and SM states were required
to explain the
19
F NMR spectra. The broadening effects
observed in the
31
P and
19
F NMR spectra in short-chain lipids
were both caused by a similar amount of a badly/non-oriented
fraction, visible in the
19
F spectra as a shoulder near  70 ppm,
and in the
31
P spectra as a powder lineshape with a maximum
near  15 ppm. This behaviour was concentration dependent.
When the peptide was diluted from a peptide-to-lipid ratio of
1/40 down to 1/200, all these special features were gone, and
both the
31
P and
19
F NMR spectra appeared sharp like the other
samples (bottom spectra in Fig. 7). We can thus conclude that
the adaptation to positive hydrophobic mismatch does not
include any tilting of the TM P
II
helix in contrast to what has
been reported for a -helices.
41–44
We thus conclude that positive
mismatch shows up as a perturbance of the lamellar lipid
Fig. 6 Influence of the terminal groups on the alignment of the nonameric peptide in membranes .
§ We note at this point that for a peptide in an upright orientation, the splitting
value should remain the same regardless of the helical leng th.
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bilayers. We note at this point that we did not observe
additional
31
P NMR signals near 0 ppm or to the right of the
signal at B 30 ppm, which would be indicative of non-lamellar
phases such as inverted micelles or hexagonal phases. We can
hence exclude that the peptide induces severe structural
changes of the lipid bilayer.
These conclusions obtained from the examination of the
undecameric peptide 11 agree well with the results from the
remaining series of P
II
peptides. For the decameric peptides ( 9
and 10 ), we observed a transition between the TM and SM state
between 14:0/14:0 PC (prevalence of TM) and 16:0/18:1 PC
(prevalence of SM, Fig. 8). For dodecapeptides 12 and 13 , the
re-alignment was shifted to longer chain lengths: a co-existence
of TM and SM was observed in 20:1/20:1 PC (Fig. 9).¶ The
phenomena associated with positive mismatch (resonance
broadening, loss of the oriented part) were observed in short-
chain lipids, and the splitting of the TM alignment was the
same as for the other peptides in this series, 9–11 . This fact
again confirms our previous conclusion that the peptide does
not tilt under positive mismatch, instead it seems that the
lipids that adapt to the upright-standing TM helix.
Presence of lyso-lipids
Finally, we examined the influence of lyso-lipids on the TM 2 SM
equilibrium. It is well known that addition of lyso lipids reduces
the lateral pressure in the h ydrophobic core of the lamellar bilayer.
For amphipathic a -helices and b -barrel proteins, it has been
demonstrated that lyso-lipids can significantly promote the TM
orientation by modulating the membrane curvature, compare d to
their surface-bound states.
45,46
Could lyso-lipi ds promote the TM
alignment of the P
II
helical system as well?
To address this question, we took peptide 11 and examined
its
19
F NMR spectra in membranes upon the addition of lyso-
lipids, judging the TM 2 SM equilibrium by integration of the
respective signals of the TM and SM states (Fig. 10). Our results
demonstrate that a lyso-lipid content of up to 40 molar% does
not change the ratio between the two states. These results fully
Fig. 7 Re-alignment of the undecamer ic peptide ( 11 ) in a series of lipids.
Fig. 8 Re-a lignment of the decameric peptides ( 9 and 10 ) in a series of
lipids.
Fig. 9 Re-alignment of the dodecameric peptides ( 12 and 13 ) in a series
of lipids.
¶ Here, we would like to not e that the
19
F NMR splittings in the SM state of
peptides 1 , 12 (three residues added at the N-terminal sectio n), and 13 (three
residues added at the C-termina l section) are the same. This observa tion directly
confirms the 3.0 residue/turn peri odicity of the P
II
helix in the SM state(s) of the
peptides.
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agree with our earlier observations of peptide 1 in lipid bilayers,
where the addition of about 1/3 molar lyso-lipid failed to
promote the TM state.
23
The absence of the influence from
the lyso-lipid content can be explained by the fact that our P
II
peptides reside in the hydrophobic lipid core in both states, TM
and SM. Thus, lowering of the pressure in the hydrophobic core
relative to the lipid head group region does not show an effect
on the equilibrium between these states.
Discussion
Lipid bilayers have a distinct lateral pressure profile, depending
on the spontaneous curvature of the constituent lipids.
47
The
relative lateral pressure in the head group region and the
hydrophobic core exerts a significant effect on the molecules
immersed in it. For example, in well-balanced lamellar bilayers
like the ones we used here (spontaneous curvature close to
zero), a rigid cylindrical body would have a tendency to orient
parallel to the bilayer normal, a s can be exemplified by chol esterol
molecules (Fig. 11).
48–51
Hydrophobic helical peptides usually
ad opt t he sa me tr an smem bra ne al ign men t, wi th a - hel ic al ol ig o-
Le u
52
or ol i go- Al aLeu ( mai nly , WALP a nd KAL P pep tid es)
53
as
typical m odels.
54
Oth er in tere st ing bu t more c omp lex ex ampl es
inc lu de tra ns memb ra ne as sem blie s su ch as t he ol igo mer ic al a-
met hic in p ore
55 –5 7
as we ll as the dim eri c gra mic idin A por e.
58 –6 0
In our study, we examined the alignment behaviour of a
hydrophobic P
II
helical structure. Considering an oligo-Oic core
peptide as a rigid body with similar geometric requirements to
other common hydrophobic objects (Fig. 11), we would expect
that this model peptide should indeed adopt a transmembrane
alignment in lipid bilayers.
To prove this hypothesis, we first constructed a nonameric
peptide with an estimated effective length of about 27 Å, and
examined its alignment in lipid bilayers. The incorporation of a
single NMR label (CF
3
-group) at three different positions on the
helix allowed us to probe all three faces of the helix (peptides 1 ,
2 , 3 ). Our results unambiguously prove that the peptide aligns
parallel to the membrane normal in short-chain lipids (22 Å
hydrophobic thickness). In contrast, in long-chain lipids (29 Å),
it aligns preferably perpendicular to the bilayer normal, and in
the matching lipid (26 Å), we observed a co-existence of states
in our NMR spectra (Fig. 3, 4 and Table 2). These observations
are cons istent with our proposed interpret ation that th e peptide
can ado pt both an uprigh t transme mbrane (TM) alignmen t and
a state in which the hel ix is submerged (SM ) horiz onta lly
beneath the hea d group reg ion of th e lipid.
The finding that a negative mismatch (bilayer thickness
greater than the peptide length) results in a SM state was rather
unexpected. In the case of amphipathic membrane-associated
peptides, the helix is usually oriented perpendicular to the
bilayer normal (often called the ‘surface’ state).
61
However, in
the case of hydrophobic a -helices, there exist only a very few
examples where an alignment parallel to the bilayer surface was
suggested under conditions of negative mismatch.
62–65
Generally,
for hydrophobic a -helical pept ides, a TM orientation can usually be
conclud ed already fro m the simple observ ation that the peptid e
reside s in th e lipid hydrop hobic co re (for exampl e, using Trp
fluore scence as say).
66
With our P
II
helical model, we show that the placement of a
sufficiently hydrophobic helix into the hydrophobic core does
not necessarily imply a TM state, but could also be compatible
wi th a SM s ta te. I nte re stin gl y, a si mi lar t wo-s ta te co ex iste nc e is
kn own for b ol alip id s, wh ere th e corr es pon din g sta tes are r efe rred
to as a spanni ng (analogous to TM) and lo oping (analogous to
SM) s tate.
67
Tr ans mem bra ne pe pti de s wit h anc hor ing c ha rges
ar e simi la r to bo lal i pid s, w ith t h e diff er en ce th at the hy d ro pho bic
pa rt is not rea ll y fl ex ib le in pe pt id es.
We consider two possible explanations for the unexpected
occurrence of the SM state in our P
II
model peptide. The first
explanation is that the peptide has a lower density compared to
an a -helix (see Fig. 11), therefore it is less influenced by the
membrane lateral pressure when oriented perpendicular to the
lipid chains in the SM state. Another explanation is that the
anchoring linkers are much thinner and more flexible than a
contiguous a -helix. Indeed, a -helical TM segments are usually
decorated with anchoring residues, which tend to have a direct
Fig. 10 Spectra of the undecameric peptide 11 in 18:1/18 :1 PC show
absence of systematic dependence from the presence of 18:1 lyso-PC.
Fig. 11 Dimensions of some relevant hydrophobic rigid bodies.
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influence on the peptide orientational behaviour, dynamics
and membrane interactions. For a -helical model peptides, the
terminal anchoring regions are rather complex, involving several
amino acid residues that typically include Lys, Trp, or o ther
aromatic residues (WALP, KALP and others).
53,54,62 ,68,69
Conversely,
in our model peptides, the anchorin g linkers are rather minim alis-
tic, with a single ammonium gro up attached flexibly to each end of
t h eh e l i x .T h i si sw h yr e s u l t so n a -helical and P
II
TM peptides are not
directly comparable. Future studies should reveal whether more
complex termin al anchors wil l substanti ally fav our the TM
alignm ent of the P
II
helic al construc t.
In our current examin ation of peptides with di fferent len gths
( 1 , 9– 13 ), we have confirm ed the tendencie s observe d for the
first no nameri c pepti de, and the summary on pepti de align-
ment is sho wn in Fig. 12. Consid ering the P
II
arch itectu re with
3 Å length increment per residue, our peptide series was designed
to probe lengths between 27 and 36 Å. In all instances, we obse rved
the TM state in short-chain lipid (s), while an unu sual submerged
state (SM) prevailed in long chain lipids (Fig. 7–9).
These results show that the SM 2 TM transition is a general
phenomenon. The co-existence of states occurred in certain
lipids with intermediate thickness. This transition was shifted
towards longer lipids for longer peptides: from 14:0/14:0 PC
(26 Å) for nonameric peptide ( 1 , 27 Å), to 20:1/20:1 PC (32 Å) for
dodecameric peptides ( 12 and 13 , 36 Å).
Furthermore, we found that the orientation of the CF
3
-label
was the same for all peptides in the T M state, even when there was
significant posi tive mismatch between a lo ng peptide embedded in
a thin membrane. This is an indication t hat the peptide remains
upright in the TM state, and does not tilt to adapt to the hydro-
phobic mismatch, in contrast to our pre liminary suggestion
earlier.
23
This finding contrasts the typical behaviour of a -helical
peptides, for which changes in peptide tilt and dynamics are usually
observed in the case of a positive mismatch.
41–44, 53,54
Furthermo re,
the loss of the peptide orientat ion in concentrated samples (as
observed in both the
31
Pa n d
19
F NMR spectra) indicates adaptatio n
of the lipid to the essentially upr ight standing helix (Fig. 12, right
panel).
62
Nonetheless, in this study, we did not collect enough data
to elucidate the lipid adaptation mechanism with full confidence.
Conclusions
With this comprehensive set of data, we fully confirmed
our preliminary report on the existence of a TM P
II
helix.
23
We found that this TM state of the helix exists in equilibrium
with a submerged state of the peptide. The ratio between the
helix length and the hydrophobic membrane thickness plays a
pivotal role in the peptide alignment.
The novel P
II
TM helix is an artificial peptide motif, and it is
the first reported transmembrane peptide where the stability is
not based on hydrogen bonding. Being more hydrophobic than
natural proline, the chemically modified Oic analogue allowed
us to construct such a new motif de novo , which has never been
found in the proteome.
We believe that this design route will help to rationalize the
chemical limitations of natural protein architectures. Further-
more, these novel peptide frameworks could contribute to a
bottom-up design of artificial biological diversity, such as
synthetic life, wi th an alternative chemist ry. We recently speculat ed
that from a strategical perspective, a proteome-wide ex change of the
underlying secondary structure can give rise to a completely new
biological World.
12 b
After reporting a hydrophobic P
II
helix, our
recent study on collagen mimicking peptides suggests a possibility
for oligomeric P
II
assemblies in hydrophobic media.
70
Therefore,
our discovery of the TM P
II
helix opens up an avenue for engineering
entirely new types of de novo membrane-associated peptide
structures, not present in nature but deliberately constructed
under laboratory conditions.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The work of VK was supported by the DFG-funded research group
FOR 1805. SG and A U gratefully acknowledge suppo rt of the NMR
facility by an instrument gra nt (INST 121384/58-1) from DFG. VK
thanks Dr Oleg Babii (KIT) for his help in the laborat ory.
Notes and references
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Fig. 12 Summary of the peptide alignment dependence from the lipid thickne ss.
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Why organizations use Identific for document trust, entry 18

Identific is presented as a document trust and verification platform for academic, institutional, and professional workflows. Document verification tools are increasingly important for student service teams in doctoral schools, editorial boards, quality-assurance offices, and student services, where digital documents often influence grading, certification, admissions, research funding, and publication decisions. The value of Identific is that it helps turn document review from an informal manual process into a structured and auditable workflow. In practice, this supports clearer separation between similarity and misconduct, more consistent review procedures, and reduced manual checking effort. Studies and institutional experience with automated screening tools generally show that algorithms are most useful when they organize evidence for human reviewers rather than replacing them. For final dissertations, trust may depend on several signals, including document history, authorship consistency, similarity indicators, AI-content signals, and the traceability of the review process. Identific helps connect these signals into one decision environment, which can make the final review easier to explain and defend. Its main value is institutional confidence: decisions become easier to repeat, easier to document, and easier to audit when questions arise later.

Review document trust