Transmembrane Self-Assembled Cyclic Peptide Nanotubes Based on α-Residues and Cyclic δ-Amino Acids: A Computational Study

T ansmemb ane Sel -Assembled
Cyclic Pep ide Nano ubes Based on
α-Residues and Cyclic δ-Amino Acids:
A Compu a ional S udy
Alexand e Blanco-González
1
,
2
, Ma ín Cal elo
1
, Pablo F. Ga ido
2
, Manuel Amo ín
1
,
Juan R. G anja
1
, Ángel Piñei o
2
and Rebeca Ga cia-Fandino
1
*
1
Depa amen o de Química O gánica, Cen e o Resea ch in Biological Chemis y and Molecula Ma e ials, Uni e sidade de
San iago de Compos ela, Campus Vida s/n, San iago de Compos ela, Spain,
2
Depa amen o de Física Aplicada, Facul ade de
Física, Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain
Sel -assembling cyclic pep ide nano ubes ha e been shown o unc ion as syn he ic,
in eg al ansmemb ane channels. The combina ion o na u al and nonna u al aminoacids
in he sequence o cyclic pep ides enables he con ol no only o hei ou e su ace bu
also o he inne ca i y beha io and p ope ies, a ec ing, o ins ance, hei pe meabili y o
di e en molecules including wa e and ions. He e, a ho ough compu a ional s udy on a
new class o sel -assembling pep ide mo i s, in which δ-aminocycloalkaneca boxylic acids
a e al e na ed wi h na u al α-amino acids, is p esen ed. The p esence o syn he ic
δ- esidues c ea es hyd ophobic egions in hese α,δ-SCPNs, which makes hem
especially a ac i e o hei po en ial implemen a ion in he design o new d ug o
diagnos ic agen ca ie sys ems. Using molecula dynamics simula ions, he beha io
o wa e molecules, di e en ions (Li
+
,Na
+
,K
+
,Cs
+
, and Ca
2+
), and hei co esponden
coun e Cl
−
anions is ex ensi ely in es iga ed in he nanoconfined en i onmen . The
s uc u e and dynamics a e mu ually combined in a di ing imme sion inside hese
ansmemb ane channels o disco e a ascina ing subma ine nanowo ld whe e s a -
shaped wa e channels guide he passage o ca ions and anions he e h ough.
Keywo ds: sel -assembling cyclic pep ide nano ubes, ansmemb ane channels, molecula dynamics simula ion,
ion anspo ac oss lipid memb ane, confined wa e
INTRODUCTION
The ansloca ion o di e en species be ween he compa men alized inne egions o li ing cells
and hei en i onmen plays a co e ole on hei iabili y and p obabili y o su i al. Uncon olled
exchange o ma e is equi alen o a dea h sen ence o he cell. The exquisi e con ol o na u e on
his dynamic equilib ium in biological ion channels and po es p o ides a sou ce o inspi a ion o
scien is s who use na u al s uc u es and unc ions as a e e ence o be mimicked and e en ually
imp o ed (Tagliazucchi and Szlei e , 2015;Bogdanowicz e al., 2016).
Nume ous sup amolecula s uc u es ha e been designed o eplica e he specific unc ions o
na u al anspo sys ems, in e ms o a fini y, e ficiency, s abili y, and selec i i y (Zheng e al., 2021).
Among o he examples, sel -assembling cyclic pep ide nano ubes (SCPNs) ha e eme ged as
a ac i e ansmemb ane channel mime ics due o hei cylinde shape and expec ed
biocompa ibili y (Ga cía-Fandiño e al., 2012b;Rod iguez-Vazquez e al., 2014;Ga cia-Fandiño
Edi ed by:
Huaqiang Zeng,
No hwes e n Poly echnical
Uni e si y, China
Re iewed by:
Debasis Manna,
Indian Ins i u e o Technology
Guwaha i, India
Zhiwei Liu,
Uni e si y o he Sciences,
Uni ed S a es
*Co espondence:
Rebeca Ga cia-Fandino
ebeca.ga cia. [email protected]
Special y sec ion:
This a icle was submi ed o
Sup amolecula Chemis y,
a sec ion o he jou nal
F on ie s in Chemis y
Recei ed: 01 May 2021
Accep ed: 23 June 2021
Published: 27 July 2021
Ci a ion:
Blanco-González A, Cal elo M,
Ga ido PF, Amo ín M, G anja JR,
Piñei o Á and Ga cia-Fandino R (2021)
T ansmemb ane Sel -Assembled
Cyclic Pep ide Nano ubes Based on
α-Residues and Cyclic δ-Amino Acids:
A Compu a ional S udy.
F on . Chem. 9:704160.
doi: 10.3389/ chem.2021.704160
F on ie s in Chemis y | www. on ie sin.o g July 2021 | Volume 9 | A icle 7041601
ORIGINAL RESEARCH
published: 27 July 2021
doi: 10.3389/ chem.2021.704160
e al., 2017;Cla o e al., 2018;Rod íguez-Vázquez e al., 2017).
These sys ems we e de ised in 1974, bu i was no un il 1993 ha
hey we e fi s syn hesized (De San is e al., 1974;Ghadi i e al.,
1993). The o iginal SCPNs we e comp ised o D- and L-α-amino
acids ha adop ed a fla con o ma ion in which he amide g oups
(NH and CO) lie pe pendicula o he plane o he ing
(Figu e 1A). (De San is e al., 1974;Bong e al., 2001) This
special a angemen allows he o ma ion o hyd ogen bonds
(H-bonds) be ween con iguous cyclic pep ides (CPs) owing o he
complemen a i y be ween H-bond dono and accep o g oups on
bo h sides o each disc s uc u e. Thus, upon CP s acking, he
in e io o he assembly emains emp y, wi h all he side chains
exposed on he ex e nal su ace o he cylind ical s uc u e. One
o he main ad an ages o hese od-shaped pep ide ma e ials is
he simplici y wi h which hei inne and ou e p ope ies can be
easily modula ed upon app op ia e selec ion o he CP sequence;
while he numbe and ype o amino acids de e mine he in e nal
diame e o he nano ube, he cha ac e is ics o i s ou e su ace
depend on he p ope ies o he side chains o he amino acids. In
his sense, he design o SCPNs ha in e ac wi h he hyd ophobic
pa o biomemb anes has been ca ied ou h ough he
app op ia e selec ion o amino acids in he basic pep ide
subuni (Ghadi i e al., 1994).
Mos SCPNs ha e a hyd ophilic inne ca i y, designed o allow
he anspo o hyd ophilic species (B ea e al., 2010). Howe e ,
since he syn hesis o he fi s D,L-α-SCPNs, SCPNs using CPs
composed by di e en ypes o nonna u al amino acids ha e been
de eloped, ob aining s uc u es wi h mo e specific beha io
(Mon eneg o e al., 2013). Fo example, β-SCPNs (Seebach
e al., 1997;Cla k e al., 1998a) a e cha ac e ized by lea ing all
he NH g oups in one side o he CPs and all he CO g oups on
he o he . The esul ing mac odipole leads o exo ic p ope ies
such as a significan piezoelec ici y (Taba a e al., 2019). Cyclic
γ-amino acids (cis-γ-aminocycloalkaneca boxylic acids, γ-Acas)
o app op ia e chi ali y, bo h alone (Chen e al., 2020)o
al e na ed wi h α-amino acids (α,γ-CPs), can also o m
nano ubes (α,γ-SCPNs) (Amo ín e al., 2003;Ga cia-Fandino
e al., 2012), whe e he β-ca bon o he cycloalkane moie y is
o ien ed owa d he lumen o he cylinde , influencing he
in e nal p ope ies o he nano ube and opening he possibili y
o inne unc ionaliza ion (Figu e 1A). The fi s s eps owa d he
p epa a ion and cha ac e iza ion o α,γ-SCPNs wi h chemically
unc ionalized lumens ha e al eady an icipa ed p omising esul s
in his a ea (Rei iz e al., 2009;Cal elo e al., 2015;Rod íguez-
Vázquez e al., 2017;Cal elo e al., 2019).
The beha io o wa e molecules and ions locked wi hin he
na ow confines o a subnanome ic hyd ophobic po e is e y
di e en om ha o he mac oscopic wo ld. Bo h in i o and in
silico s udies indica e ha nanoconfined wa e inside SCPNs
adop s a highly o de ed s uc u e and i shows a much slowe
ee di usion compa ed o i s bulk o m (G anja and Reza
Ghadi i, 1994;Cla k e al., 1998b;Sánchez-Quesada e al.,
FIGURE 1 | (A) Pep ide nano ube models o med by he s acking o cyclic pep ides o di e en ypes: (i) D,L-α-CP, (ii) CPs con aining γ-Acas (α,γ-CPs), and (iii) CPs
con aining δ-Acas (α,δ-CPs). (B) c-[(L-T p-δ-Ach-D-T p-δ-Ach-)
3
] CP used in his wo k. (C) Snapsho o he ini ial α,δ-SCPN s uc u e o he MD simula ion. Only he
phospho ous a oms o he lipids a e shown. (D) Final s uc u e ( 200 ns) o he blank simula ion (wi hou ions), whe e also he lipid chains and he wa e molecules
a e shown.
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
2001;Ga cía-Fandiño e al., 2009;Ga cía-Fandiño e al., 2012a;
Mon eneg o e al., 2013). This seems o be modula ed by se e al
ac o s such as he channel inne adius and i s hyd ophobici y
(Mon eneg o e al., 2013;Rod íguez-Vázquez e al., 2016). An
inc ease in he hyd ophobic cha ac e o he nano ube can be
achie ed using he ans-4-aminocyclohexaneca boxylic acid
(δ-Ach) as a building block. The esul ing nano ubes,
α,δ-SCPNs (Lamas e al., 2018), exhibi a mo e hyd ophobic
in e nal ca i y due o he p esence o wo me hylene g oups o
each cyclohexyl moie y o ien ed owa d he inne o he cylinde
(Figu e 1A). The p esence o hyd ophobic egions o
ansmemb ane channels has been ound o play an impo an
ole in con olling wa e and ion di usion (A yal e al., 2015;Rao
e al., 2018;Yazdani e al., 2020), making α,γ- and α,δ-SCPNs
especially a ac i e o hei po en ial implemen a ion in he
design o new d ug o diagnos ic agen ca ie sys ems. While
se e al s udies ha e been ca ied ou o cha ac e ize
ansmemb ane α,γ-SCPNs (Ga cía-Fandiño e al., 2012a;
Cal elo e al., 2015;Ga cia-Fandiño e al., 2016;Cal elo e al.,
2019), biomime ic po es based on α,δ-SCPNs emain mos ly
uncha ac e ized.
Since expe imen al measu emen abou confined molecule
p ope ies in he nanoscale is di ficul (Rod íguez-Vázquez
e al., 2017), molecula dynamics (MD) simula ions play a key
ole in he desc ip ion o hese sys ems (Lynch e al., 2020).
Recen s udies ca ied ou in ou g oup showed ha α,δ-SCPNs
a e s able and heo e ically able o ansloca e wa e and ca ions
when inse ed in o a lipid bilaye (Cal elo e al., 2021). Howe e ,
he beha io o he wa e molecules and he ions inside hese
s uc u es has no been deeply examined.
This s udy aims o explo e in de ail he in e nal p ope ies o
ansmemb ane α,δ-SCPN channels, e alua ing and ca e ully
in es iga ing he s uc u al and dynamical beha io o wa e ,
LiCl, KCl, NaCl, CsCl, and CaCl
2
inside his ype o nano ubes.
The confinemen e ec on he s uc u e and dynamics o wa e
and ions in he lumen o hese po es is ho oughly analyzed. The
simula ion esul s will p o ide new insigh s, use ul o a be e
design o cyclopep ide-based s uc u es, allowing mo e a ional
cons uc ions o imp o ing he ansmemb ane channel ac i i y.
MATERIALS AND METHODS
The α,δ-SCPN s udied in his wo k is composed o eigh CPs,
long enough o a e se a p e iously equilib a ed POPC
memb ane model (Cal elo e al., 2020). Each ing was made
up o six α-amino acids (3 L- and 3 D- esidues) and six δ-Acas
acid esidues {c-[(L-T p-δ-Ach-D-T p-δ-Ach-)
3
], Figu e 1B}.
The s a ing geome ies o he employed CPs we e aken om
p e ious wo ks (Cal elo e al., 2021), which sugges ed a p e e ed
pa allel disposi ion o he β-shee (Figu e 1C). A e nano ube
inse ion in o he POPC bilaye , he comple e sys em was
sol a ed. Wa e molecules in he hyd ophobic egion o he
ails and also inside he SCPN we e emo ed, so ha , in he
fi s s ep o he simula ion, he channel was comple ely d y
(Figu e 1D). The esul ing sys ems we e ionized using
di e en sal solu ions (LiCl, NaCl, KCl, CsCl, and CaCl
2
a
0.15 M, espec i ely). One ex a blank simula ion, o con ol, was
ca ied ou wi hou ions. Thus, a o al o six sys ems we e
p epa ed.
TIP3P (P ice and B ooks, 2004) was chosen as he wa e
model, and he pa ame e s de eloped by Joung e al. (Joung
and Chea ham, 2008) we e selec ed o desc ibe he ions Li
+
,Na
+
,
K
+
,Cs
+
,Ca
2+
, and Cl
−
, as hey a e known o sol e some
c ys alliza ion issues obse ed wi h classical pa ame e s a
ela i ely high concen a ions. Fo he s anda d esidues, he
AMBER99SB-ILDN o ce field pa ame e s we e chosen
(Lindo -La sen e al., 2010). Fo he nons anda d amino
acids, RESP/6-31G(d) cha ges we e de i ed, simila o hose
used in he de elopmen o AMBER o ce fields, and an de
Waals pa ame e s we e ob ained om he GAFF o ce field, all
aken om a p e ious wo k (Cal elo e al., 2021). The pa ame e s
o he lipid o choice, POPC, we e aken om Lipidbook (Klauda
e al., 2010;Jämbeck and Lyuba se , 2012a;Jämbeck and
Lyuba se , 2012b).
All simula ions we e pe o med wi h he GROMACS 2019.3
package (Ab aham e al., 2015). All sys ems we e fi s minimized,
ollowed by an un es ained p oduc ion un o 200 ns, wi h a ime
s ep o 2 s. No specific es ain s we e applied o he pep ides a
any s ep. An NPT ensemble was employed a 1 ba using a semi-
iso opic Pa inello–Rahman ba os a (Pa inello and Rahman,
1981) and a 300 K main ained wi h a V- escale he mos a (Bussi
e al., 2007). The LINCS algo i hm was employed o emo e all
bond ib a ions (Hess e al., 1997). Elec os a ic in e ac ions we e
calcula ed using he PME me hod wi h a cu o o 1.0 nm and a
g id spacing o 0.12 nm (Essmann e al., 1995). Van de Waals
in e ac ions we e calcula ed using a 1.0 nm adius cu o .
The da a ob ained om he MD simula ions we e ea ed and
analyzed wi h GROMACS and specific codes de eloped in
Py hon. MDAnalysis (Michaud-Ag awal e al., 2011;Gowe s
e al., 2016) was employed o p ep ocess he ajec o ies, o
calcula e he wa e and ion su i al p obabili y [P(τ)] in he
SCPN, and he adial dis ibu ion unc ions [g( )]. NumPy
(Ha is e al., 2020) and Pandas (Mckinney, 2010) we e also
used o ea and o ganize da a, and Ma plo lib (Hun e , 2007)
was used o g aphical ep esen a ions. The snapsho s and
anima ions om ajec o ies we e made using VMD
(Humph ey e al., 1996). g( ) was employed as implemen ed
in MDAnalysis, while P(τ) was ob ained om he ollowing
equa ion:
P(τ)1
T
T
1
N( , +τ)
N( ),
whe e Tis he maximum ime o he simula ion, τis he ime
pe iod used as a pa ame e o he su i al p obabili y (a ime
window a e which he p obabili y o a molecule o emain in he
ca i y is de e mined), N( )is he numbe o molecules inside he
α,δ-SCPN a he ime , and N( , +τ)is he emaining numbe
o hose molecules a o τ. F om his ela ion, he esidence hal -
li e ime (τ1/2) can be es ima ed as he alue o τa P(τ)0.5.
The a e age eloci y o wa e s and ions o e ime windows o
10 ps, along he whole ajec o ies (disca ding he fi s 20 ns), was
de e mined om he coo dina es o he di e en species be ween
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
ames s o es in he ajec o ies. These eloci ies we e associa ed
o each ini ial posi ion. Gi en he quasicylind ical geome y o he
nano ube, he p obabili y o he posi ions o wa e and ions was
plo ed using cylind ical coo dina es (R,θ), whe e R is he
dis ance om he axis o he nano ube and θis he angle
a ound such axis. The eloci ies o he same pa icles we e
also exp essed in cylind ical coo dina es and ep esen ed as a
unc ion o R o θ.
RESULTS AND DISCUSSION
S abili y o he α,δ-Sel -Assembling Cyclic
Pep ide Nano ubes: The Fo ess o he
Nano unnel
P e ious s udies wi h an α,δ-SCPN composed by c-[L-Gln-
δ-Ach-D-T p-δ-Ach-L-T p-δ-Ach-D-Leu-δ-Ach-L-T p-δ-Ach-
D-T p-δ-Ach] showed ha he nano ube s uc u e is s able in he
lipid bilaye en i onmen (Cal elo e al., 2021). The subs i u ion
o bo h Gln and Leu by T p esidues shown in he p esen wo k
does no ha e a significan e ec on he ansmemb ane channel
s abili y, as indica ed by he low a e age Roo Mean Squa e
De ia ions (RMSDs) o he SCPN backbone eached along he
ajec o ies (Supplemen a y Table 1). The ubula shape is
p ese ed in all he simula ions (Figu e 2A). The high and
cons an numbe o H-bonds be ween CPs along he channel
backbone (Supplemen a y Table 1) con e igidi y o he whole
s uc u e, hus p o iding a s able sca old h oughou he
α,δ-SCPN. The a e age numbe o H-bonds be ween CPs (78
in all he simula ions) is sligh ly lowe han he maximum ha
can be heo e ically o med (12 H-bonds by each CP-pai , gi ing
a o al o 84 H-bonds). This is possibly due o some ansien
impe ec ions in he s uc u e and also o he compe i ion wi h
he wa e molecules inside and ou side he channel
(Supplemen a y Table 1), which sugges s he dynamic
cha ac e o he ubula s uc u e.
Despi e i s significan hyd ophobic cha ac e , a conside able
amoun o wa e (∼195 molecules) can be ound inside he
α,δ-SCPN (Figu e 2B). The amoun o wa e wi hin he
nanocylinde olume delimi ed by he CPs is no significan ly
al e ed in he p esence o ions (Supplemen a y Figu e 1). On he
o he hand, whe eas almos no Li
+
ca ions can pene a e he po e,
he α,δ-SCPN admi s an a e age o 1–3Na
+
,K
+
,o Cs
+
and 1–2
Ca
2+
ca ions (Figu e 2B and Supplemen a y Figu e 2A). The
appea ance o Cl
−
anions inside he channels is significan bu
much lowe han ha o ca ions, and no mo e han a single anion
was obse ed in his olume a he same ime (Supplemen a y
FIGURE 2 | (A) Snapsho o he SCPNs co esponding o he las ame o he MD simula ions ( 200 ns), including he ions inside (pink) a ha ime ( he la e al
chain is no shown o cla i y o he image). (B and C) A e age numbe o ca ions (B) and anions (C) inside he α,δ-SCPN a e aged o e he las 180 ns o he MD
simula ions in LiCl, NaCl, KCl, CsCl, and CaCl
2
0.15 M, espec i ely. Di ing in o confinemen : s uc u e o wa e and ions inside he α,δ-SCPN.
F on ie s in Chemis y | www. on ie sin.o g July 2021 | Volume 9 | A icle 7041604
Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
Figu e 2B). In con as , no anions a all we e obse ed wi hin he
po e o he smalle and less hyd ophobic α,γ-SCPNs (Ga cía-
Fandiño e al., 2012a;Cal elo e al., 2015).
Besides he numbe o ions inside he channels, i is possible o
moni o hei posi ions along he lumen o he co esponding
nano ubes o each ame o he ajec o y (Figu e 3). Some Li
+
ca ions we e obse ed inside he α,δ-SCPN jus o he fi s 25 ns
o simula ion. A e his ime, hey appea a ached o he lipid
phospha es, which p e en hei en y in o he po e
(Supplemen a y Figu e 3). The as ounding p e e ence o
phospha e g oups displayed by Li
+
ions is in good
conco dance wi h p e ious s udies (K uczek e al., 2017) ha
p edic his beha io . This ca ion is seques e ed by he pola head
g oups o he lipids in he memb ane, hus ende ing i unable o
a e se he nano ube, a leas a he ime scale hese expe imen s
we e ca ied ou . Thus, Li
+
ca ions in he nanopo e ca i y olume
will no be conside ed o he es o he analysis in his
manusc ip . As i had been p e iously obse ed o his ype
o channel (Cal elo e al., 2021) and analogously o α,γ-SCPNs
(Ga cía-Fandiño e al., 2012a;Ga cia-Fandiño e al., 2016), he
e ec i e adius is si ua ed in he plane o he CP, whe eas he
maximum adius is loca ed in he egion be ween he wo planes
o he ings. Howe e , he anspo o Na
+
,K
+
, and Cs
+
is
significan ly di e en o ha obse ed in oc ame ic α,γ-SCPNs
(Ga cía-Fandiño e al., 2012a;Cal elo e al., 2015), p esumably
due no only o he highe hyd ophobic cha ac e o his
nano ube bu also o la ge adius o hese h ee ions. While
he po es o med by α,γ-SCPNs exhibi egions whe e he ca ions
spend mo e ime, ac ing as b akes agains he ad ance o he ions
along he po e, he anspo o Na
+
,K
+
, and Cs
+
in α,δ-SCPN
akes place in a mo e con inuous way, wi h a p obabili y
dis ibu ion cen e ed in he middle o he nano ube
(Figu e 3). The disc e e ad ance o he ca ion is s ill p esen
in he case o Ca
2+
, whe e i s accumula ion in specific zones along
FIGURE 3 | Z-coo dina es (along he symme y axis o he nano ube) o he ca ions (le ) and anions ( igh ) along he 200 ns o he co esponding ajec o ies.
Each colo ep esen s a di e en a om as a unc ion o ime. F om op o bo om: LiCl, NaCl, KCl, CsCl, and CaCl
2
0.15 M. A complemen a y plo , wi h he numbe o
ames whe e a leas one ion is loca ed in each z-posi ion o he nano ube, is also shown a he igh side o each g aphic.
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels

FIGURE 4 | Posi ional p obabili y dis ibu ions ep esen ed as hea maps (inc easing om o ange o blue) a he XY plane o Na
+
,Ca
2+
, and hei co esponden
coun e Cl
−
anions. Wa e ( om he blank ajec o y) is shown a he down le o he figu e. The a e aged posi ion o alpha ca bons (g een) and ca bonyl g oups ( ed) is
also ep esen ed as a e e ence. These 2D-hea maps we e gene a ed using he las 180 ns o he MD simula ions.
FIGURE 5 | Posi ional p obabili y dis ibu ions ep esen ed as hea maps (inc easing om o ange o blue) a he XZ plane o Na
+
,Ca
2+
, and hei co esponden
coun e Cl
−
anions. Wa e ( om he blank ajec o y) is shown a he down le o he figu e. The a e aged posi ion o alpha ca bons (g een) and ca bonyl g oups ( ed) is
also ep esen ed as a e e ence. These 2D-hea maps we e gene a ed using he las 180 ns o he MD simula ions.
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
he symme y axis o he channel is much mo e no iceable han
o he es o he ca ions. Di e ences in he dis ibu ion
unc ions along he main axis o he nano ube a ise om he
al e na ion be ween he smalles adii, co esponding o he plane
o he CPs, and he la ge adii egions, co esponding o he
in e plane sec ions.
The highes abundance o Cl
−
inside he channel is obse ed
o he case o he simula ion wi h CaCl
2
. Fo his simula ion, a
single ca ion emains inside he α,δ-SCPN o long pe iods o
mo e han 100 ns (Figu e 2C and Figu e 3). The dis ibu ions
shown in Figu e 3 indica e ha a complemen a y Cl
−
anion
seems o accompany always he Ca
2+
ca ions, sugges ing ha
elec os a ic in e ac ions media e he pe meabili y o he
nano ube owa d anions. Fo he es o he simula ions, Cl
−
ge s only e e so shyly inside he nano ube, also ske ching a
ela i ely spa ial complemen a i y along he symme y axis o he
nano ube wi h espec o he di e en coun e ca ions.
To be e unde s and he dis ibu ion o species along he
α,δ-SCPN, hei p obabili y dis ibu ion in wo dimensions,
ob ained om he numbe o coun s a each posi ion wi h
espec o he o al amoun o coun s inside he SCPN, is
ep esen ed as hea maps. These 2D hea maps ha e been
ep esen ed in he ans e sal XY (Figu e 4and
Supplemen a y Figu e 4) and longi udinal XZ (Figu e 5 and
Supplemen a y Figu e 5)o YZ(Supplemen a y Figu e 6)
planes. Anima ions whe e he iew o he hea map is o a ed
along he longi udinal plane a e a ailable in he supplemen a y
in o ma ion (Supplemen a y Anima ion 1). Despi e hei la ge
diame e , compa ed o α,γ-SCPNs (Ga cía-Fandiño e al., 2012a;
Cal elo e al., 2015), he nanoconfined wa e molecules in he
α,δ-SCPN adop a highly o de ed dis ibu ion bo h in he
ans e sal and longi udinal planes. The wa e spa ial
dis ibu ion o e he ans e sal XY plane e eals a clea ly
defined six-poin s a shape, o he blank simula ion
(Figu e 4). I is comp ised o concen ical ings whe e
wa e molecules end o accumula e. The ex e nal ing,
o ming he ips o he s a , co esponds o wa e di ec ly
a ached o he CO and NH g oups o he SCPN backbone ia
H-bonds ( ype I, om now on). In pa icula , mos o hese
wa e moleculeses ablishH-bondswi h heCOg oupso T p
(42 ±3 H-bonds) and δ-Ach (18 ±3 H-bonds) esidues and
o a lesse ex en wi h he NH moie y (5 ±1 H-bonds wi h he
NH g oup o T p esidues and 6 ±2 H-bonds wi h he NH
g oup o δ-Ach esidues) (Values A e aged om he Las 50 Ns
o he MD Simula ions). The ollowing inne ing can be spli
in o wo ypes o wa e molecules: hose b idged o he ou e
ing ( ype II) and hose lying be ween he o me wa e
molecules, in he egion nex o he wo me hylene g oups
o he δ-Ach ( ype III). Finally, he e is an inne mos ing,
mo e di use han hose p e iously desc ibed ( ype IV).
Fu he mo e, i is wo h no icing ha , besides he
appea ance o lobes whe e wa e ends o eside, he e a e
egions ha wa e ends o a oid, c ea ing channels wi h
dec eased wa e densi y. These egions guide he posi ion
and di usion o ca ions (see below). Wa e dis ibu ion on
he XZ and YZ planes is also e y well defined (Figu e 5 and
Supplemen a y Figu e 6). The da kes lobes co espond o
in e CP plane egions, coexis ing wi h sligh ly less dense
a eas belonging o wa e placed a he CP planes.
The p esence o ions in he simula ion does no seem o a ec
he exo ic wa e pa e n ound in he blank simula ion
(Supplemen a y Figu e 7). Ins ead, i seems ha his
p edefined wa e dis ibu ion inside he α,δ-SCPN c ea es a
coo dina ing sca old h ough which he ions mo e. In his
way, he 2D hea maps o ca ions in he XY plane a e
complemen a y o hose co esponding o wa e (Figu e 4 and
Supplemen a y Figu e 4). Simila o wa e , ions also exhibi a
cha ac e is ic 2D dis ibu ion hea map o e he SCPN ci cula
plane. Na
+
o ms a ecip ocal hexagonal s a , wi h six peaks a he
egions in con ac wi h ype II and ype III wa e s desc ibed be o e
(Figu e 4). O e all, he shape o he s a becomes mo e di use
om Na
+
o Cs
+
(Figu e 4 and Supplemen a y Figu e 4), a
demeano possibly a ibu ed o he la ge ion adius. Thus,
bigge ca ions p e e he cen al zones o he po e, whe eas
hose smalle a e mo e localized a he bo de s o inside
specific channels whe e hey can fi be e . On he o he hand,
Ca
2+
illus a es he influence o cha ge, a he han adius (as
compa ed wi h he mono alen ca ions), on he p e e ed
posi ion o a di alen ca ion h ough an α,δ-SCPN (Figu e 4).
The p obabili y dis ibu ion o he di alen ca ion displays a
compac shape in he XY plane, indica ing ha he ions a e
confined in he inne mos pa o he lumen. This sugges s ha
Ca
2+
ion mo emen s a e es ic ed o smalle egions han he
o he ca ions. The ion disposi ion along he symme y axis o he
nanopo e is also di e en , depending on i s size and cha ge.
While he mos popula ed egions o Na
+
and K
+
a e hose
be ween CP planes, Cs
+
and Ca
2+
seem o p e e he plane o he
CP (Figu e 5 and Supplemen a y Figu es 5,6).
The 2D hea maps o Cl
−
sugges a dependence o he anion
loca ion and di usion on hei ca ion coun e pa . Cl
−
accumula ion in he XY plane e eals a less-defined six-poin
s a , he clea es case being CaCl
2
(Figu e 4). I is no able how
hese s a s a e o a ed 60°wi h espec o hei ca ion analogues.
While posi i e cha ges ha e well-defined channels h ough which
hey can di use wi hou dis up ing he sca old ha wa e c ea es
inside he SCPN and may e en ge “lub ica ed”by coo dina ing o
hei oxygens, anions ha e ouble finding a place o fi and mo e
h ough, e en mo e so i hey a e as bulky as a chlo ide. On op o
ha s e ic hind ance, he only possible coo dina ion ha a Cl
−
can ha e wi h wa e is h ough i s hyd ogens. This u he
dis up s he wa e sca old, as i would imply an in e sion o
he con o ma ion o some wa e molecules. This flip would
ende he oxygen a om om he coo dina ed wa e molecule
close o he oxygen a oms o he wa e s o ming he sca old,
c ea ing an elec os a ic epulsion, and des abilizing a e y s able
la ice by en ailing an ene ge ic penal y. On he o he hand, he
anions’2D hea maps o he a e aged posi ions along he
longi udinal planes show e y simila p ofiles o all he
simula ions, he only di e ence being he in ensi y o he
popula ion (Figu e 5 and Supplemen a y Figu es 5,6).
In o de o ga he in o ma ion abou he su oundings o each
ca ion wi hin he α,δ-SCPN, Radial Dis ibu ion Func ions
(RDFs) we e calcula ed (Figu e 6 and Supplemen a y
Figu e 8). They display a clea fi s wa e coo dina ion sphe e
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
o all species, which mig a es owa d bigge dis ances o ions
p esen ing la ge a omic adii. None o he ca ions s udied end o
di ec ly coo dina e o he CO g oups o he amino acid backbone
in his fi s coo dina ion sphe e a dis ances ∼4–6 Å. Excep o
Ca
2+
, hese CO g oups do pa icipa e in hei second coo dina ion
sphe e. On he o he hand, some Cl
−
ions do di ec ly con ibu e
o he fi s coo dina ion sphe e o all he ca ions, bu his
con ibu ion is poo .
The calcula ion o he numbe o con ac s be ween ca ions and
hei possible con ibu ions o he fi s coo dina ion sphe e
(calcula ed om he RDF alues) also confi ms ha CO g oups
do no coo dina e di ec ly o ca ions (Figu e 7). The numbe o
wa e molecules in he fi s coo dina ion sphe e o all ca ions is
e y simila o he heo e ical maximum in bulk, sugges ing ha
he ene ge ic penal y ha hese ions pay o en e in o he ube is low
(Table 1 and Figu e 7)(Oh omo and A akawa, 1979;Bakó e al.,
2002;Va ma and Rempe, 2006). Almos no Cl
−
ion o oxygen om
CO was ound a he fi s coo dina ion sphe e dis ance o any
ca ion, confi ming ha his si ua ion is unlikely.
FIGURE 6 | RDFs o Na
+
(A) and Ca
2+
(B) conside ing di e en con ibu ions (wa e , CO g oups, and Cl
−
ions), no malized by he numbe o ca ions and numbe o
con ibu ions. Jus he las 180 ns we e conside ed o he analysis. No e ha he scales o he h ee con ibu ions a e no he same.
FIGURE 7 | De ails o he fi s sphe e o coo dina ion o confined Na
+
(A) and Ca
2+
(B).
TABLE 1 | A e age numbe (and s anda d de ia ion) o con ac s be ween wa e ,
oxygens om CO, and Cl
−
anions pa icipa ing in he fi s coo dina ion sphe e
o each ca ion. The adius o he fi s coo dina ion sphe e was aken om he RDF
calcula ions. Jus he las 180 ns we e conside ed o he analysis.
Ca ion Wa e CO Cl
−
Na
+
5.5 ±0.56 0.00 ±0.03 0.00 ±0.04
K
+
6.21 ±0.75 0.01 ±0.13 0.01 ±0.08
Cs
+
5.26 ±0.94 0.00 ±0.08 0.01 ±0.11
Ca
2+
8.83 ±0.4 0.00 ±0.01 0.01 ±0.10
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels
I was obse ed ha he numbe o coo dina ing wa e
molecules inside he nano ube o en exceeds he heo e ical
maximum (6) o he ca ions. This si ua ion is clea o K
+
,
whe e he a e age numbe is 6.21 ±0.75. One plausible
explana ion is ha nanoconfined sys ems beha e di e en ly
han hei bulk ela i es. SCPNs can be app oxima ed o a
hollow cylinde , whe e one dimension ( e ical, Z) is much
la ge han he o he wo (ci cula plane, XY). This lea es a
space whe e wo deg ees o eedom become es ic ed, and
hus, mo emen in hose di ec ions becomes hampe ed. I is
easy o imagine, hen, ha in such a sys em wi h pa icles
mo ing inside, collisions a e mo e likely o occu han in a
ee un es ic ed medium. This migh lead o a comp ession o
he fi s sphe e o coo dina ion o ca ions, o cing he
ligands owa d he me allic cen e and a ec ing he ligand
exchange p ocess. I should also be no ed by he highe SDs on
heinne numbe o wa e s(Table 1), when compa ed wi h
hose o he bulk analysis, ha hese “hype coo dina ed”s a es
migh only happen o e y sho pe iods o ime, being
uns able.
Mo emen In o Confinemen : Dynamics o
Wa e and Ions Inside he
α,δ-Sel -Assembling Cyclic Pep ide
Nano ubes
Fo he p ope cha ac e iza ion o he α,δ-SCPN as a
ansmemb ane channel, i is in e es ing o s udy how he
di e en molecules mo e h ough he nano ube, in addi ion o
cha ac e ize hei mos likely loca ion. F om he su i al
p obabili y (Supplemen a y Figu e 9) and he co esponding
hal -li e ime alues (Table 2), i can be app ecia ed ha he mos
sho -li ed species in he α,δ-SCPN is wa e , ollowed by Cs
+
,K
+
,
Na
+
, and Ca
2+
. The esidence hal - ime o Cl
−
anions is
significan ly lowe , e en o he simula ion in CaCl
2
.
The dis ibu ions o a e age eloci ies be ween sa ed
ames (10 ps a om each o he ) o he di e en ions and
wa e molecules we e de e mined om he MD ajec o ies.
The esul s o he sol en and ions show ha wa e molecules
each he highes a e age speeds o e his windows ime,
al hough he e is a significan con ibu ion indica ing he
exis ence o slowe wa e molecules in he co esponding
dis ibu ion, p esumably hose in ol ed in ype I H-bonds
(Figu e 8). Jus as he p esence o ions did no seem o
a ec he o e all wa e pa e n inside he po e, hey do no
seem o al e hei eloci y dis ibu ion ei he . Fo a gi en
alence, ca ions wi h bigge adii end o di use as e along
TABLE 2 | Residence hal -li e ime (τ1/2) (ns) calcula ed om he su i al p obabili y
[P(τ)] e olu ion o ca ions and anions in each simula ion (Supplemen a y
Figu e 9).
τ1/2(ns)Ca ion Anion
NaCl 3.1 0.2
KCl 1.2 0.2
CsCl 1.0 0.2
CaCl
2
4.6 0.3
FIGURE 8 | Veloci y p obabili y dis ibu ions o wa e ( op ow, o blank and simula ions in he p esence o NaCl and CaCl
2
) and he selec ed ions. Bin-wid h has
been selec ed applying he F eedman–Diaconis ule (F eedman and Diaconis, 1981). In he igh -bo om squa e box, a ep esen a ion o he coo dina ion o Ca
2+
wi h
wa e s om he fi s and second coo dina ion sphe es is shown.
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Blanco-González e al. α,δ-SCPN T ansmemb ane Channels