Conjugating Catalytic Polyproline Fragments with a Self-Assembling Peptide Produces Efficient Artificial Hydrolases

Article abstract of DOI:10.1021/acs.biomac.9b01620

A polyproline fragment containing a catalytic dyad of His-His or Ser-His was coupled with a self-Assembling peptide MAX1 to design new hydrolases (H2H5 and H2S5) for catalyzing ester hydrolysis. Circular dichroism measurements indicated that the peptides

Full text of DOI:10.1021/acs.biomac.9b01620

Subscriber access provided by BOSTON UNIV
Article
Conjugating Catalytic Polyproline Fragments with a Self-
Assembling Peptide Produces Efficient Artificial Hydrolases
Kuei-Yen Huang, Chi-Ching Yu, and Jia-Cherng Horng
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b01620 • Publication Date (Web): 17 Jan 2020
Downloaded from pubs.acs.org on January 19, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 36
Biomacromolecules
Page 1
1
2
3
4
5
6
7
8
9
Conjugating Catalytic Polyproline Fragments with a Self-
Assembling Peptide Produces Efficient Artificial Hydrolases
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
a,b
Kuei-Yen Huang , Chi-Ching Yu , and Jia-Cherng Horng *
aDepartment of Chemistry, National Tsing Hua University, Hsinchu,
Taiwan 30013, R.O.C.
bFrontier Research Center on Fundamental and Applied Sciences of
Matters, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C.
*To whom correspondence should be addressed. Department of Chemistry, National
Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C. phone: +886-3-5715131 ext
3
5635, fax: +886-3-5711082, e-mail: jchorng@mx.nthu.edu.tw
ACS Paragon Plus Environment

Biomacromolecules
Page 2 of 36
Page 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
A polyproline fragment containing a catalytic dyad of His-His or Ser-His was
coupled with a self-assembling peptide MAX1 to design new hydrolases (H2H5 and
H2S5) for catalyzing ester hydrolysis. Circular dichroism measurements indicated that
the peptides change their conformation from coils to β-sheets when pH increases from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
to 10. IR spectra also displayed the vibration modes corresponding to their β-
structures at pH 9.0. TEM and AFM measurements showed that in solution, the
designed peptides self-assemble into network fibrils having significantly increased
catalytic efficiency on ester hydrolysis. On p-nitrophenyl acetate (p-NPA) substrate,
1 1
the designed peptides exhibit high catalytic efficiency at pH 9.0 (k /K = 12.1 M s
cat
M
1 1
for H2H5, 13.3 M s for H2S5), and their efficiency is even better at pH 10.0
1 1
1 1
(
k /K = 24.3 M s for H2H5, 99.4 M s for H2S5). Additionally, H2H5 and
cat M
H2S5 also display good activity on catalyzing the hydrolysis of p-nitrophenyl-(2-
phenyl)-propanoate (p-NPP) and p-nitrophenyl methoxyacetate (p-NPMA).
Combining the polyproline based catalytic scaffold with a self-assembling peptide
generates an efficient hydrolase, providing a new design for effective artificial
enzymes.
Keywords: polyproline, hydroxyproline, self-assembling, β-structure, catalytic dyad,
hydrolase
ACS Paragon Plus Environment

Page 3 of 36
Biomacromolecules
Page 3
1
2
3
4
5
6
7
8
9
Introduction
The activities of natural enzymes with complicated tertiary or quaternary
structures are limited under specific conditions, such as small molecule or ion
binding, pH, temperature, light, and pressure. Even if they have great catalytic
efficiencies in certain environments, the limit of availability and stability still remains
a problem. Thus, the development of artificial enzymes with incredible catalytic
efficiencies and multiple functions has become an attractive topic in biochemical
research. A variety of strategies have been applied to design peptide-based artificial
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
enzymes, including α-helical coiled-coils and barrels,2-4 short helices, -hairpins,
1
5
6, 7
8
and β-peptide bundles. In addition, supramolecular assembling approaches have also
been widely used in developing artificial enzymes because the assembled constructs
9
possess switchable properties and microenvironments for catalysis. For instance,
proline-based peptide hydrogels have been used as catalysts for direct aldol
coupling,10, 11 histidine-based β-sheet amyloids or fibrils12-17 and metal-induced
assemblies18-22 have been used as a framework to catalyze ester hydrolysis, and
assembled nanomaterials with Ser/His/Asp triads enhance catalytic activity.23
Among the reported catalytic peptide assemblies for ester hydrolysis, metal-
9
chelated higher-order constructs exhibit the highest activities. In a recent study, Ulijn
1
7
and coworkers created a switchable hydrolase, VK2H, by incorporating an
oligopeptide into a self-assembling peptide MAX1 that was previously designed by
ACS Paragon Plus Environment

Biomacromolecules
Page 4 of 36
Page 4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Schneider et al.24, 25 In alkaline conditions, this peptide can form fibrils and hydrogels,
and it has a relatively high catalytic efficiency on the hydrolysis of p-nitrophenyl
acetate (p-PNA) despite containing only one histidine as the catalytic residue. These
studies strongly suggest that a higher-order structure can dramatically enhance the
catalytic activity of a peptide-based catalyst. In our recent study, we utilized the
polyproline helix as a scaffold to design a series of catalytic oligoproline peptides for
ester hydrolysis, and we found that the (2S,4R)-4-hydroxyproline (Hyp) based
peptides have higher activity than proline-based ones because Hyp residues can
strengthen the polyproline II structure and their hydroxyl groups may stabilize the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
reaction intermediates. In this study, the most PPII structured peptide Hyp-H3H6
exhibited more than a 2-fold increase in catalytic efficiency over that of the proline-
based H3H6. However, the catalytic activity is still well below those derived from
9
peptide assemblies. To improve the activity of polyproline-based hydrolases, herein
we attached the MAX1 peptide to the C-terminus of a hydroxyproline rich
oligopeptide (Figure 1) as a new catalyst design for ester hydrolysis. Various
spectroscopic methods were used to characterize the designed peptides and showed
that the peptides could self-assemble into -structured fibrils at alkaline pH. Under the
alkaline conditions, these newly designed peptides exhibit good catalytic activity on
ester hydrolysis.
ACS Paragon Plus Environment

Page 5 of 36
Biomacromolecules
Page 5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. The design of catalytic oligoproline/self-assembling peptide constructs for
ester hydrolysis: (A) The sequence contains an oligoproline fragment (catalytic) and a
MAX1 peptide (assembling); (B) The diagram of expected assembled structures: the
active residues are shown as sticks and the assembled -sheets are shown in magenta.
Experimental Section
Materials
Reagents for peptide synthesis were purchased from Aldrich-Sigma Chemical,
Alfa Aesar, Fluka, Acros, or Novabiochem, and used without further purification.
Fmoc-protected amino acids, O-benzotriazole-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HBTU), and hydroxybenzotriazole (HOBt) were obtained from
Advanced ChemTech, AnaSpec, CreoSalus or AgeneMax. 4-Nitrophenyl acetate (p-
ACS Paragon Plus Environment

Biomacromolecules
Page 6 of 36
Page 6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NPA) was purchased from Acros. p-Nitrophenyl-methoxy-acetate (p-NPMA) was
27
synthesized according to the previous report, while p-nitrophenyl-(2-phenyl)-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28
propanoate (p-NPP) was prepared using the method described in the literature. Mass
spectra were obtained by MALDI-TOF-MS spectrometer (Bruker Daltonics Autoflex
III Smartbeam LRF200-CID spectrometer). -Cyano-4-hydroxycinnamic acid
(
HCCA) was used as the matrix for MALDI-TOF measurements.
Peptide synthesis
Peptides were prepared on a 0.1-mmol scale by standard solid-phase methods
and Fmoc-chemistry protocols using Fmoc-protected amino acids and HBTU
mediated coupling reactions on an automated PS3 peptide synthesizer (Protein
Technologies Inc.). Use of a Rink amide resin generated an amidated C-terminus
upon cleavage from the resin with a solution of 95% TFA/2.5%
triisopropylsilane/2.5% H O (v/v). Each peptide had an acetylated N-terminus. The
2
peptides were purified by reverse-phase HPLC with a Dr. Maisch semipreparative
C18 column. H O/acetonitrile gradients with 0.1% (w/v) TFA as the counterion were
2
used as the eluting solvent system. The identities of peptides were confirmed by
MALDI-TOF mass spectrometry (Table S1 and Figures S1 and S2 in the Supporting
Information). The peptides were more than 90% pure according to HPLC analysis
(Figure S3 in the Supporting Information).
ACS Paragon Plus Environment

Page 7 of 36
Biomacromolecules
Page 7
1
2
3
4
5
6
7
8
9
Circular dichroism (CD) spectroscopy
CD experiments were conducted with an Aviv Model 410 CD spectrometer. Far-
UV CD spectra were recorded using a 1-mm pathlength quartz cuvette with a peptide
concentration of 50 M. Buffered solutions of 15 mM Tris-HCl with pH values of 5.5
to 10.0 were used. Temperature dependent far-UV CD measurements for 50 M
H2H5 were performed at temperatures of 5 °C to 95 °C in intervals of 5 °C.
Attenuated total reflection Fourier transform infrared (ATR-FTIR)
spectroscopy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A FTIR spectrometer (Tensor27, Bruker) operating in continuous scan mode and
coupled with a multipass attenuated total reflection (ATR) from BioATR Cell II
(Bruker) was used for measurements. The IR absorption spectra of samples dried
under N purge in the sample compartment of the ATR were collected. The peptide
2
–1
concentration was 1 mM. The spectral resolution was 4 cm with 64 scans averaged
in each spectrum.
Atomic force microscopy (AFM)
The AFM samples were prepared from 30 μM H2H5 or H2S5 solution (pH 9.0
Tris buffer). Before measurements, a 10-μL droplet of the H2H5 or H2S5 solution
was dropped and dried on a polished silicon wafer at room temperature. AFM images
were collected in tapping mode through the Bruker Dimension Icon SPM system. The
ACS Paragon Plus Environment

Biomacromolecules
Page 8 of 36
Page 8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AFM probe was adopted from the Nanoworld NCSTR series, in which the resonance
frequency and spring constant are 160 KHz and 7.4 N/m respectively. As to the
scanning parameters of the SPM system, the scan rate of the AFM tip was 0.5 Hz and
the resolution was 256  256 pixels. The morphology was analyzed in Nanoscope
software.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Transmission electron microscopy (TEM)
A 7 μL droplet of 30 M H2H5 or H2S5 solution (pH 9.0 or pH 10.0) was
dropped on a copper grid and held at room temperature for 1 min. Then, after removal
of the liquid, a 7 μL droplet of 3 % phosphotungstic acid was added and dyeing was
allowed to proceed for 2 mins. The grids were left covered in a Petri dish to dry at 60

C in an oven. TEM micrographs were collected on a Hitachi HT7700 transmission
electron microscope with an acceleration voltage of 120 kV.
Preparation of peptide stock solutions
Purified and lyophilized peptides were dissolved in buffered solution, 15 mM
Tris for pH 7.4−9.0, and CAPS for pH 10.0, to make a 1 mM or 200 M peptide stock
solution. The concentration of peptides was determined by the UV absorbance at 280
nm in 6 M guanidine hydrochloride solution at pH 6.5, using an extinction coefficient
−1
−1
of 1480 M cm for tyrosine.
Determination of the catalytic efficiency on ester hydrolysis
ACS Paragon Plus Environment

Page 9 of 36
Biomacromolecules
Page 9
1
2
3
4
5
6
7
8
9
The catalytic activities of the designed peptides were measured using 4-
nitrophenyl acetate (p-NPA), p-nitrophenyl-methoxy-acetate (p-NPMA) and p-
nitrophenyl-(2-phenyl)-propanoate (p-NPP) as the substrate. The hydrolytic product,
p-nitrophenol having an intense absorption at 405 nm, allowed us to determine the
reaction by monitoring the absorbance at 405 nm and 25 °C. A JASCO V-630
spectrophotometer associated with an STR-773 water thermostatic cell holder and
stirrer was used for measurements. A volume of 80 or 40 μL of the freshly prepared
peptide stock solution was added to (1920  x) or (1960  x) μL of buffer solution as
the blank solutions. After the initial absorbance (blank solution) was recorded as the
background signal, a volume of x μL of 20 mM p-NPA, 2 mM or 20 mM p-NPMA,
and 2 mM p-NPP stock in acetonitrile was subsequently added to obtain a final p-
NPA concentration of 100−1600 M, p-NPMA concentration of 40400 M, and p-
NPP concentration of 20100 M (the final acetonitrile content was less than 5% in
all reaction mixtures). Since p-NPMA and p-NPP hydrolyzed faster than p-NPA and
high concentrations caused the absorbance over the instrumental limit rapidly, lower
concentrations were used to more precisely measure the hydrolysis kinetics of p-
NPMA and p-NPP. In addition, the time-dependent absorbance of the substrate-only
solution was also measured as the self-hydrolysis blank. The absorbance was recorded
every 2 s for 100−200 s and the self-hydrolysis blank absorbance was subtracted from
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Biomacromolecules
Page 10 of 36
Page 10
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the measured data. Initial rates were determined from linear fits of the first 20 seconds
of the plots of absorbance versus time, and the recorded values were averages of
duplicate measurements. The extinction coefficients of the hydrolysis product at
different pH values were experimentally determined by measuring the absorbance of
p-nitrophenol in the specified buffers and by taking the average of at least three
measurements. The reported kinetic parameters (k and K ) were derived by fitting
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cat
M
1
퐾푀
1
1
the data to the Lineweaver-Burk or double-reciprocal equation: 푉
=
+
,
(
)
푉푚푎푥 [푆]
푉푚푎푥
where V is the reaction rate, Vmax = k [E] is the maximal velocity, k is the catalytic
cat
cat
rate constant, K is the binding constant, k /K is the enzymatic efficiency, and [E]
M
cat
M
and [S] are the enzyme and substrate concentrations, respectively.
Results and discussion
Peptide design
In a PPII helix, each turn contains three residues, and thus the residues at
positions i and i + 3 will be on the same face. According to the structural features, we
previously designed and synthesized a series of polyproline peptides and showed that
26
they exhibit catalytic activity on ester hydrolysis. Among the polyproline peptides,
those with His-His or His-Ser pair incorporated had decent activity, and particularly
the replacement of Pro to Hyp increased the efficiency for the His-His pair (Hyp-
26
H3H6). Therefore, in this work we introduced His or Ser residues into the i and i + 3
ACS Paragon Plus Environment

Page 11 of 36
Biomacromolecules
Page 11
1
2
3
4
5
6
7
8
9
positions to form a His-His or His-Ser catalytic dyad and designed two new MAX1
conjugated peptides, H2H5 and H2S5 (Figure 1A). In the designed peptides, one
glycine residue was inserted between MAX1 and the oligoproline as a spacer, while a
Tyr-Gly sequence was incorporated into the N-terminus for concentration
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
determination. According to our design and as shown in Figure 1B, the MAX1
peptide fragment, comprising a D-proline in the β-turn, was expected to self-assemble
into -sheets, as reported in the previous studies,24, 25 and the N-terminal Hyp-rich
oligopeptide would be exposed to solvent and form a PPII-like helix. In addition, to
examine if the assembled structure from MAX1 would be the critical factor affecting
catalytic activity, we also prepared two control peptides, Hyp-H3H6 ((Hyp) -His-
2
(
Hyp) -His-(Hyp) -Gly-Tyr) and Hyp-S3H6 ((Hyp) -His-(Hyp) -Ser-(Hyp) -Gly-
2 5 2 2 5
Tyr), for comparison. Each peptide had an acetylated N-terminus and an amidated C-
terminus. The peptides were chemically synthesized via solid-phase methods.
Characterizations of Peptide Assemblies
Upon the preparation of the peptides, we first conducted pH-dependent circular
dichroism (CD) measurements on the H2H5 and H2S5 peptides to characterize their
secondary structures. As shown in Figure 2, both H2H5 and H2S5 form random coils
at neutral and acidic pH, but they exhibit the characteristics of -structures when the
pH values are greater than 9.0. This observation is similar to what was found for the
ACS Paragon Plus Environment

Biomacromolecules
Page 12 of 36
Page 12
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
peptides MAX1 and VK2H.17, 25 Since the Lys sidechains will be protonated at low
pH, the repulsion between the positive charges will prohibit the peptides from forming
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

-sheets. It was noted that the change in the CD spectra from pH 9.0 to pH 10.0 was
very small for H2H5 but quite significant for H2S5, indicating that H2S5 forms a
more highly packed -structure at pH 10.0. The peptides were further characterized
by attenuated total reflection (ATR) FTIR spectroscopy. As shown in Figure 3, at pH
9
1
.0, the ATR-FTIR spectra of H2H5 and H2S5 display amide I bands at 1625 and
1
1
674 cm , and the characteristic band around 1530 cm , suggesting that the peptides
fold into -sheets,29, 30 which is consistent with the CD measurements. Although the
peptides do not exhibit intense IR signals at low concentrations, the characteristic
bands can still be observed, indicating that the peptides have a relatively high
propensity to form -structures.
ACS Paragon Plus Environment

Page 13 of 36
Biomacromolecules
Page 13
1
2
3
4
5
6
7
8
9
Figure 2. pH-dependent far-UV CD spectra of (a) H2H5 and (b) H2S5 at 25 C. The
peptide concentration was 50 M.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. FT-IR spectra for H2H5 and H2S5 with a concentration of 50 M and 1
mM at pH 9.0.
2
5
Since the MAX1 peptide could self-assemble into fibrils at alkaline pH, we also
used transmission electron microscopy (TEM) to examine if our designed peptides
could self-assemble into higher-order structures. As shown in Figure 4, H2H5 and
H2S5 both self-assemble into network fibrils at pH 9.0, showing that the additional
Hyp-rich peptide fragment does not alter the assembling property of MAX1.
Additionally, atomic force microscopy (AFM) further showed the fibrous structure of
the assemblies (Figure 4). Based on the assembled structure of the MAX1 sequence,
the catalytic dyad of His-His or His-Ser on the fibrils is expected to be solvent-
exposed, allowing it to participate in the hydrolysis of substrates. Such an
arrangement may serve as a good scaffold for catalyzing ester hydrolysis, as
ACS Paragon Plus Environment

Biomacromolecules
Page 14 of 36
Page 14
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
24
suggested by a previous study. To compare the assembled structures at different pH
conditions, we also took the TEM images of H2H5 and H2S5 at pH 10.0. As shown in
Figure S4 of the Supporting Information, the assemblies of H2H5 become film-like at
pH 10.0 while H2S5 assembles into more packed rod-like structures upon increasing
pH from 9.0 to 10.0, which is consistent with the CD measurements. The difference in
assembled topology of H2H5 and H2S5 could be due to the residue variance on the
Hyp-rich peptide. The -sheets induced by MAX1 might be similar but the small
hydroxyl group of Ser and the bulky imidazole group of His could affect the packing
in their large scale assembled constructs, resulting in various morphology as shown in
Figure S4 of the Supporting Information.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. TEM (left) and AFM (right) images of the assembled fibrils for (A) H2H5
and (B) H2S5 at pH 9.0.
ACS Paragon Plus Environment

Page 15 of 36
Biomacromolecules
Page 15
1
2
3
4
5
6
7
8
9
Evaluation of Catalytic Activity for the Designed Peptides
To evaluate the catalytic activity of our designed peptides, we first used p-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitrophenyl acetate (p-NPA) as the substrate to measure the hydrolytic rates. Since the
hydrolytic product, p-nitrophenol (Scheme S1 in the Supporting Information), has an
intense absorbance at 405 nm, the initial hydrolytic rate can be determined by UV-Vis
spectroscopy. As shown in Figure S5 of the Supporting Information, at pH 9.0, the
hydrolytic reaction is much faster in the presence of H2H5 or H2S5 than with control
peptides (Hyp-H3H6 and Hyp-H3S6), showing that H2H5 and H2S5 exhibit decent
catalytic efficiency. As shown in Figure S6B of the Supporting Information, the
double-reciprocal plots of the initial rate versus substrate concentration are linear for
H2H5 and H2S5, indicating they follow Michaelis-Menten kinetics. The curves were
fit to the Lineweaver-Burk equation to calculate the kinetic parameters. In contrast,
for the control peptides Hyp-H3H6 and Hyp-H3S6, there were no significant signal
differences after subtracting the self-hydrolysis blank and their double-reciprocal plots
could not be obtained, indicating their catalytic activity is relatively low. This
observation strongly suggests that the assembled structure makes a significant
contribution to the catalytic activity. As shown in Table 1, the k /K values for
cat
M

1
1
1 1
H2H5 and H2S5 are 12.1 M s and 13.3 M s respectively. Compared to the
VK2H peptide, which contains an HSG triplet and a MAX1 peptide and has a k /K
cat
M
ACS Paragon Plus Environment

Biomacromolecules
Page 16 of 36
Page 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1 1
17
value of 19.18 M s on p-NPA hydrolysis, H2H5 shows a comparable catalytic
efficiency under the same condition. The results suggest that the His-His or His-Ser
pair on the Hyp-rich fragment of our designed peptide can form a catalytic dyad.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. The catalytic parameters on ester hydrolysis for the designed peptides at 25
C.

3
1
1 1
peptide
H2H5
substrate
pH
7.4
k (10 s )
K (mM)
k /K (M s )
cat m
cat
m
p-NPA
2.10  0.01
37.2  0.57
9.98  0.01
69.8  1.04
7.59  0.01
0.78  0.01
17.4  0.01
47.4  0.14
38.9  0.49
7.27  0.01
2.99  1.26
3.07  1.28
0.41  0.04
0.15  0.04
0.13  0.01
0.75  0.24
1.31  0.19
0.48  0.03
0.19  0.07
0.05  0.01
0.71  0.29
12.1  5.03
24.3  2.62
452  110
58.1  3.41
1.04  0.34
13.3  0.38
99.4  6.55
203  71
9
.0
1
1
0.0
9.0
9.0
7.4
p-NPMA
p-NPP
H2S5
p-NPA
9
.0
0.0
9.0
9.0
p-NPMA
p-NPP
145  30
The parameters were derived from fitting the data to the Lineweaver-Burk equation.
The deviations of k and K were the standard errors of fitting and were used to
cat
M
calculated the deviation of k /K with the equation σ
= (푘 /퐾 ) ×
푐푎푡 푀
cat
M
푘cat/퐾M
2
2
(σ푘푐푎푡/푘 ) + (σ /퐾 ) .
푐푎푡 퐾푀 푀
We also measured the catalytic activities of H2H5 and H2S5 at pH 7.4 and pH
10.0 because their assembly was pH-dependent and could be correlated with the
activity. Since p-NPA was known to rapidly hydrolyze under alkaline conditions, we
also monitored the time-dependent absorbance for the p-NPA only solution at pH 10.0
(Figure S7 in the Supporting Information). Compared to the p-NPA only solution, the
ACS Paragon Plus Environment

Page 17 of 36
Biomacromolecules
Page 17
1
2
3
4
5
6
7
8
9
hydrolytic reaction is much faster in the presence of H2H5 or H2S5, showing that our
designed peptides do catalyze the reaction at pH 10.0. As shown in Figure S6A of the
Supporting Information, Table 1, and Figure 5B, both H2H5 and H2S5 exhibit much
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1 1
1 1
less activity (k /K = 0.71 M s and 1.04 M s ) at pH 7.4 than at high pH
cat
M
values, again demonstrating that the assembled -structure from MAX1 is crucial to
enhance their catalytic efficiency. At pH 10.0 both H2H5 and H2S5 exhibit greater
catalytic activity than that at pH 9.0 (Figure 5 and Table 1). Compared to that at pH
9
.0, H2H5 has a twofold increase in its catalytic activity at pH 10.0 (k /K = 24.3
cat M
1 1
1 1
M s ). Strikingly, H2S5 has a k /K value of 99.4 M s at pH 10.0, which is a
cat
M
more than 7-fold increase from that at pH 9.0. Since CD measurements show that
H2S5 has more intense -structure characteristic signals at pH 10.0 than at pH 9.0 and
TEM images reveal that it assembles into even more packed and dense structures
upon increasing pH from 9.0 to 10.0, its kinetic data strongly suggest that the catalytic
activity is highly correlated with the assembled construct. To the best of our
knowledge, the hydrolase activity of H2S5 on p-NPA at pH 10.0 is the highest among
the reported peptide-based esterases which do not contain metals or cofactors.1, 9
ACS Paragon Plus Environment

Biomacromolecules
Page 18 of 36
Page 18
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. (A) The esterase activity for the designed peptides on catalyzing p-NPA
hydrolysis at pH 10.0 and 25 °C, with solid lines showing the best fits to the
Lineweaver-Burk equation. (B) The esterase activities of H2H5 and H2S5 at different
pH values and 25 °C. The measurements were conducted with a peptide concentration
of 40 μM.
The enhanced catalytic activity observed on H2H5 and H2S5 supports our
hypothesis that the MAX1 peptides assemble into a high-order structure to facilitate
the arrangement of the catalytic residues on the Hyp-rich peptides to serve as an
active site. In the assembled construct, the Hyp-rich peptide might not fold into a
typical PPII helix but the catalytic residues (His and Ser) could adjust their sidechain
conformation to reduce the distance within the dyad to favor the electron transfer
during hydrolytic reactions. Although our original design was to form a catalytic dyad
from the same peptide, we could not exclude the possibility that the dyad was actually
ACS Paragon Plus Environment

Page 19 of 36
Biomacromolecules
Page 19
1
2
3
4
5
6
7
8
9
formed from inter-strand residues. It is likely that the Hyp-peptides are relatively
close upon the assembly of MAX1 peptides to allow the catalytic residues on adjacent
strands to form catalytic dyads. In each case, the MAX1 assembly provides a great
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
scaffold and support to facilitate the formation of catalytic dyads. The open active site
is also critical for good activity as illustrated in the previous studies.26, 31, 32 In our
designed peptide, the rigidity of Hyp residues may make the environment of
polyproline peptides loosened and less ordered in the assembled -structure, which
can allow the His-His or His-Ser dyad highly exposed to solvent and substrates to
facilitate the catalytic reactions.
To gain more insight into the thermal stability of our designed peptide, we
further conducted temperature-dependent CD measurements on H2H5. As shown in
Figure S8 of the Supporting Information, H2H5 maintains a -structure even at 95 C
and pH 9.0, indicating that the assembled structure is quite robust. Additionally, we
also measured its catalytic activity on p-NPA hydrolysis at 37 °C (Figure S9 in the
Supporting Information), and found that H2H5 had an even higher catalytic efficiency
1 1
with a k /K value of 31.3 M s . The results demonstrate that H2H5 is very stable
cat
M
and still active even at a high temperature. Moreover, for H2H5, the k value is
cat
1
1
higher at 37 °C (0.546 s ) than at 25 °C (0.0372 s ), and the K value is also greater
M
at 37 °C (17.5 mM) than at 25 °C (3.07 mM), indicating that increasing the
ACS Paragon Plus Environment

Biomacromolecules
Page 20 of 36
Page 20
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
temperature will speed the reaction but reduce the substrate affinity.
Besides p-NPA, we also used p-nitrophenyl-methoxy-acetate (p-NPMA) and p-
nitrophenyl-(2-phenyl)-propanoate (p-NPP) as the substrates to examine the hydrolase
activity of H2H5 and H2S5 on other esters. Since p-NPMA and p-NPP can quickly
hydrolyze in aqueous solution even without catalysts, we monitored their reaction in
the absence of the designed peptides as the control for comparison. As shown in
Figures S10 and S11 of the Supporting Information, the hydrolysis of p-NPMA and p-
NPP in the presence of H2H5 or H2S5 is faster than that without these peptides,
indicating that H2H5 and H2S5 do have catalytic activity on the hydrolysis of these
two substrates. In contrast, Hyp-H3H6 and Hyp-H3S6 do not display catalytic activity
because the time-dependent absorbance for the solution with either peptide is almost
identical to the substrate only blank. The results further support that the assembled
construct plays a key role in making the peptide active on catalyzing ester hydrolysis.
As shown in Figure 6 and Table 1, H2H5 and H2S5 both exhibit good activity on
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1 1
1 1
catalyzing the hydrolysis of p-NPMA (k /K = 452 M s for H2H5, 203 M s
cat
M
1 1
1 1
for H2S5) and p-NPP (k /K = 58.1 M s for H2H5, 145 M s for H2S5). The
cat
M
data also show that H2H5 and H2S5 both has better catalytic activity on p-NPMA and
p-NPP than p-NPA under the same condition due to their greater affinity for these two
ester substrates. This implies that we may modulate the catalytic amino acid
ACS Paragon Plus Environment

Page 21 of 36
Biomacromolecules
Page 21
1
2
3
4
5
6
7
8
9
sequences or dyads in the future to develop efficient artificial hydrolases on different
substrates.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Esterase activity for the designed peptides on catalyzing (A) p-NPMA and
(B) p-NPP at pH 9.0 and 25 °C. The solid lines indicate the best fits to the
Lineweaver-Burk equation. The peptide concentration was 4 μM for the
measurements.
Conclusions
In this work, we have successfully conjugated a polyproline fragment containing
a catalytic dyad (His-His or His-Ser) into the self-assembling peptide MAX1 and
shown that the designed peptides can form -structured fibrils under alkaline
conditions and exhibit high catalytic activity on ester hydrolysis. Since the assembled
structure is mainly from the MAX1 moiety, the catalytic dyad should be exposed to
ACS Paragon Plus Environment

Biomacromolecules
Page 22 of 36
Page 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
solvent as the active site. With the support of the assembled construct, H2H5 and
H2S5 containing the catalytic Hyp-based sequence can serve as efficient hydrolases,
and their pH-dependent conformations are correlated with the catalytic activity.
Through the assistance of a self-assembling peptide, our newly designed polyproline-
based hydrolases exhibit great improvement in catalyzing ester hydrolysis.
Particularly, as compared to the metal or cofactor-free peptide-based hydrolases
reported in the literature, H2S5 has the greatest catalytic efficiency on p-NPA
hydrolysis at pH 10.0. In conclusion, we have demonstrated that combining a
polyproline-based dyad with a self-assembling peptide can create a good and effective
scaffold to catalyze hydrolytic reactions. Our results provide a new, valuable approach
and can be applied to the development of artificial enzymes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Acknowledgements
We are grateful for the financial support of the Ministry of Science and
Technology of Taiwan (MOST 107-2113-M-007-002 and MOST 108-2113-M-007-
0
12). This work was also financially supported by the “Frontier Research Center on
Fundamental and Applied Sciences of Matters” from the Featured Areas Research
Center Program within the framework of the Higher Education Sprout Project
(107QR001I5) by the Ministry of Education (MOE) in Taiwan. We thank the NTHU
instrument center for TEM measurements and the NCHU instrument center for AFM
ACS Paragon Plus Environment

Page 23 of 36
Biomacromolecules
Page 23
1
2
3
4
5
6
7
8
9
experiments. We also thank Ms. Pei-Yu Hung for her help on the synthesis of p-
NPMA and p-NPP, and Prof. Li-Kang Chu for providing the ATR-FTIR instrument.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Supporting Information
HPLC chromatograms, MALDI-TOF spectra, temperature-dependent CD
spectra, UV-monitored hydrolytic reaction plots, additional TEM images, and
additional Lineweaver-Burk plots are included. This material is available free
charges via the Internet at http://pubs.acs.org.
Conflict of Interest
The authors declare no conflict of interest.
References
(
1) Korendovych, I. V.; DeGrado, W. F. Catalytic Efficiency of Designed Catalytic
Proteins. Curr. Opin. Struct. Biol. 2014, 27, 113-121.
2) Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.; Woolfson, D. N.
(
Installing Hydrolytic Activity into a Completely De novo Protein Framework. Nat.
Chem. 2016, 8, 837-844.
(
3) Zastrow, M. L.; Peacock, A. F.; Stuckey, J. A.; Pecoraro, V. L. Hydrolytic
Catalysis and Structural Stabilization in a Designed Metalloprotein. Nat. Chem. 2012,
, 118-123.
4
(4) Zastrow, M. L.; Pecoraro, V. L. Influence of Active Site Location on Catalytic
ACS Paragon Plus Environment

Biomacromolecules
Page 24 of 36
Page 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Activity in De novo-Designed Zinc Metalloenzymes. J. Am. Chem. Soc. 2013, 135,
895-5903.
5) Bezer, S.; Matsumoto, M.; Lodewyk, M. W.; Lee, S. J.; Tantillo, D. J.; Gagne, M.
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
R.; Waters, M. L. Identification and Optimization of Short Helical Peptides with
Novel Reactive Functionality as Catalysts for Acyl Transfer by Reactive Tagging.
Org. Biomol. Chem. 2014, 12, 1488-1494.
(6) Matsumoto, M.; Lee, S. J.; Gagne, M. R.; Waters, M. L. Cross-Strand Histidine-
Aromatic Interactions Enhance Acyl-Transfer Rates in Beta-Hairpin Peptide
Catalysts. Org. Biomol. Chem. 2014, 12, 8711-8718.
(7) Matsumoto, M.; Lee, S. J.; Waters, M. L.; Gagne, M. R. A Catalyst Selection
Protocol That Identifies Biomimetic Motifs From -Hairpin Libraries. J. Am. Chem.
Soc. 2014, 136, 15817-15820.
(8) Wang, P. S.; Nguyen, J. B.; Schepartz, A. Design and High-Resolution Structure
3
of a  -Peptide Bundle Catalyst. J. Am. Chem. Soc. 2014, 136, 6810-6813.
(
9) Zozulia, O.; Dolan, M. A.; Korendovych, I. V. Catalytic Peptide Assemblies.
Chem. Soc. Rev. 2018, 47, 3621-3639.
10) Díaz-Oltra, S.; Berdugo, C.; Miravet, J. F.; Escuder, B. Study of the Effect of
(
Polymorphism on the Self-Assembly and Catalytic Performance of an L-Proline
Based Molecular Hydrogelator. New J. Chem. 2015, 39, 3785-3791.
ACS Paragon Plus Environment

Page 25 of 36
Biomacromolecules
Page 25
1
2
3
4
5
6
7
8
9
(11) Tena-Solsona, M.; Nanda, J.; Díaz-Oltra, S.; Chotera, A.; Ashkenasy, G.;
Escuder, B. Emergent Catalytic Behavior of Self-Assembled Low Molecular Weight
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Peptide-Based Aggregates and Hydrogels. Chem. Eur. J. 2016, 22, 6687-6694.
(12) Garcia, A. M.; Kurbasic, M.; Kralj, S.; Melchionna, M.; Marchesan, S. A
Biocatalytic and Thermoreversible Hydrogel from a Histidine-Containing Tripeptide.
Chem. Commun. 2017, 53, 8110-8113.
(13) Zhang, C.; Xue, X.; Luo, Q.; Li, Y.; Yang, K.; Zhuang, X.; Jiang, Y.; Zhang, J.;
Liu, J.; Zou, G.; Liang, X. J. Self-Assembled Peptide Nanofibers Designed as
Biological Enzymes for Catalyzing Ester Hydrolysis. ACS Nano 2014, 8, 11715-
1
1723.
(14) Huang, Z.; Guan, S.; Wang, Y.; Shi, G.; Cao, L.; Gao, Y.; Dong, Z.; Xu, J.; Luo,
Q.; Liu, J. Self-Assembly of Amphiphilic Peptides into Bio-Functionalized
Nanotubes: A Novel Hydrolase Model. J. Mater. Chem. B 2013, 1, 2297-2304.
(15) Singh, N.; Conte, M. P.; Ulijn, R. V.; Miravet, J. F.; Escuder, B. Insight into the
Esterase Like Activity Demonstrated by an Imidazole Appended Self-Assembling
Hydrogelator. Chem. Commun. 2015, 51, 13213-13216.
(16) Maeda, Y.; Javid, N.; Duncan, K.; Birchall, L.; Gibson, K. F.; Cannon, D.;
Kanetsuki, Y.; Knapp, C.; Tuttle, T.; Ulijn, R. V.; Matsui, H. Discovery of Catalytic
Phages by Biocatalytic Self-Assembly. J. Am. Chem. Soc. 2014, 136, 15893-15896.
ACS Paragon Plus Environment

Biomacromolecules
Page 26 of 36
Page 26
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(17) Zhang, C.; Shafi, R.; Lampel, A.; MacPherson, D.; Pappas, C. G.; Narang, V.;
Wang, T.; Maldarelli, C.; Ulijn, R. V. Switchable Hydrolase Based on Reversible
Formation of Supramolecular Catalytic Site Using a Self‐Assembling Peptide. Angew.
Chem. Int. Ed. 2017, 56, 14511-14515.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(18) Al-Garawi, Z. S.; McIntosh, B. A.; Neill-Hall, D.; Hatimy, A. A.; Sweet, S. M.;
Bagley, M. C.; Serpell, L. C. The Amyloid Architecture Provides a Scaffold for
Enzyme-Like Catalysts. Nanoscale 2017, 9, 10773-10783.
(19) Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stohr, J.; Smith, T. A.; Hu, X.;
DeGrado, W. F.; Korendovych, I. V. Short Peptides Self-Assemble to Produce
Catalytic Amyloids. Nat. Chem. 2014, 6, 303-309.
(
20) Heier, J. L.; Mikolajczak, D. J.; Böttcher, C.; Koksch, B. Substrate Specificity of
an Actively Assembling Amyloid Catalyst. Pept. Sci. 2017, 108, e23003.
21) Lengyel, Z.; Rufo, C. M.; Moroz, Y. S.; Makhlynets, O. V.; Korendovych, I. V.
(
Copper-Containing Catalytic Amyloids Promote Phosphoester Hydrolysis and
Tandem Reactions. ACS Catal. 2018, 8, 59-62.
(22) Luong, T. Q.; Erwin, N.; Neumann, M.; Schmidt, A.; Loos, C.; Schmidt, V.;
Fändrich, M.; Winter, R. Hydrostatic Pressure Increases the Catalytic Activity of
Amyloid Fibril Enzymes. Angew. Chem. Int. Ed. 2016, 55, 12412-12416.
(23) Wang, M.; Lv, Y.; Liu, X.; Qi, W.; Su, R.; He, Z. Enhancing the Activity of
ACS Paragon Plus Environment

Page 27 of 36
Biomacromolecules
Page 27
1
2
3
4
5
6
7
8
9
Peptide-Based Artificial Hydrolase with Catalytic Ser/His/Asp Triad and Molecular
Imprinting. ACS Appl. Mater. Interfaces 2016, 8, 14133-14141.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
24) Nagy-Smith, K.; Moore, E.; Schneider, J.; Tycko, R. Molecular Structure of
Monomorphic Peptide Fibrils within a Kinetically Trapped Hydrogel Network. Proc.
Natl. Acad. Sci. USA 2015, 112, 9816-9821.
(25) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger,
J. Responsive Hydrogels from the Intramolecular Folding and Self-Assembly of a
Designed Peptide. J. Am. Chem. Soc. 2002, 124, 15030-15037.
(
26) Hung, P.-Y.; Chen, Y.-H.; Huang, K.-Y.; Yu, C.-C.; Horng, J.-C. Design of
Polyproline-Based Catalysts for Ester Hydrolysis. ACS Omega 2017, 2, 5574-5581.
27) Knowlton, R. C.; Byers, L. D. Acyl Substituent Effects on Ester Aminolysis. J.
Org. Chem. 1988, 53, 3862-3865.
28) Richter, F.; Blomberg, R.; Khare, S. D.; Kiss, G.; Kuzin, A. P.; Smith, A. J.;
(
(
Gallaher, J.; Pianowski, Z.; Helgeson, R. C.; Grjasnow, A.; Xiao, R.; Seetharaman, J.;
Su, M.; Vorobiev, S.; Lew, S.; Forouhar, F.; Kornhaber, G. J.; Hunt, J. F.;
Montelione, G. T.; Tong, L.; Houk, K. N.; Hilvert, D.; Baker, D. Computational
Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis. J. Am. Chem.
Soc. 2012, 134, 16197-16206.
(29) Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. ATR-FTIR: A
ACS Paragon Plus Environment

Biomacromolecules
Page 28 of 36
Page 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
“
Rejuvenated” Tool to Investigate Amyloid Proteins. Biochim. Biophys. Acta 2013,
828, 2328-2338.
30) Naik, S. S.; Savin, D. A. Poly(Z-lysine)-Based Organogels: Effect of Interfacial
Frustration on Gel Strength. Macromolecules 2009, 42, 7114-7121.
31) Kaplan, J.; DeGrado, W. F. De novo Design of Catalytic Proteins. Proc. Natl.
Acad. Sci. USA 2004, 101, 11566-11570.
32) Ting, Y.-H.; Chen, H.-J.; Cheng, W.-J.; Horng, J.-C. Zinc(II)–Histidine Induced
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
(
Collagen Peptide Assemblies: Morphology Modulation and Hydrolytic Catalysis
Evaluation. Biomacromolecules 2018, 19, 2629-2637.
ACS Paragon Plus Environment

Page 29 of 36
Biomacromolecules
Page 29
1
2
3
4
5
6
7
8
9
Table of Contents Graphic
The conjugation of a polyproline-based catalytic sequence and a self-assembling
peptide produces a highly active hydrolase.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment