Curvature of Buckybowl Corannulene Enhances its Binding to

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C: Physical Processes in Nanomaterials and Nanostructures

Curvature of Buckybowl Corannulene Enhances its Binding to Proteins Shuangli Du, Hongyu Wang, Yueyue Yang, Xi-Zeng Feng, Xueguang Shao, Christophe Chipot, and Wensheng Cai J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b10302 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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The Journal of Physical Chemistry

Curvature of Buckybowl Corannulene Enhances its Binding to Proteins Shuangli Du,†,○ Hongyu Wang,‖,‡,○ Yueyue Yang,‖,‡ Xizeng Feng,*,‖,‡ Xueguang Shao,†,‡,# Christophe Chipot,¶,┴, and Wensheng Cai*,†,# †

Research Center for Analytical Sciences, College of Chemistry, Tianjin Key Laboratory of

Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China ‖

Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences,

Nankai University, Tianjin 300071, China ‡

State Key Laboratory of Medicinal Chemical Biology, Tianjin 300071, China

#

Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, 300071, China

¶

L or to r Int rn t on l Asso

LPCT, UMR 7019 Un v rs t ┴

CNRS n Un v rs ty o Ill no s t Ur n

Lorr n CNRS, V n

Department of Physics, University of Ill no s t Ur n

C mp

n,

uvr -lès-Nancy F-54500, France C mp

n, 1110 W st Gr n Str t,

Urbana, Illinois 61801, United States ○

Contributed equally to this work

ACS Paragon Plus Environment

1

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

ABSTRACT: Corannulene, a polycyclic aromatic hydrocarbon, has garnered much attention on account of its promising applications n

-p r orm n

l tron s. Investigations of its

potential biological applications have, however, remained hitherto scarce. In the present contribution, to explain the distinctive interaction of the geodesic corannulene with proteins, the adducts formed by lysozyme (LSZ) and two carbon materials, namely corannulene:LSZ and perylene:LSZ, were prepared and investigated using a variety of experimental and computational approaches. We find that LSZ binds the two ligands at its active site, forming stable complexes. Interestingly, although corannulene and perylene have very similar structures, standard binding free-energy calculations demonstrate that the former possesses a much greater binding affinity for LSZ compared with the latter, which can be ascribed to its t r - m ns on l π- owl urv tur

and unique charge distribution, enhancing its electrostatic interaction with LSZ.

Corannulene s oun to

mor

t v than perylene in n

tn t

prot n

t v ty. In

addition, corannulene hardly affects the fluorescence of LSZ. The present work indicate that endowed with a urv

π-surface and large dipole moment, corannulene is a promising ligand,

capable of binding through a variety of intermolecular interactions a broad range of proteins, modulating their biological or catalytic activity, yet without affecting their fluorescent properties.


2

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INTRODUCTION C r on n nop rt l s, w m t r ls,1-3 or n r s r

s

nolo

l ppl

t

ot r

orts

l n

v

xtr

v

tun l ,

t or t

n r t low st uno up

t ons o

ts

ntr u n

pol mom nt,14,

t

su

n

15

ompl x r ox

olo

t ons. M ny v

s t rou

prop rt s o

or nnul n

su st tut on.9, 16, 17 Mor ov r,

xplo t

row n num r o stu

n

ss ul synt s s o

mol ul r or t l llows t m t r ls

n

r t r st s

ts pot nt l ppl

n tur o t

m t r ls,21, 22 s nsors, n opto l tron

l nv st

t s nov l m t r ls,

n w st r m t r l. T

p n n on t

ott st

l prop rt s.9-11

u ky owl.9, 16-18 Opto l tron

s.19, 20 In sp t o t

-p r orm n

s on o t

n l or tory r s r m

l r

mult un t on l

v lopm nt o mol ul r l tron

l trons. H n , or nnul n - s

tr ns r

t

m sts to nv st

tow r t

p r p ry o t

,

t

ont mpor ry

r t

ou ly

π-sur

t v ty, m k

s nsp r

n r port

vn

on v

r

un t on l z n t

m r

poly r n , poss ss s C5v symm try.12, 13 T

s

n

v

nt y rs. Am

t ons u to ts sup r or p ys o s

v

n ustry n r

to

-p r orm n

os nsor,6-8

n

n

or nnul n

n

t ons n

prom s n

or nnul n , su

prop rt s

ppl

s,4, 5

v

m

ons

Cor nnul n , o

oun

p otovolt

r s n

or nnul n

v

n

or t

ommo t

our

s no

r -

s on or nnul n v

nt

r

o

s,18, 23, 24 xp r m nt l n

l un t ons n potential biological applications r

l m t .25 Du to ts notor ous

l ty to r ul t

s

s

not ns l t

l t r tur .29-31 In t n notu s35-37 n

l st r p n

v r ty o

mo l prot n on w 7, 38, 39

s, on t

o

m

lr

ount o ts n tur l

xplor t on o t stru tur s n

t ons,25-27 lysozym (LSZ) un n t o

un t ons o LSZ

7, 28

n

xt ns v

ull r n s,32-34 sm

r on

r m rk l


3


pro r ss. C lv r s m

t l.

monstr t

t tt

t-p rtur t on n lys s.34 In

l-s

st n t

t on t

mult -w ll

morp olo

n r port

r

ls p o t

u t orm

nt work, w

n n o

y LSZ w t C60, us n NMR r v l

t t or nnul n

LSZ ryst l, omp r

n p ryl n ,11 su

r on n notu , r p n

on v poly r n . How v r,

1:1

Page 4 of 29

w t non- un t on l z

st n o

un qu prop rty o t

r m nt o C60,40, 41 w t LSZ

or nnul n ,

s

sn v r

t rto.

In t s stu y, t

m

su str t on ot t xp r m nt l n

n sm w r y or nnul n stru tur

n

t v ty o LSZ w r

omput t on l ppro

pl n r poly y l

rom t

n LSZ sso p r

s. To un rs or t

t

t rou

to t om n t on o

un qu prop rt s o

y ro r on, p ryl n ,42, 43 poss ss n

or nnul n ,44 w s lso nv st

t , n t

or nnul n ,

stru tur s m l r to t t o

or omp r son purpos s.

EXPERIMENTAL AND COMPUTATIONAL METHODS Samples Preparation. T LSZ n w t r to

LSZ solut on w s pr p r

y

ssolv n

or nnul n (synt s z

l.44) n p ryl n to 1mM LSZ solut on w t t t on or 60 m n w t

LSZ solut on orm n

mx

solut ons w r o t n

t rou

o t

ssolv n

on ntr t on o 0.2 mM. S mpl s o t s two

LSZ:perylene) w r pr p r

ultr son

y

pro

n n

sto

u t. T

m xtur

t 5000

mounts o

u ts (LSZ:corannulene and n prov

y Juríč k t

r l t ons p t 5:1. A t r

t , or nnul n /p ryl n w s

solut on ont n n t ntr u t on o t

om tr

ppropr t

sp rs

nt

omo n ous n st l or 10 m n n

oll t on

sup rn t nt.34


4

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Absorption, Fluorescence and Circular Dichroism Spectra Study. UV-V s stu

s w r

m sur

p r orm

y luor s n

ro sm (CD) r ul r

y S

sorpt on

m zu 2401 UV-V s sp trop otom t r. Fluor s n

sp trop otom t r. T

w s

x t t on w v l n t w s 280 nm. C r ul r

t us n qu rtz uv tt o 1 mm p t l n t w s oll t

rom JASCO 810

ro sm sp tropol r m t r.

Effect of Carbon Materials on the Antibacterial Activity of LSZ. 200 µL samples of LSZ and LSZ with two carbon materials were added into the Oxford cup that prepositioned on the medium. All the plates were incubated at 37 ºC for 6 to 8 h. T m sur

y m

r

o

nt

t r lrn sw r

J so tw r .

Activity Assay. 4-methylumbelliferyl β-D-N,N',N"-triacetylchitotrioside hydrate stock solution was prepared by dissolving it in methanol:water (1:1) and stored at -20 ºC. The substrate concentration was calculated from the absorbance at 316 nm (molar extinction coefficient of 12.3 mM-1). The samples were prepared by mixing 27.4 μL of 1 mM LSZ or the other two adducts (LSZ:perylene and LSZ: or nnul n ) w t 1.7 μL o 87 μM su str t

n 2 mL o 25 mM

ammonium acetate buffer (pH 4.6). Then, all the samples were incubated for 30 min at 42 ºC. The reactions were eventually stoppe

y

n 240 μL o 1 M N OH. T

r l s o

r

4-

methylumbelliferone was recorded by emission fluorescence spectra after diluting the samples by 20000600 times in pure water, using an exciting wavelength of 360 nm and measuring the emission at 455 nm. Statistical analysis. D t us n Gr p p s n

Pr sm so tw r

rv

rom t l st t r

y ANOVA. T

n p n nt r pl

v lu s o p < 0.05 w r

t s w r

ons

r

n lyz

st t st

lly

nt.


5


Page 6 of 29

Molecular Docking and Dynamics Simulations. AutoDock 4.2 was used to syst m t s r

t

poss l binding poses of corannulene and perylene with LSZ.45 In

lly

t on, ll the

MD simulations reported here were conducted using the parallel, scalable MD program NAMD 2.1246 with the CHARMM General Force Field47 and the TIP3P water model.48 Quantum Mechanical Calculations. Geometry optimizations of corannulene and perylene were done using ab initio second-order Møller-Plesset perturbation theory (MP2) with 6311++G(d,p) basis set, followed by vibrational frequency calculations to verify the reasonability of the optimized structure. The electrostatic potential and Merz–Kollman (MK)49 charges were calculated at the same level of theory. The luor s n -qu n

n m

n sm was explored by

time-dependent density functional theory (TD-DFT). All the QM calculations were performed by Gaussian 09 software.50 Standard Binding Free-Energy Calculations. A stepwise strategy was employed to estimate the standard binding free energy of the complex. T ppr

ly

ur y o t s m t o

s ons

r

to

r t n t t o MM-PBSA.51 The unbinding of LSZ-ligand complex was divided

into eight subprocesses, wherein one degree of freedom is characterized at a time. The potential of mean force (PMF) for each subprocess can be achieved by a one-dimensional free-energy calculation, which was carried out using the extended adaptive biasing force (eABF) method implemented in the Colvars module of the NAMD package.52, 53 The degrees of freedom of the ligand can be characterized by the RMSD of the ligands with respect to its conformation in the bound and unbound states, the three Euler angles, the two spherical angles and the separation of the ligand from the protein. The corresponding contributions to the standard binding free energy are denoted by

,

,

,

,

,

,

, and

𝑟

p

, respectively.


6

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The standard binding free energy is t n +

+

𝑟

p

+

+

v n y .

= n

+

+

+

+

calculated analytically. A new VMD

plug-in, named BFEE (binding free-energy estimator), was used to generate the input files and do the post-treatment for the standard binding free energy calculation.54 Instantaneous values of t

or

w r

ru

n

ns o w t

qu l to 1˚, 0.05 Å, n 0.1 Å, or t

n ul r, RMSD,

and separation PMFs, respectively. Harmonic angular and RMSD restraints of ligands were enforced in each free-energy calculation by means of harmonic potentials with a force constant equal to 0.1 kcal/(mol·degree2) and 100 kcal/(mol·Å2), respectively. D t l o t v l l nt

M t o s S t on n t

m t o olo y s

Support n In orm t on.

RESULTS AND DISCUSSION Interaction of Corannulene and Perylene with LSZ. To investigate the properties of adsorption of the two carbon materials, the absorption spectra of LSZ in an isolated state, and LSZ with two carbon materials were determined. As shown in Figure 1a, the absorbance of LSZ at 280 nm increases in different degrees after mixing with each of the two carbon materials, indicative of the formation of a protein:carbon-material complex. Furthermore, the maximum absorption intensity of LSZ:corannulene is found to increase more than that of LSZ:perylene. The change in the absorption intensity is generally consistent with the strength of the protein:ligand interaction.55 Corannulene, therefore, exhibits a stronger adsorption potency than perylene on LSZ. The UV-vis spectra showing all the visible spectral range for all samples n

t t t t r s no s tt r n

nt

solut on u to or nnul n /p ryl n

r

t s (s own


7


n F ur S1). SDS-PAGE (s own n F ur S2) n t

t r

s mpl s  LSZ lon , LSZ w t

t st tt r

Page 8 of 29

r no ov l nt

u ts n

or nnul n , n LSZ w t p ryl n .

Figure 1. ( ) Absorption spectra of LSZ, LSZ:corannulene, LSZ:perylene, corannulene and perylene. (b) Fluorescence spectra of LSZ, LSZ:corannulene and LSZ:perylene. B n n s t o or nnul n

n p ryl n

n LSZ n t

n l stru tur after the 1-μs MD s mul t on o t

optimal binding pose selected by AotuDock. ( ) LSZ:corannulene, (d) LSZ:perylene. All t

Trp


8

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


r s u s o LSZ r s own n r . Molecular orbital energy diagram to show the relative energetic dispositions of the frontier orbitals of the fluorophore Trp108 and the two ligands. The change of fluorescence before (left panel) and after (right panel) the binding of corannulene (e) and perylene (f). popul r mol ul r o k n pro r m AutoDo k56 w s mploy

T t

poss l

LSZ:l

n

or

n n s t s of corannulene and perylene in LSZ. T ompl x w r

s l t

rom t

yn m s (MD) s mul t ons w r su s qu ntly

out or

simulation time amounts to 7 s. Detail on the s l t provided in t

Support n

In orm t on (F ur

S3

s r

o

top t n pos s or

s or n - un t on- s rr

syst m t

r nk n . Mol ul r

o k n pos . The aggregate

pos s and the MD simulations is n

Table S1). Analysis of the MD

trajectories shows that nine of the ten binding poses are not robust, the ligand eventually dissociating from the protein after simulation typically less than 400 ns. Only the top-ranked pose was found to be robust in a 1-s MD simulation. The final structures of the two complexes, LSZ:corannulene and LSZ:perylene, are depicted in F ur 1c and d. The binding site of each ligand is found to be located in the active site of the LSZ, w

s m nly constituted by

residues Glu35, Asn46, Asp52, Gln57, Asn59, Trp63, Asp101, Asn103, Ala107, Trp108 and Val109.57 Detail discussion s prov

n the section about nzym t

t v ty.

Structural analysis shows that both or nnul n and p ryl n are close to the six fluorescence groups (Trp28, Trp62, Trp63, Trp108, Trp111 and Trp123, s particular, Trp108. Int r t on o the six residues w t t t

rol pl y

y t s r s u s. Our r sults n

nt r t on o Trp108 w t

t

l

n

n sw s x mn

t t t mon t

poly r n su str t s s t

F ur 1

) of LSZ, in to un rst n

s x tryptop n r s u s, t

stron st (s

F ur S5 in t


9


Support n

Page 10 of 29

In orm t on). The emission of LSZ, as reported, mainly stems from residues

Trp108.34 Therefore, binding of these ligands may lead to luor s n

qu n

n o LSZ.

Fluorescence Spectroscopy and Quantum Calculation. The fluorescence spectra for the above three samples (LSZ, LSZ:perylene and LSZ:corannulene) were measured and are reported in Figure 1b. It is apparent that LSZ shows a predominant emission band at 348 nm upon excitation at 278 nm. In addition, the peak at 448 nm corresponds to the characteristic fluorescence peak of corannulene itself. From Figure 1b, it is apparent that the fluorescence intensities of LSZ in the presence of corannulene, and LSZ in the presence of perylene, decrease omp r

wt

t

pur

LSZ solut on. T

orr spon n

qu ntum y l s (Φ)

r

0.86

(LSZ:corannulene) and 0.84 (LSZ:perylene). T

p otop ys

t m - p n nt

l pro ss s t t

nl

to luor s n

qu n

n w r

nv st

us n

ns ty un t on l t ory (TDDFT).58 Two mol ul r mo ls ons st n

Trp108 and the two ligands were constructed to analyze the luor s n -qu n T

t

n m

o

n sm.

r C rt s n oor n t s w r extracted from the structures of the complexes corresponding

to the global minimum of the separation PMF (see detailed discussion in the section of Free Energy Calculation). T

mol ul r or t l

M062X/6-31+g(d) level, s llustr t nt x t t qu n

ompl x s

n

ttr ut

l tron o tryptop n s n

o t

two l

n s n

n r y l v ls w r

n Figure 1e and f. O v ously, t to

n

r ms n t

p oto- n u tr ns rr

stron

to

n

l ul t

n luor s n

l tron tr ns r (PET) m low-ly n uno up

luor s n . In ontr st,

p rm ts the x t

n sm. T

or t l, r sult n

n n o t

s luor s n . As depicted in Figure 1f, the LUMO orbital of tryptop n s

t t o p ryl n , w

tt

two l

n n s

rt n

electron to transfer from tryptop n to p ryl n ,

leading to emission quenching. The same qu n

n m

n sm w s oun

nt

n n wt


10

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corannulene. In addition, the energy gap between the LUMO of tryptop n and perylene is larger than that between tryptop n and corannulene, resulting in a higher luor s n t

orm r. Our luor s n

l ul t ons, t r or , r t on l z

qu n

t

ov

qu n

n

or

xp r m nt l o s rv t on o

n .

Binding Free Energy of Corannulene and Perylene with LSZ. In t

pr s nt work, a

stepwise strategy for standard binding free-energy calculations using a geometrical route and a set of recently developed coarse variables were employed to evaluate the standard binding affinities of or nnul n

n p ryl n tow r LSZ.54, 59 The results are shown in Table 1, and the

corresponding one-dimensional free-energy profiles for the different contributions are provided in Figure S6 in the Supporting Information. It is apparent that or nnul n has a much higher binding affinity for LSZ than p ryl n , in agreement with the UV-Vis absorption experiment. As r port

n T l 1, t

ontr ut ons o t

(RMSD) t rms n t

v

s p r t on o t

n s rom t

st p o t

two l

un oun -, oun -st t root-mean-square deviation

n ul r t rms, Θ, Φ, Ψ, θ, φ r LSZ, . ., t

pr s nt str t y, orr spon n to t

most ru 𝑟

p

lmost n l l n

l . In ontr st, t

omput t on lly nt ns v

t rm n T l 1, constitutes the main

contribution to the standard binding free energy. The representative structures at the global minimum of the separation PMF are provided in Figure S7 in the Supporting Information, which are very similar to the structures obtained in the 1-μs qu l r um s mul t ons (s

F ur 1).


11


Table 1. Contr ut ons to t

n n

r

n r y or t

or nnul n PMF S mul t on (k l/mol) T m (ns) -0.1 12 -1.1 45 -0.8 210 -0.6 128 -0.3 90 -0.3 40 -11.2 286 +0.1 10 +6.6 -7.7 821

ontr ut on

𝑟

st n r

p

Page 12 of 29

two l

n swt t

LSZ.

perylene PMF (k l/mol) -0.1 -0.6 -0.5 -0.7 -0.2 -0.2 -8.4 +0.1 +6.6 -4.0

S mul t on T m (ns) 13 131 73 57 20 20 258 6 578

To analyze the driving forces responsible for binding, the separation PMFs were decomposed into van der Waals and electrostatic terms, as depicted in Figure 2a (right panel). As can be seen, van der Waals interactions constitute the dominating driving force for the binding of LSZ either ligand. Electrostatic interactions, however, contribute differently to the binding of or nnul n n p ryl n , s n n n s, w

ntly to t

on lu

p ryl n ow n to

orm r, ut only m r n lly to t

t t or nnul n

x

ts

r t r

l tt r. On t

n n

sso t s

n ty t n t

pl n r

vor l electrostatic interactions.


12

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Figure 2. ( ) PMFs or s p r t on o t orr spon n to t

ontr ut on o

𝑟

p

l

n

( or nnul n

n

perylene)

n

LSZ,

n T l 1 (l t p n l). Free-energy decomposition of

the s p r t on PMFs of LSZ:corannulene and LSZ:perylene into LSZ-ligand electrostatic and LSZ-ligand van der Waals contributions (right panel). (b) Plot of the electrostatic potential for or nnul n

n p ryl n w t t

ront (l t) n s

(r

t) v w. T

pol mom nts o


13


or nnul n

n p ryl n

l ul t

tt

Page 14 of 29

s m l v l r 2.659 n 0.009 D, r sp t v ly. T

l t o LSZ or ompl x s (c) LSZ:corannulene, and (d) LSZ:perylene. These two ompl x s stru tur s

r

obtained after 1-μs MD simulations, residues around the binding sites are

displayed by v n

r W ls sp r s.

To delve further into the distinct adsorption in LSZ:corannulene and LSZ:perylene, the electrostatic potential (ESP) and the charge distribution of t

two l

n s (s own n F ur 2b

and F ur S8 in the Supporting Information) were calculated at the MP2/6-311++G(d, p) level of theory using the Gaussian 09 program. ESP, the molecular fingerprint, is a powerful, predictive and interpretive tool to rationalize intermolecular interactions.60, 61 As can be seen from the front view of the two structures, their ESPs are similar  the center is negative, accompanying with a positive periphery. A l r two ESPs rom t wt

rs

n symm tr

on v on

st n t on m y, ow v r,

v w. Int r st n ly nou , ts s n ul r urv tur r

s pos t v ly

str ut on. T r

onv x sur

nt r t on w t

perylene. A sp

stru tur s o t

- ll n r pr s nt t on o t

n n s t s s s own n F ur 2 r p t mor

rmly, r sult n

t v ly

tw n t

n ows or nnul n r

,w r st

. T s un qu charge distribution results in a large dipole

moment, which enhances the l trost t

t

sn

oun

n

n tt r

LSZ, omp r

ompl x s (F ur 1

. It s pp r nt t t t n n

n ty o

wt

t

pl n r

n

)n r

l t o LSZ n rrows, so s to or nnul n tow r

LSZ t n

p ryl n . Circular Dichroism Spectroscopy and Secondary Structure of LSZ. T

r ul r

ro sm (CD) spectra of LSZ and the two LSZ complexes were measured to probe possible conformational changes in the protein62. The spectrum of LSZ shows two characteristic negative


14

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bands at 209 nm and 222 nm,63, 64 which originates from p–p* transfer of peptide bond and n-p* tr ns r o p pt

on

nt

α-helix.65 Modification of the peak of LSZ depicted in F ur 3

indicate that the original secondary structure of LSZ was slightly altered by the addition of the two

r on m t r ls. T

α-helical contents of the free LSZ and of LSZ bound with carbon

materials were calculated based on the CD spectra (detail of the calculation method is provided in the METHODS section),66 as shown in F ur 3. It

n

s n t t t

α-helical content

increases moderately upon addition of the two carbon materials, which indicates that both of the two ligands can improve the stability of LSZ.67

Figure 3. Circular dichroism spectra of LSZ with and without the presence of (a) perylene, and (b) corannulene. The number denotes the corresponding α-helix rate of LSZ.

V r t on o t

secondary structure of LSZ in the three molecular assemblies (isolated LSZ,

LSZ:corannulene and LSZ:perylene) is shown in F ur 4 - . The tertiary structure of LSZ is overall preserved, albeit with a slight perturbation of some r s u s roun t

n n s t . In

addition, the α-helical content of LSZ increased slightly after binding with the two ligands, in line with the experimental measurement (see F ur 4d-f).


15


Page 16 of 29

Figure 4. Time evolution of the secondary structure over the last 100 ns simulations: pure LSZ (a), LSZ:corannulene (b) and LSZ:perylene (c). Changes of the representative α-helical that formed by residues 25-37 (black dashed lines): pure LSZ (d), LSZ:corannulene (e) and LSZ:perylene (f). Modulation of the Enzymatic Activity by the Two Carbon Materials. The active site of LSZ lies in a cleft on its surface, viz. the red area in F ur 5a and b. The active site is known to be formed by six subsites (usually labeled as A-F) accommodating six sugar moieties,34,

68

among which three subsites (B, C and D) closer to the binding site are marked in Figure 5. In order to evaluate the role played by the three subsites, int r t on o these subsites w t t


16

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l

n s s s own n F ur 5 . Clearly, the interaction of siteC with the two ligands is the

strongest, demonstrating that both corannulene and perylene are located in one of the sugarbinding sites, specifically site C, underscoring the competition between the LSZ active site and the binding site of ligand. It is, therefore, reasonable to deduce that binding of the ligands may inhibit enzymatic activity to a certain extent. Detail information is provided in t

Support n

In orm t on.

Figure 5. A t v s t o LSZ (r

r

on) n LSZ:corannulene (a) and LSZ:perylene (b).

Variation of the interactions of siteB, siteC and siteD with l simulations (c). C t lyt

n s computed over the last 100 ns

r s u s Glu35 n Asp52 ( n r n) n LSZ:corannulene (d) and

LSZ:perylene (e). Electrostatic and van der Waals interactions of the with l

t lyt

r s u s of LSZ

n s obtained from the last 100 ns simulations (f).


17


Page 18 of 29

Residues Glu35 and Asp52 at site D (see F ur 5d and e) have proven to be critical for LSZ catalytic activity.28, 69 As can be seen, both corannulene and perylene bind near the two residues. t lyt

Analysis of the interaction of these

r s u s with the ligands as depicted in F ur 5f

indicates that while van der Waals contributions are marginal, electrostatic interactions are on v

favorable. Compared to the planar perylene, t

u ky owl shows a stronger

electrostatic attraction to the two r s u s, on account of its distinctive charge distribution. It may, therefore, be concluded that or nnul n poss ss s

stron r n

t on

t on t

enzymatic activity than perylene. Enzym t - t v ty ss ys were performed to confirm the impact of the two on t

t v ty o t

D-N,N',N"-tr su str t

LSZ, w r n t

tyl

n

totr os

l v

t v ty s m sur luor s n

LSZ to r l s t

y mon tor n t

t v t s (s own n r

t r

activity inhibition. In n lu n

o

t t LSZ. S n

t

ryl, t

n

or nnul n . T

nzym t

t p n l o F ur 6 ) r m n 84.3% n 79.6%, r sp t v ly.

t on o t on, t

or nnul n on t

r m nt w t t

ryl β-

sp tr . As s own n F ur 6 , t

t on o p ryl n

r on m t r ls mp t t

t on-zon m t o w s us . As om s sm ll r

to

luor s nt mo ty 4-m t ylum ll

m ss on luor s n

r s upon

In order to further assess how n

ly n s su str t , 4-m t ylum ll

(s own n F ur S10) w s us

yt

nt ns t s

luoro n

r on m t r ls

r sults o

p t

or nnul n st t st nt

l

t r l

nt

n F ur 6 , t

t r l

t v ty o LSZ, t

m t ro t

n p ryl n , w

s

m t rs s own n r

tp n l

n

t on zon

llm rk o enzymaticmonstr t t t t

t v ty o LSZ s r t r t n p ryl n , n oo

nzym t - t v ty ss ys.


18

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. ( ) Fluor s n A t v ty o

t

r

sp tr o t

LSZ, LSZ:p ryl n

****p

Curvature of Buckybowl Corannulene Enhances its Binding to

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