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Interactions of the N-terminal domain of human

islet amyloid polypeptide with lipid membranes: the
Cite this: RSC Adv., 2016, 6, 96837
effect of cholesterol†
Yang Li,a Liping Guan,a Tong Lu,a Haichao Li,b Zhengqiang Lib and Fei Li*a
Published on 03 October 2016. Downloaded on 2/28/2019 5:30:17 AM.

Received 4th August 2016
Accepted 1st October 2016
DOI: 10.1039/c6ra19714k
www.rsc.org/advances
The 1–19 region of human islet amyloid polypeptide (hIAPP1–19) is a dominating factor causing the
interaction between hIAPP and membrane. It contains a short sequence RLANFLV that fulfils the amino-
acid arrangement of the inversed cholesterol recognition amino-acid consensus (CARC) and may
mediate a direct contact of hIAPP with cholesterol. In this study, we focused on the interaction of
hIAPP1–19 with the lipid membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
and cholesterol, and examined the role of the CARC motif in the peptide–membrane interaction. Using
31 1
differential scanning calorimetry, P-NMR spectroscopy, H-NMR titration measurement and dye
leakage assay, we demonstrated that hIAPP1–19 interacts with DPPC vesicles more strongly in the
presence of cholesterol than it does in the absence of cholesterol. The peptide–membrane interaction
promotes the domain segregation of the raft-containing membrane. The peptide is more disruptive to
the cholesterol-containing membrane than it is to the cholesterol-depleted membrane. The substitution
of the residue Phe at position 15 of hIAPP1–19 by Leu leads to a distinct decrease in the peptide–
membrane interaction in the presence of cholesterol, but the effect of the residue substitution on the
peptide–membrane interaction is very small in the absence of cholesterol. The circular dichroism data
indicated that a conversion of the structure from a random coil to an a-helix is induced by cholesterol
for both peptides and the structural conversion is more Chol-dependent for the wild-type peptide than
the F15L variant. Our findings suggest that cholesterol could facilitate the insertion and aggregation of
the N-terminal domain of hIAPP in the membrane, and the phenylalanine in the CARC motif could be
involved in the interaction of the N-terminal domain with Chol.

amyloidogenesis are controversial.11 Lines of evidence support

Introduction
Human islet amyloid polypeptide (hIAPP), also called amylin, is
a neuropancreatic hormone composed of 37 amino acids.1,2 Its
amyloid deposit has been found in the islets of Langerhans of
the pancreas of patients affected by Type 2 Diabetes Mellitus
(T2DM).2,3 Although the exact mechanism of T2DM is unknown,
hIAPP has been linked to the damage and actual death of b-cells
in islets.4–6 The interactions between hIAPP and membranes
may be a key process in the toxicity of the amyloid peptide to the
cells.
Cholesterol (Chol) is one of the important compositions in
the cellular membranes concerning neurodegenerative
diseases.7–10 However, the effects of Chol on the
a
State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
2699 Qianjin Avenue, Changchun 130012, P. R. China. E-mail: feili@jlu.edu.cn;
Fax: +86-431-85193421; Tel: +86-431-85168548
b
Key Laboratory for Molecular Enzymology & Engineering, The Ministry of Education,
Jilin University, Changchun 130012, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra19714k
the promotion role of Chol in the polymerization of amyloid
peptides,12,13 while some studies demonstrate the inhibition
effect of Chol on the amyloid brillation.14–17 Chol modulates
brillogenesis either by a direct interaction with protein or by
mediating the interactions of protein with other lipid compo-
nents (e.g. GM1).11
The molecular mechanisms that control the binding of Chol
to proteins have been studied extensively. An important cate-
gory of Chol-associated proteins include one or more specic
Chol-binding motif(s). So far two types of Chol-binding motifs
are identied. One is referred as CRAC (cholesterol recognition
amino-acid consensus) fullling an algorithm of (L/V)–X1–5–(Y)–
X1–5–(K/R), another is the reversed version of CRAC, named
CARC, fullling an algorithm of (K/R)–X1–5–(Y/F)–X1–5–(L/V).18–21
The CRAC and CARC motifs have been found in many trans-
membrane proteins. In most cases, the central aromatic resi-
dues of CRAC and CARC appear to play a key role in the protein–
Chol interactions, driven by the CH–p interaction.22 Another
main category of Chol-associated proteins are found in a broad
range of viruses and amyloid proteins.23–28 These proteins
include peptide domains that adopt a tilted orientation when

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they insert in the membrane. Tilted peptides interact with Chol
through a geometric complementarity between the apolar
domain of cholesterol and the aliphatic residues of the peptide
without a requirement of a consensus amino acid motif.18
The N-terminal domain of hIAPP from residues 1 to 19
(hIAPP1–19) is believed to be essential for the binding of hIAPP to
membranes.29–34 Unlike the full length peptide, hIAPP1–19 is
weakly brillogenic whether in solution or in the presence of
membranes.31,32 Nevertheless, previous studies have shown that
hIAPP1–19 is toxic to both articial membranes and islet
cells.32,35 The N-terminal region of IAPP adopts an a-helical
structure in the membranes containing anionic lipids36,37 and
a transmembrane topology,38 and is involved in the early stages
of oligomerization of the peptide.39
Notably, the N-terminal region of hIAPP contains a triad of
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amino-acid residues composed of R11, F15 and V17 that
constitutes a CARC-like motif. In previous studies, more
attentions have been attracted to the aromatic residue F15, and
the inuences of the residue on the membrane binding and
self-assembly of various hIAPP-derived peptides have been
explored. The results from the study of hIAPP12–18 showed that
the substitutions of F15 by A and L increase the peptide–
membrane interaction and affect the aggregation kinetics, but
not affect the amyloid formation of the peptide.40 Tu and
Raleigh also found the effects of the substitutions at F15 on the
brillation rate of hIAPP. They related the effects of different
F15 substituents on the brillation rate to the a-helical
propensity of the single site substituted hIAPP.41 Wiltzius and
coworkers reported that hIAPP forms a dimer at the early stage
of polymerization through the interaction of the helices at
residues 8–18 with each other and the aromatic rings of two F15
make an important contact in the dimer formation.42 The N-
terminal domain of hIAPP is also found to bind insulin by the
interaction between the 7–19 region of hIAPP and the 9–20
region of insulin, and it is suggested that the hIAPP–insulin
binding is mediated by the recognition of the aromatic amino
acids Phe15 of hIAPP and Tyr16 of insulin.43 Despite having
been extensively studied, the interaction of the N-terminal
region of IAPP with the membrane in the presence of Chol
and the role of the CARC or F15 in the peptide–membrane
interaction are unclear. Because of the importance of both the
N-terminal region of hIAPP and Chol in the peptide–membrane
binding and the amyloidogenesis of hIAPP, elucidating how the
N-terminal region of hIAPP interacts with the membrane in the
presence of Chol and the effects of the peptide–membrane
interaction on the phase behavior of the membrane and the
structure of the peptide are signicant for disclosure of the
mechanism underlying the membrane disruption by hIAPP.
In this study, we used DPPC as a model membrane to
examine the interactions of hIAPP1–19 and its F15L variant
hIAPP1–19/F15L with the neutral membrane in the absence and
presence of Chol. By measuring the binding affinities of the
peptides for the membrane, the phase behaviors of the
membrane and the conformational conversion of the peptides,
we revealed that Chol plays an important role in the binding of
hIAPP1–19 to the membrane. By mediating the insertion in the
membrane, the formation of the a-helical conformation, and
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likely the aggregation, Chol enhances the performance of the N-
terminal peptide of hIAPP in the disruption of the membrane.
Our results showed that the phenylalanine in the CARC motif is
involved in the interaction between hIAPP1–19 and Chol.
Results
Effects of Chol on the peptide–membrane interactions
31
P-NMR measurements of DPPC SUVs (small unilamellar lipid
vesicles) alone and DPPC SUVs containing 15% and 20% Chol
were performed at a lipid-to-peptide ratio (L/P) of 20 : 1 in Tris–
HCl buffer (pH 7.4). A single peak from the resonance of the
phosphorus atom on the head-groups of the lipid vesicles was
observed in the spectra. Compared with the 31P-NMR spectrum
of DPPC vesicles alone, the signal of DPPC vesicles containing
Chol was broadened with increasing Chol concentration
(Fig. 1A). When hIAPP1–19 was incorporated into the vesicles,
the signal of DPPC vesicles was broadened further either in the
absence or in the presence of Chol. However, the signal
broadening induced by the peptide was more pronounced in
the presence of Chol than that in the absence of Chol. The
signal even disappeared at a 20% Chol concentration (Fig. 1B).
The incorporation of hIAPP1–19/F15L into DPPC SUVs also
resulted in a broadening of the 31P-NMR signals both in the
absence and presence of Chol (Fig. 1C). However, the effects of
the hIAPP1–19/F15L on the signal of DPPC vesicles were obviously
smaller than those of hIAPP1–19 in the presence of Chol, while
the effect of the variant on the signal of DPPC vesicles was
similar to that of the wild-type peptide in the absence of Chol,
suggesting that there may be an interaction between hIAPP1–19
and Chol and F15 may play a role in the peptide–Chol
interaction.
The observation of the difference in the interactions of
hIAPP1–19 and hIAPP1–19/F15L with the Chol-containing DPPC
membrane in the 31P-NMR measurements impelled us to
explore the role of Chol in the peptide–membrane interactions
further. For this purpose, we performed the 1H-NMR titration
experiments and evaluated the peptide–membrane binding
affinity. The 1H-NMR signals of the peptides broadened grad-
ually with the increase in the lipid-to-peptide ratio. The
Fig. 1 31P-NMR spectra of DPPC SUVs containing various percentages
of Chol in the absence (A) and presence of hIAPP1–19 (B) and
hIAPP1–19/F15L (C) measured in Tris–HCl buffer at pH 7.4, 50
C.
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intensities of the proton signals at the resonances of Hd on Table 1 Dissociation constants (Kd) of the interactions of hIAPP1–19
His18, Hb on Asn14 and Hh on Arg11 were used to calculate the and hIAPP1–19/F15L with DPPC SUVs containing various percentages of
Chol in Tris–HCl buffer at pH 7.4, 50
C
dissociation constants (ESI†). The dependences of the intensi-
ties (I/I0) upon the lipid-to-peptide ratios at various concentra- Peptide Chol (%) Kd (mM)
tions of Chol were plotted and the dissociation constants (Kd) of
the peptide–membrane binding were obtained by the curve hIAPP1–19 0 8.12  0.18
tting (Fig. 2 and Table 1). The data in Fig. 2C and Table 1 5 2.16  0.25
10 0.41  0.02
showed that the dissociation constants decrease with 15 0.21  0.11
increasing Chol concentration for the two peptides, and the Kd 20 0.15  0.07
values of hIAPP1–19 are smaller than those of hIAPP1–19/F15L at 30 0.034  0.0011
the same Chol concentrations. In contrast, the Kd values of the hIAPP1–19/F15L 0 8.62  0.37
two peptides in the absence of Chol are very close. This indi- 5 5.23  0.43
10 2.06  0.95
cates that the differences in Kd values of different peptides arise 15 0.66  0.13
mainly from the interactions between the peptides and Chol, 20 0.37  0.06
but not the interactions between the peptides and DPPC 0.088  0.0013
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component. The 1H-NMR results further conrm that Phe15
has a contribution to hIAPP1–19–Chol interaction.
It should be noted that all the Kd values of F15L variant ob-
tained at various Chol concentrations are smaller than that of
the peptide interacting with the Chol-depleted membrane, even
though the direct interaction between the peptide and Chol
30
could be reduced or eliminated due to the absence of the
aromatic residue. This suggests that in addition to the direct
contact, Chol could affect the interaction of hIAPP1–19 with the
membrane by other mechanism.

Fig. 2 (A and B) Dependences of the 1H-NMR intensities of the peptides on the lipid-to-peptide ratios at various percentages of Chol: (A) hIAPP1–19
and (B) hIAPP1–19/F15L. (C) The logarithm values of the dissociation constants Kd of the peptides at various Chol concentrations obtained by the fitting
of the curves in A and B. The data were obtained in Tris–HCl buffer of the DPPC/Chol SUVs incorporated with the peptides at pH 7.4, 50
C.

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Fig. 3 DSC thermograms of DPPC vesicles alone and DPPC vesicles containing various quantities of Chol in the absence (A) and presence of
hIAPP1–19 (B) and hIAPP1–19/F15L (C).

Effects of the peptides on the domain segregation of the Chol-
containing DPPC membrane
The thermotropic phase behaviors of DPPC SUVs alone and
DPPC SUVs mixed with 15% and 20% Chol in PBS buffer at pH
7.4 were examined by DSC measurements. As described in
previous study,44–46 the thermogram of DPPC SUVs alone dis-
played a pre-transition from a lamellar gel phase (Lb0 ) to
a rippled gel phase (Pb0 ) at 34.5
C and a main transition from
a rippled gel phase to a liquid-crystalline phase (La) at 41.6
C.
In the presence of Chol, the pre-transition was eliminated,
while the main endothermic transition of DPPC was broadened
dramatically. Moreover, an asymmetric prole consisting of two
overlapping transitions was observed in the presence of 15%
Chol. A deconvolution treatment to the asymmetric prole
showed a sharper peak at a lower transition temperature and
a broader one at a higher transition temperature. The sharper
one was assigned to the melting of Chol-poor domain and
another to the melting of Chol-rich domain (Fig. 3A and Table
2). In the presence of 20% Chol, the domain segregation was not
clearly observed. Nevertheless, we deconvoluted the DSC
endotherm prole using two-component tting and obtained
the thermodynamic parameters as listed in Table 2.
When hIAPP1–19 was incorporated with DPPC vesicles
(Fig. 3B), the pre-transition temperature Tp was decreased by 4.8


C and the main transition temperature Tm was decreased by
1.3
C along with the decreases in the cooperativity (DT1/2) and
the melting enthalpy change (DH). This indicates that hIAPP1–19
interacts with DPPC vesicles, by which the lipid chain packing is
disturbed. However, when hIAPP1–19 was incorporated with the
DPPC vesicles containing 15% and 20% Chol, more distinct
separation of the main transition peaks was observed in the
DSC proles (Fig. 3B) compared with the thermogram of DPPC/

Table 2 The phase transition data of DPPC vesicles mixed with different percentages of Chol in the absence and presence of hIAPP1–19 and
hIAPP1–19/F15L

Chol-poor domain Chol-rich domain

DH DH DHt
System Chol (%) Tm (
C) DT1/2 (
C) (kcal mol1) Tm (
C) DT1/2 (
C) (kcal mol1) (kcal mol1) DTm (
C)

DPPC/Chol 0 41.6 0.97 6.1 6.1 0

15 40.1 2.75 2.6 41.9 4.40 1.5 4.1 1.8
20 40.4 5.77 2.5 42.9 5.70 0.9 3.4 2.6
DPPC/Chol/hIAPP1–19 0 40.3 1.28 5.7 5.7 0
15 39.6 1.91 2.2 42.2 4.44 2.8 5.0 2.6
20 38.3 3.15 1.3 41.4 4.41 2.2 3.5 3.1
DPPC/Chol/hIAPP1–19/F15L 0 41.0 1.86 5.5 5.5 0
15 38.7 1.60 2.0 40.9 5.18 2.5 4.5 2.2
20 38.6 5.45 1.7 41.5 7.12 1.3 3.0 2.9

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Fig. 4 CD spectra of hIAPP1–19 (A) and hIAPP1–19/F15L (C) in PBS and hIAPP1–19 (B) and hIAPP1–19/F15L (D) in DPPC vesicles with various percentages
of Chol.
Chol system without the peptide (Fig. 3A). A deconvolution
treatment revealed that the differences between the main
transition temperatures (DTm) of the Chol-poor and Chol-rich
domains are 2.6
C and 3.1
C in the presence of 15% and
20% Chol, respectively, which are larger than those of DPPC/
Chol systems without the peptide (1.8 and 2.6, respectively,
see Table 2). Moreover, the incorporation of hIAPP1–19 with
DPPC/Chol membrane led to a decrease in the enthalpy change
of the gel to liquid-crystalline phase transition in the Chol-poor
domain and an increase in the enthalpy change in the Chol-rich
domain compared with the results of the Chol/DPPC systems.
This suggests that the incorporation of hIAPP1–19 with the lipid
bilayers may induce a redistribution of Chol by mediating more
Chol molecules clustering in the Chol-rich domain, and/or
induce a more intensive perturbation to the lipid packing in
the Chol-poor domain than it does to the Chol-rich domain by
selectively interacting with specic regions of the ra-
containing membrane.
When hIAPP1–19/F15L was incorporated with DPPC vesicles,
the main transition peak was broadened and shied to a lower
Tm by 0.6
C along with a decrease in the enthalpy change, while
the pre-transition temperature was unexpectedly increased by
about 1.3
C (Fig. 3C). The change in Tp may be associated with
a change in the ripple periodicity or a change in the membrane
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curvature induced by the peptide.35 The incorporation of
hIAPP1–19/F15L with lipid membrane may decrease the ripple
periodicity by inserting in membrane at a proper position, most
likely at the position between the head-groups and the aliphatic
chains. In the presence of Chol, the effects of hIAPP1–19/F15L on
the proles of the main transitions were small. No obvious
differences between the thermograms of the peptide-free and
peptide-containing vesicles were observed both at 15% and 20%
Chol. However, a deconvolution by a two-peak tting revealed
a peak separation with DTm of 2.2
C (15% Chol) and 2.9
C
(20% Chol) that are smaller than those of DPPC/Chol/hIAPP1–19
systems, but larger than those of DPPC/Chol systems. A
decrease in DH of the Chol-poor domain and an increase in DH
of the Chol-rich domain compared with the data of DPPC/Chol
vesicles were also observed in the DPPC/Chol/hIAPP1–19/F15L
systems. The results suggest that hIAPP1–19/F15L disturbs the
distribution of Chol and/or affects the packing of the DPPC/
Chol membrane less severely than hIAPP1–19 does.
Effects of Chol on the secondary structures of the peptides
Both hIAPP1–19 and hIAPP1–19/F15L were unstructured in bulk
solution, as shown in the CD spectra (Fig. 4A and C). Similar
results were also obtained for the peptides incorporated with
the Chol-depleted DPPC vesicles. However, when the peptides
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Table 3 The secondary structure data of hIAPP1–19 and hIAPP1–19/F15L

incorporated with DPPC SUVs containing various percentages of Chol
in PBS at pH 7.4

Secondary structure (%)

Peptide Chol (%) Helix Strand Turn Unordered

hIAPP1–19 0 18.7 18.2 14.7 48.4

15 24.4 17.4 16.3 41.9
20 46.4 16.2 17.1 20.3
30 51.9 15.5 14.5 18.1
hIAPP1–19/F15L 0 18.9 19.5 13.8 47.8
15 24.0 18.9 15.8 41.3
20 40.7 15.6 18.2 25.5
30 45.8 16.1 15.3 22.8
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were incorporated with the Chol-containing DPPC vesicles,

a conversion from a random structure to an a-helical structure
was observed in the CD spectra of both peptides (Fig. 4B and D).
The data obtained by the secondary structure analysis showed
that the content of the a-helical structure increases with
increasing percentage of Chol, and the increase in the helicity is
dramatic from 15% Chol to 20% Chol (Table 3). This suggests
that Chol facilitates the formation of a-helix for both peptides.
Chol-induced formation of a-helical structure is also observed
for Ab in model membranes.14 Compared with the results of
hIAPP1–19, the a-helix contents of hIAPP1–19/F15L were lower at
20% and 30% Chol, implying that the variant inserts in the
membrane less deeply and/or aggregates less effectively than Fig. 5 Kinetics of the membrane permeabilization induced by hIAPP1–19
the wild-type peptide in the presence of higher percentages of (A) and hIAPP1–19/F15L (B). The peptides of 75 mM were added to calcein
Chol. containing DPPC/Chol LUVs at the lipid-to-peptide ratio of 20 : 1.

Effect of Chol on the peptide-induced membrane disruption

Previous studies have shown that hIAPP1–19 can disrupt POPG
vesicles to a similar extent as full-length IAPP without forming
amyloid bers.32,35 In order to gain insight into the role of Chol
in the membrane disruption and the effect of F15L on the
activity of the peptide in disrupting membrane, we performed
the dye leakage assays of DPPC LUVs (large unilamellar lipid
vesicles) in the absence and presence of Chol (Fig. 5). The
results demonstrated that both hIAPP1–19 and hIAPP1–19/F15L
can disrupt the membranes consisting of DPPC alone and
DPPC/Chol mixture. However, the performances of the
peptides in the membrane disruption were distinct in the
presence and absence of Chol. The peptides induced more
amount of dye leakage in the presence of Chol, particularly in
the presence of 20% Chol, than they did in the absence of
Chol, suggesting a promotion role of Chol in the peptide–lipid
interaction and the activities of the peptides in disrupting the
lipid membranes.
Moreover, compared with the wild-type peptide, the F15L
mutant was less efficient in inducing dye leakage through the
membrane. Lines of evidence from previous studies have shown
that amyloid peptides disrupt the membrane by their toxic
oligomers. Therefore, our results could suggest that Chol
promotes the formation of toxic oligomers more efficiently for
the wild-type peptide than for the F15L variant.
Discussion
The interactions of the amyloid proteins/peptides with
membranes play an important role in the formation of amyloid
brils and lead to membrane damage.47,48 The insertion of the
N-terminal region in membrane, most likely as a monomer, is
the initial step of hIAPP–membrane interaction.30 The N-
terminal region of hIAPP is also involved in the self-
association of hIAPP.39 Because the 1–19 region of hIAPP has
a weak brillogenic propensity either in solution or at
membranes and disrupts lipid membranes similarly to full-
length hIAPP, the peptide fragment has been used as a struc-
ture mode to investigate the early aggregation and membrane-
disrupting mechanism of hIAPP.37 Interestingly, hIAPP1–19
includes a sequence fragment RLANFLV in which the arrange-
ment of amino-acid residues R11, F15 and V17 is consistent
with the CARC motif. Therefore, the N-terminal fragment of
hIAPP may be also involved in the interaction of the peptide
with Chol. In this study, we examined the interactions of
hIAPP1–19 with the DPPC membrane in the absence and pres-
ence of Chol in order to gain an insight into the mechanisms
underlying the effects of Chol on the binding and disruptive
activity of hIAPP1–19 to the membranes. The role of CARC in the
peptide–membrane interaction was also explored by the

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substitution of the aromatic residue in CARC by the aliphatic
residue leucine.
Our results showed that the binding affinity of hIAPP1–19 for
the DPPC membrane is very weak (8.12  103 M), but it
increases with the increase in the concentration of Chol (e.g., 3.4
 105 M at 30% Chol). Chol concentration also facilitates the
formation of a-helical structure of the peptide, while in the Chol-
depleted DPPC membrane, the peptide is unstructured. The
structural conversion of the peptide induced by Chol could be
elucidated by the topological change of the peptide from lying at
membrane surface to inserting into the hydrophobic region of
the membrane. In the absence of Chol, the peptide could bind to
the surface of the neutral membrane with a lower affinity, which
could not induce a dened secondary structure. The presence of
Chol leads to the formation of micro-domains in the DPPC
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membrane, which could facilitate the insertion of hIAPP1–19 in
the membrane deeply. Chol is also an important factor affecting
the membrane insertion and secondary structure of b-amyloid
peptide (Ab 1–40).14 With the contrary of our result, the study of L
Caillon and coworkers demonstrated that Chol does not have any
signicant effect on the binding of full length hIAPP to DOPC
vesicles (the binding affinities are 1.4  103 M and 2.9  103
M, respectively, in the absence and presence of 30% Chol).49 The
difference in the Chol effect could be partly associated with
a stronger aggregation propensity of full length hIAPP than
hIAPP1–19 fragment. The pre-oligomerization of full length hIAPP
may occur before binding to the membrane, which may impair
the interaction of the N-terminal fragment with Chol.
The DSC data demonstrated that the domain segregation
associated with Chol heterogeneous distribution in the
membrane is enhanced instead of being reduced by the incor-
poration of hIAPP1–19. This indicates different interactions of
hIAPP1–19 with membrane components or domains. The
incorporation of hIAPP1–19 with the DPPC membrane contain-
ing Chol resulted in a distinct decrease in the melting enthalpy
change of the Chol-poor domain and an increase in the melting
enthalpy change of the Chol-rich domain, suggesting that the
peptide preferentially partitions into the Chol-poor domain of
the membrane, alternatively, the peptide renders further clus-
tering of Chol in the membrane. R. Winter and coworkers have
evidenced the preferential partitioning of IAPP into the uid
lipid phase (Chol-poor domain) using the membrane systems of
both giant unilamellar lipid vesicles (GUVs) of
DOPC : DPPC : Chol 1 : 2 : 1 and pancreatic INS-1E b-cells.50,51
They suggested that IAPP is mostly absorbed at the rim of the
domains. Both the experimental and molecular dynamics
simulation results demonstrate that the addition of Chol in PC
membranes leads to an increase in the membrane thick-
ness.52,53 The enrichment of Chol in ra domains leads to
differences in the thickness of the Chol-rich (lipid-ordered lo)
domains compared with the Chol-poor (lipid-disordered ld)
domains in the bilayers.54 As a result, there is a signicant strain
on the boundary between the lo and ld domains, and this
boundary region is more vulnerable to membrane disrupting
agents.55 The binding in the boundary diminishes energetic
costs and relieves the associated tension.54 Therefore, the
interaction of hIAPP1–19 with the heterogeneous DPPC
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membrane may be explained by the preferential insertion of the
peptide in the border between the Chol-poor and Chol-rich
domains, where Chol promotes the peptide–membrane inter-
action likely by hydrophobic effects.39,53 The existence of ra
induces a curvature strain on membrane, which could also
facilitate the binding of peptide to membrane.56
Previous study has showed that Chol stimulates the aggrega-
tion of hIAPP into compact 200–500 nm clusters both in the
neutral and anionic lipid membranes, while hIAPP forms pore-
like globular oligomers with 25–35 nm in size in the membrane
without Chol.47 The Chol-induced clustering of peptide could also
occur in the DPPC/Chol system of hIAPP1–19. By promoting the
formation of toxic oligomers of the peptide, Chol renders the
peptide more disruptive to the membrane. Our DSC results
showed that the peptide mainly affects the packing of the Chol-
poor domains, but less affects the packing of the Chol-rich
domains, suggesting that the peptide could accumulate predom-
inantly at the side of the Chol-poor domain of the boundary, but
not the side of the ra. The preferential interaction of the peptide
with specic regions of the membrane could further restrict the
exibility of the peptide and therefore facilitate the clustering of
the peptide (Fig. 6). The results of the dye leakage assays showed
that the amount of dye leakage increased with increasing Chol
percentage in the DPPC LUVs. This may be attributed to the
increases both in the amount of toxic oligomers and in the length
of the boundary regions at the higher cholesterol concentration.
The direct interaction between hIAPP1–19 and Chol could
occur when the peptide accumulates at the border of the
domains. The CARC motif RLANFLV corresponding to the N-
terminal 11–17 region of hIAPP may be involved in the
peptide–Chol interaction. The in silico studies revealed that
Chol binds to CARC motif via the b-face, leaving the a-face
exposed.57 The contacts between the a-face derived by the van
der Waals interaction may mediate the clustering of Chol to
form micro-domain in lipid membrane. The self-recognition
properties of Chol in turn favor the oligomerization of the
CARC-containing peptide. The residue Phe at position 15 is one
of a triad of essential amino acid residues in the CARC motif.
Our study indicated that compared with the wild-type hIAPP1–19,
Fig. 6 Schematic depiction of the mode of the interaction between
hIAPP1–19 and DPPC membrane in the absence and presence of Chol.
RSC Adv., 2016, 6, 96837–96846 | 96843

RSC Advances
the F15L variant has a distinctly lower binding affinity for the
Chol-containing DPPC membrane, whereas the binding affini-
ties of the two peptides for the Chol-depleted DPPC membrane
are very similar. This suggests that Phe15 may play a role in the
interaction of hIAPP1–19 with Chol. The F15L substitution may
also decrease the effect of the N-terminal peptide on the lipid-
based segregation of Chol and the formation of a-helical
structure of the peptide in the Chol-containing membrane. The
membrane damage induced by hIAPP1–19 is also reduced by the
substitution, even though the extent of the effect seems small.
These changes in the performance of the peptide disturbing the
membrane property and in the secondary structure of the
peptide derived by the F15L substitution may be ascribed partly
to the changes in the peptide–Chol interaction.
In the interaction of the peptide with Chol, the aromatic
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residue in the CARC motif stacks classically onto one of the
sterane rings of Chol by CH–p interaction.58 The branched
aliphatic residues Leu or Val are required by the need to
accommodate the crevices and asperities of the Chol molecules
by numerous van der Waals contacts between these residues and
Chol.58 The presence of the basic residue Lys or Arg is crucial for
accommodating the OH group of Chol at the membrane surface,
even if there is no direct interaction between Chol and K/R.
Because the CARC motif is typically involved in the Chol-
binding sites of the transmembrane domains that are mainly
a-helical,18 the presence of the CARC motif in hIAPP may imply
the formation of the a-helix conformation at least in the region of
11–17 of hIAPP1–19 and an insertion of hIAPP1–19 in the
membrane with the cationic group emerging at the membrane
surface and the aromatic and hydrophobic residues contact with
the apolar zone of Chol. The NMR data obtained in the presence
of DPC-micelles revealed that residue 7–17 in hIAPP1–19 is
involved in an a-helix at a neutral pH and buried in the micelles
deeply.38 Our CD results showed that the percentage of the a-
helical structure in hIAPP1–19 is about 52% in the presence of
30% Chol in the DPPC membrane and the content is smaller at
a lower concentration of Chol. This could suggest that the CARC
motif is involved in the a-helical structure in the Chol-containing
DPPC membrane, considering that the disulde bond between
C2 and C7 prevents the formation of a-helical structure in the N-
terminal region of hIAPP1–19. The absence of the aromatic ring in
hIAPP1–19/F15L decreases the helical content and membrane
insertion of the peptide likely by the decrease or even the elimi-
nation of the direct peptide–Chol interaction, leading to the
decrease in the effect of the peptide on the membrane.
Experimental section
Materials
The peptides corresponding to the residues from 1 to 19 of
hIAPP (hIAPP1–19) and those with a substitution of phenylala-
nine at position 15 by leucine (hIAPP1–19/F15L) were synthesized
by Shanghai Science Peptide Biological Technology Co., Ltd
(Shanghai, China). The cysteines at positions 2 and 7 in both
peptides were oxidized to form a disulde bond and the C-
termini of the peptides were amidated. The purity of the
peptides was assessed to be higher than 95% by high-
96844 | RSC Adv., 2016, 6, 96837–96846
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Paper
performance liquid chromatography and mass spectroscopy.
Lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was
purchased from Avanti Polar Lipid, Inc. (Alabaster, AL). Other
chemical agents were obtained from Sigma-Aldrich (St. Louis,
MO). All chemical agents were used directly without further
purication.
Preparation of small unilamellar lipid vesicles (SUVs) and
large unilamellar lipid vesicles (LUVs)
DPPC and Chol powders were separately dissolved in 200 mL
chloroform/methanol (2 : 1 v/v) co-solvent. Appropriate volume of
DPPC solution was mixed with certain volume of Chol solution
according to desired percentage of Chol in total lipids. The
solution mixture was evaporated by a stream of nitrogen, and the
resulting lipid lm was kept under vacuum overnight to remove
residual organic solvents. The dried lipid lm was hydrated with
buffer solution and vortexed for several seconds. The vesicle
suspension was sonicated for 1 h at 10
C above the phase tran-
sition temperature of the lipids, by which the SUVs were obtained.
The liposome samples were used immediately aer preparation.
To study the dye leakage of membrane, we prepared LUVs
containing calcein. The dried lipid lm was hydrated with 1 mL
Tris–HCl buffer containing 70 mM calcein. To increase solu-
bility of calcein, 5 M NaOH was added until the solution turned
transparent. The solution was incubated for 1 h at 50
C. Then
the solution was freeze-thawed 5 times and extruded through
polycarbonate lter (0.1 mm pore size) for 10 cycles. To eliminate
the nonencapsulated calcein, the LUVs solution was dialyzed in
Tris–HCl buffer over night through a membrane with a cut-off
of 1000 Da.
Preparation of peptide-SUVs samples
Peptide was dissolved in 1,1,1,3,3,3-hexauoro-2-propanol
(HFIP) at a concentration of 1 mg mL1. The solution was
sonicated in water bath for 15 min to break up any preformed
aggregates of peptide. An appropriate volume of peptide stock
solution was added in a mixture of DPPC and Chol that were
dissolved previously in a chloroform/methanol (2 : 1 v/v) co-
solvent at a certain percentage of Chol. The solution was
further treated following the method described above in the
preparation of SUVs. By this method, the samples of peptide
incorporated with SUVs in buffer solution were obtained.
Differential scanning calorimetry (DSC) measurements
DSC scans were carried out using a Microcal VP-DSC calorim-
eter (MicroCal, Inc., Northampton, MA). Both the sample and
reference were degassed by vacuum for 10 min and heated from
20 to 60
C with a heating rate of 0.5
C min1. To ensure the
reproducibility and accuracy of the experiments, three inde-
pendent samples were measured. The results were analyzed
aer buffer subtraction and baseline correction using Microcal
Origin soware. The SUV samples of 1.5 mM DPPC/Chol mixed
with 75 mM peptide (L/P ¼ 20 : 1) dissolved in 25 mM phosphate
buffer (pH 7.4) containing 50 mM NaCl were used. The mole
percentages of Chol composition in DPPC/Chol mixtures were
varied from 0, 15% to 20%.
This journal is © The Royal Society of Chemistry 2016

Paper
NMR spectroscopy
1 31
H- and P-NMR spectra of the peptide/SUV samples were
performed on a Bruker Avance 600 spectrometer (Bruker Bio-
Spin, Fällanden, Switzerland) at 50
C. The SUV samples of
DPPC/Chol and DPPC/Chol/peptide (L/P ¼ 20 : 1) mixtures
dissolved in 10 mM Tris–HCl buffer (pH 7.4) containing 100
mM NaCl and 10% D2O were used in all NMR experiments. 31P
spectra were collected with scans of 2592 and a relaxation delay
of 1.5 s. The 31P chemical shi of H3PO4 was used as a reference
chemical shi. A total of 300 mM peptide and 6 mM lipids (DPPC
and Chol) were used in the 31P-NMR experiments.
1
H-NMR titration experiments of the peptide incorporated
with SUVs were performed at different Chol concentrations: 0,
5, 10, 15, 20 and 30 mol%. By increasing the concentration of
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lipid component from 0 to 15 mM at a xed Chol : DPPC ratio
and peptide concentration (300 mM), a series of 1H-NMR spectra
of peptide in DPPC/Chol SUVs with different lipid-to-peptide
ratios were obtained. All spectra were collected with scans of
512 and a relaxation delay of 3 s. DSS (2,2-dimethyl-2-sila-
pentane-5-sulfonate-d6) was used as an internal standard.
The intensities of the peptide signals were measured at
different L/P ratios and the dissociation constants (Kd) of the
peptide binding to SUVs were estimated by eqn (1) derived from
a simple bimolecular binding equilibrium:49,59
 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
0 1


2
Kd Kd
B þxþ1  þ x þ 1  4xC
B P0 P0
B
I ¼ I0 B1 
2 C
@ A
where I and I0 are the observed and maximum intensity of the
peptide 1H resonance signals, respectively, P0 is the concen-
tration of peptide, and x represents the lipid-to-peptide ratio.
Circular dichroism (CD) spectroscopy
CD measurements were performed on a PMS-450 spec-
tropolarimeter (Biologic, France) at room temperature. Samples
used in CD measurements were prepared similar to those used in
DSC measurements. A 25 mM phosphate buffer solution (pH 7.4)
without salt was used in the preparation of the CD samples. The
sample solutions were added into a quartz cuvette with 0.5 mm
path length. The spectra were scanned from 190 nm to 260 nm
with a step of 1 nm in an interval of 10 s. The background collected
from the peptide-free sample was subtracted. Each spectrum rep-
resented an average of three independent experiments and
a smoothing algorithm was used. The absorbance was expressed
as molar ellipticity (q) in a unit of deg cm2 dmol1. The concen-
trations of peptide and lipids were 75 mM and 1.5 mM, respectively.
The secondary structure contents were calculated by the
CDPro soware package using the program CONTIN/LL. A
reference set of SMP56 including 56 proteins was used in the
analyses of CD data.
Membrane leakage assays
The membrane leakage assays were performed on a uores-
cence spectrophotometer RF-5301 PC (Shimadzu, Japan) at
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View Article Online
RSC Advances
room temperature. The uorescence spectra were excitated at
495 nm with emission wavelength from 530 nm to 650 nm. The
spectra were scanned with an excitation slit of 3 nm and an
emission slit of 3 nm. Each spectrum was an average of three
time scans. The standard deviations were estimated based on
three separate measurements. The 100% leakage (I100) was ob-
tained by the calcein-LUVs with 0.2% Triton X-100.
The data was analyzed using eqn (2) (ref. 40, 60 and 61):
I  I0
% Dye leakage ¼ (2)
I100  I0
where I0 is the uorescence intensity of the calcein-LUVs
without peptide and I the observed uorescence intensity of
the calcein-LUVs in the presence of peptide.
Conclusion
Using the ra-containing DPPC/Chol vesicles as a model
membrane system, we detected the promotion effects of Chol
on the binding affinity of hIAPP1–19 for the membrane and on
the disruptive activity of the peptide to the membrane. By the
substitution of F to L at position 15, we revealed that the CARC
motif composed of a triad of amino acid residues R11, F15 and
V17 in hIAPP1–19 plays a role in the peptide–Chol interaction.
The peptide variant devoid of the aromatic residue that contacts
with the sterane rings of Chol by CH–p interaction binds to the
C ra-containing membrane more weakly and exerts a smaller
C (1)
effect on the Chol redistribution in the neutral membrane. In
addition to the direct interaction of the peptide with Chol, the
preferential insertion of the peptide in the boundary region of
the Chol-rich and Chol-poor domain in the membrane also
plays a role in the Chol-associated peptide–membrane interac-
tion. Chol induces the conversion of the secondary structure of
the peptide from a random coil to an a-helix. By exerting
specic conformational effects on the peptide in the
membrane, Chol could restrict the exibility of the peptide and
result in the formation of the toxic oligomers.
Acknowledgements
We thank Dr Chunyu Wang for the help in NMR experiments.
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