Covalent Catalysis by Cross β Amyloid Nanotubes

Article abstract of DOI:10.1021/jacs.9b13517

The binding pockets of extant enzymes feature precise positioning of amino acid residues that facilitate multiple complex transformations exploiting covalent and non-covalent interactions. Reversible covalent anchoring is extensively used as an efficient tool by Nature for activating modern enzymes such as esterases and dehydratases and also for proteins like opsins for the complex process of visual phototransduction. Here we construct paracrystalline amyloid surfaces through the self-propagation of short peptides which offer binding pockets exposed with arrays of imidazoles and lysines. As covalent catalysis is utilized by modern-day enzymes, these homogeneous amyloid nanotubes exploit Schiff imine formation via the exposed lysines to efficiently hydrolyze both activated and inactivated esters. Controls where lysines were mutated with charged residues accessed similar morphologies but did not augment the rate. The designed amyloid microphases thus foreshadow the generation of binding pockets of advanced proteins and have the potential to contribute to the development of functional materials.

Full text of DOI:10.1021/jacs.9b13517

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Communication
Covalent Catalysis by Cross # Amyloid Nanotubes
Baishakhi Sarkhel, Ayan Chatterjee, and Dibyendu Das
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b13517 • Publication Date (Web): 21 Feb 2020
Downloaded from pubs.acs.org on February 22, 2020
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Covalent Catalysis by Cross  Amyloid Nanotubes
Baishakhi Sarkhel, Ayan Chatterjee and Dibyendu Das*
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Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur 741246,
India
Supporting Information Placeholder
ABSTRACT: The binding pockets of extant enzymes feature
precise positioning of amino acid residues that facilitate multiple
complex transformations exploiting covalent and non-covalent
interactions. Reversible covalent anchoring is extensively used as
an efficient tool by Nature for activating modern enzymes such as
esterases, dehydratases and also for proteins like opsins for the
complex process of visual phototransduction. Here we construct
paracrystalline amyloid surfaces through the self-propagation of
short peptides which offer binding pockets exposed with arrays of
imidazoles and lysines. As covalent catalysis is utilized by modern-
day enzymes, these homogenous amyloid nanotubes exploit Schiff
imine formation via the exposed lysines to efficiently hydrolyse
both activated and inactivated esters. Controls where lysines were
mutated with charged residues accessed similar morphologies but
did not augment rate. The designed amyloid microphases thus
foreshadow the generation of binding pockets of advanced proteins
and have the potential to contribute to the development of
functional materials.
Nature accesses vastly orthogonal chemical strategies to regulate
complex multi-step chemical processes catalyzed by the highly
evolved present day enzymes.1 These extant biocatalysts, products
of millions of years of evolution, feature well-defined three-
dimensional architectures created from folding of long polymers of
amino acids and offer specific binding pockets for small molecular
guests.1-3 Precise positioning of the amino acid residues in the
active site is critical for catalysis through a sequence of cascade
reactions.1-4 Involvement of multiple active site residues is required
for the complicated tasks of manipulation of protonation events,
polarizing and activating the nucleophiles and electrophiles and so
forth. Notably, in the regulation of complex biological processes,
reversible covalent bond formation has also been extensively used
as an efficient tool by Nature.5-8 Direct covalent bonding for the
enzyme or cofactor with the substrates has been seen including the
formation of covalent intermediates such as acyl enzyme or Schiff
base intermediate.7 Enzymes such as serine protease, protein kinase
and phosphatase utilize transient covalent bonds to improve
substrate affinity and lower the activation energy.5-8 Further, for the
beautiful phenomenon of visual phototransduction, reversible
Imine formation is exploited by opsin where the lysine side chain
bonds reversibly with a chromophoric aldehyde.7 Also, the
biosynthetic shikimate pathway followed by dehydratases proceeds
through imine formation between lysine side chain and the
carbonyl group of the substrate. Mutating lysine results in sharp
decline of activity, suggesting the importance of the reversible
covalent bond.8
Figure 1. Schematic representation of the hydrolysis reaction
facilitated through imine formation. a, b) Blue and green tube
represents Im-KL and Im-RL nanotubes, c) structures of amyloid
sequences.
repetitive structures of cross  amyloid folds have been argued as
the primitive protein folds by works of Ulijn, Lynn, and
Korendovych.9-12 These offer solvent exposed binding surfaces to
interact with diverse guests.13 Herein, we report catalytic potential
of one such amyloid phase, which exploits the process of covalent
catalysis with exposed lysine side arms and imidazoles to show
hydrolytic activity and cleave both activated and inactivated esters
(Figure 1).
We asked whether a short amyloid-based peptide can use such
covalent bonding between the catalyst and substrate to amplify the
catalytic rates. Short peptides capable of accessing inherent
We used peptide fragment 17LVFFA21 from the nucleating core of
amyloid β 1-42, recognized as the fibrillar aggregates of
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Alzheimer’s disease.14-15 This pentapeptide core has been known to
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access diverse morphologies.14-15 The sequence H2N-LVFFA-
COOH was condensed with lysine and leucine residues at the N-
and C-termini respectively. Further, the N terminal lysine residue
was condensed with imidazole acetic acid leading to the sequence
of Im-KLVFFAL-NH2 (Im-KL, Figure 1). It was expected that the
lysine coupled with imidazole group would be exposed to the
solvent upon assembly enabling interaction with substrate. While
the imidazole can assist in hydrolysis, the exposed lysine residues
would be used for imine bond formation and thus can augment rates
by covalent catalysis. This sequence assembled into homogenous
nanotubular structures (diameter 50±10 nm) as observed under
transmission electron microscopy (TEM, Figure 2a) and atomic
force microscopy (AFM, Figure 2d, average height of nanotubes
was 7.6 ±0.7 nm, Figure S1, SI). The -sheet conformation of Im-
KL assemblies was confirmed from circular dichroism (CD) and
FTIR spectroscopy (Figure 2f, S2, SI). Powder X-ray diffraction
(PXRD) had reflection at d-spacing of 4.65 Å and 10.62 Å
corresponding to the distances of H-bonded -strands and -sheet
laminates respectively (Figure 2f).14b To probe that indeed the
charged lysines coupled with imidazoles are exposed to solvent,
negatively charged Au-NP was added to the assemblies. Ordered
arrays of nanoparticles were observed to be bound to the nanotubes
(Figure S3, S4a). Control done with cationic AuNP did not result
in specific binding (Figure S4b). To probe the binding capabilities
of these nanotubes, we used a small hydrophobic fluorescent dye
(coumarin 343). The  strands arranged in antiparallel fashion
expose both the lysines and leucines side chains to solvent and thus
can bind non-polar substrates.12d,14b,15a Confocal fluorescence
microscopy revealed fluorescent structures when incubated with
coumarin 343, thus supporting the binding capability of these
assemblies (Figure 2e, Figure S5, SI for details and control).
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Figure 2. TEM micrographs of a) Im-KL b) Im-RL, c) Im-OL,
AFM of d) Im-KL, e) Confocal microscopy image of coumarin-
343 (ex = 488 nm) bound Im-KL, f) CD spectra and PXRD (inset)
of Im-KL.
An analogous substrate lacking the keto group (2) was synthesized
(Figure 3a). Notably, Im-KL showed only a modest enhancement
in activity with 2 when compared to Im-RL ([vi]Im-KL=6.2±0.1 M
min-1, [vi]Im-RL= 3.1±0.5 M min-1) which supports the activating
role of the lysines for the keto containing substrates (Figure 3b).
The hydrolysis reactions followed typical Michaelis-Menten
kinetics (Figure S7, Table S2). The substrate affinity of the lysine
nanotubes towards the keto containing substrate was much higher
compared to the control arginine exposed nanotubes as reflected
from the Km value of 1 for Im-KL which was 3 fold lower
compared to Im-RL. The catalytic efficiency of Im-KL for the keto
containing substrate was also following a similar trend (Table S2).
Reduction in presence of excess NaBH3CN was done to trap the
kinetically unstable imine of 1 with Im-KL to the corresponding
amine which was confirmed by Mass spectrometry (SI). To further
investigate the role of side chain of the amino acid at 16th position,
we mutated the cationic lysine with negatively charged glutamic
acid (Im-EL, Figure 1). Im-EL did not assemble to form
homogenous nanotube but instead accessed sheet like
morphologies (Figure S8). Activity was found to be significantly
lower due to lack of the primary amine for the imine bond and also
due to possible increment of pKa of neighboring imidazoles (Figure
3c).
We investigated the role of the lysine exposed Im-KL nanotubes to
assist in the hydrolase like activity. Keto ester 4-nitrophenyl 4-
oxopentanoate (1) featuring carbonyl group was synthesized with
the expectation that it can form iminium intermediate upon binding
to Im-KL (Figure 3a). As control, the lysine residue of the Im-KL
group was mutated with arginine, generating the sequence Im-RL
(Im-RLVFFAL). Im-RL would not be able to form such imine
bonds due the presence of non-nucleophilic guanidine moiety in
place of a primary amine (Figure 1). Im-RL assembled to form
nanotubes with similar diameter of 46±4 nm, presumably due to the
similar protonation states of lysine and arginine (Figure 2b). CD
indicated similar -sheet like conformation in assembled state of
Im-RL (Figure S6s). Significantly, Im-KL showed an order of
magnitude higher activity for 1 ([vi]Im-KL=5.6 ±0.5 M min-1,
Figure 3c) compared to the control Im-RL ([vi]Im-RL=0.49±0.07
M min-1, ca. 11 fold at pH=8, Table S1, Figure 3b) suggesting the
important role of lysine residues (no acylated Im-KL peptide was
observed from HPLC).
To check the role of side chain length of the amine and also to
investigate the effect of neighboring strands of the cross β
assembly, lysine (four methylenes) was mutated with its shorter
congener ornithine (3 methylenes) to generate Im-OL (Im-
OLVFFAL). Im-OL assembled to form morphologically similar
nanotubes of diameter 40±5 nm (Figure 2c). Higher activity was
observed for Im-OL compared to Im-RL ([vi]=3.6±0.6 M min-1,
Figure 3c) further supporting the importance of amines.
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Figure 3. a) Chemical structure of substrates b) Bar diagram of the ratio of hydrolysis rates by Im-KL and Im-RL for 1 and 2. c) Bar
diagram of hydrolysis rate of 1 by Im-KL, Im-RL, Im-EL, Im-OL and Ac-KL in presence of imidazoleacetic acid, [Peptide]= 1mM,
[Substrate]= 50 µM.d) Grids showing the amine accessible surface of Im-KL and Im-OL nanotubes with the average conformational ranges
indicated by blue and red circles respectively.
However, the activity for Im-OL was lower than Im-KL, indicating
the assistance from the exposed amines from the proximal strands
of the cross β structures. PXRD patterns suggested larger accessible
surface for the lysines versus the ornithines (Figure 3d and Figure
S9, see SI for details). Further, Im-KL nanotubes were
disassembled with hexafluoroisopropanol (HFIP, SI). Disassembly
of secondary structure was suggested from loss of CD signals
(Figure S10a, SI) and absence of any distinct morphologies in TEM
(Figure S10b, SI). The disassembled Im-KL aged for ca. 1 h
showed substantially lower activity compared to the nanotubes
of Im-KL and added imidazole acetic acid separately to Ac-KL
(Ac-KLVFFAL, assembles to form homogenous nanotubes11-12
and did not have measurable intrinsic hydrolase activity).
Compared to Im-KL, the activity dropped to 14-fold, indicating the
importance of a surface with arrays of imidazoles and lysines
([vi]=0.40±0.09 M min-1, Figure 3c, Table S1, imidazole acetic
acid was expected to be near to the positively charged nanotubes).
For spectroscopic investigation of the imine formation, fluorescent
aldehyde 6-hydroxy 2-naphthaldehyde (Ar-CHO) was used.
Naphthaldehyde backbone is known for its binding to amyloid
phases as it features in molecular histological stains like Congo
Red.15b Ar-CHO registered quenching upon addition to Im-KL,
suggesting the formation of Schiff base as the lone pair of imine
nitrogen is known to quench through nonradiative channel (Figure
S11, SI).17 However, when Ar-CHO was incubated with the
control nanotubes of Im-RL, the fluorescence intensity registered
an enhancement as expected from the binding of the dye to the
(Table S1). Significantly, disassembled Im-RL showed similar
activity to that found for disassembled Im-KL, thus
suggesting the role of the neighboring amines generated
from assembled structures for covalent catalysis (Figure 3d,
Table S1). We detached the residues responsible for the activity
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hydrophobic environment of the nanotubular surfaces (Figure S11,
demonstrate advanced traits such as covalent catalysis. The
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SI). Further, hydrophobic fluorophores such as ANS and pyrene
which lack the carboxyl group, showed enhancement of intensities
upon binding to the Im-RL and Im-KL nanotubes (Figure S12,
SI).18
microenvironment featuring lysines and imidazoles shows higher
substrate affinity and accelerates catalytic activity. Importantly,
these peptide nanotubes promoted the hydrolysis of inactivated
esters, thus opening up the possibility of designing new materials
capable of performing catalysis on expanded substrate scope. The
simplicity of the peptide sequences which offers multiple amino
acid residues on the paracrystalline phases foreshadows the
prebiotic origins of binding pockets of advanced proteins. This
observation also supports the covalent hypothesis of involving
covalent bond formation in intermediate as exploited by Nature for
evolved enzymes.
We expected that this assistance of a covalent linkage can broaden
the scope of the substrates that are kinetically more stable. Hence,
we investigated the ability of the Im-KL nanotubes to hydrolyse
less activated esters. We acetylated the phenolic group of Ar-CHO
to form 3 (Figure 3a). Interestingly, Im-KL showed facile
hydrolysis of 3 as monitored by UV-Visible spectroscopy (6.6±0.8
M min-1, followed at 310 nm, Figure S13, S14, SI). This rate was
7.9 fold higher compared to Im-RL nanotubes (0.83±0.08 M min-
1) thus underpinning the importance of covalent linkage for the
development of an efficient catalytic system (Figure 4a). The
hydrolytic activity for Im-KL could also be reflected from the time
course generation of fluorescent entangled networks in
epifluorescence microscopy, signifying the formation of surface-
bound fluorescent Ar-CHO (Figure 4b). As a control, 4 was
synthesized which will form 2-naphthol after hydrolysis (Figure
3a). Due to the symmetrically forbidden n→ transitions (low  of
527 M-1cm-1), fluorescence spectroscopy was used to monitor the
product formation.19 The rates for 4 for Im-KL was similar to that
shown by Im-RL which further underpinned the importance of the
covalent anchoring of the substrates ([vi]Im-KL=0.79±0.09 M min-
1, [vi]Im-RL=0.52±0.09 M min-1, Figure 4a, Figure S15). From
HPLC with Im-KL, the conversions were found to be 21 % after
30 min for 3, whereas for 4, it was only 2%, thus further stressing
the effectiveness of the reported system (Figure S16-S17). In order
to compare the activity of Im-KL with native enzyme carbonic
anhydrase (CA), a standard substrate 4-nitrophenyl acetate was
used. Although the catalytic efficiency was around 2 orders of
magnitude lower (2.4±0.3 M-1sec-1) than CA, on the basis of
molecular mass, the activity was only 22.7-fold lower than the
evolved enzyme.20
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Experimental procedures, details of
kinetic analysis, synthesis and characterization of molecules.
AUTHOR INFORMATION
Corresponding Author
*dasd@iiserkol.ac.in
ORCID Dibyendu Das:0000-0001-6597-8454
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
D.D. is thankful to Nano Mission Grant (SR/NM/NS-1082/2015),
DST, GOI for financial assistance. BS and AC acknowledge
CSIR, India. We thank Syed Pavel Afrose for his valuable help.
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