An Artificial Chaperone Serves a Dual Role in Regulating the Assembly of
Peptides through Phase Separation
Wang Li1, Yang Zhou1, Sheng
He1, Congsen Wang1, Peichen
Shi1, Suixu Li1, Xinchang
Wang2, Liulin Yang1*, Xiaoyu
Cao1*, and Zhongqun Tian1
W. Li, Y. Zhou, S. He, C. S. Wang, P. C. Shi, S. X. Li, Prof. L. L.
Yang, Prof. X. Y. Cao, and Prof. Z. Q. Tian
1State Key Laboratory of Physical Chemistry of Solid
Surface, Key Laboratory of Chemical Biology of Fujian Province,
Collaborative Innovation Center of Chemistry for Energy Materials
(iChEM), Innovation Laboratory for Sciences and Technologies of Energy
Materials of Fujian Province (IKKEM), College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen 361005, P. R. China
E-mail:
llyang@xmu.edu.cn,
xcao@xmu.edu.cn
Prof. X. C. Wang2 School of Electronic Science and Engineering
(National Model Microelectronics College), Xiamen University, Xiamen
361005, P. R. China
Keywords : artificial chaperone, catassembly, supramolecular
polymerization, peptide assembly
Abstract: In biological systems,
molecular assembly primarily relies on the assistance of molecular
chaperones. Inspired by nature, strategies like
‘chaperone-assisted assembly’ and
‘catalyzed assembly’ have been proposed towards the sophisticated
control of molecular assembly. Nonetheless, significant challenges
remain in the rational design of such systems, calling for a deep
understanding of underlying principles. Herein, we demonstrate an
artificial chaperone serves a dual role, i.e. catalyst in low dosages
and inhibitor in high dosages, in regulating the supramolecular
polymerization of peptides. Low dosages of carboxymethyl cellulose, as
the chaperones, catalyse the assembly of Aβ16-22peptides into fibrils through multi-step phase separation, while high
dosages trap the peptides into coacervate intermediates and therefore
inhibit the fibrillation. Consequently, the quantity of chaperone does
not follow the intuition that ‘more is better’ for catalyzing assembly,
but instead has an optimal molar ratio. Investigation reveals that the
interplay and evolution of electrostatic and hydrophobic interactions
between chaperones and peptides are the keys to achieving these
processes. This study provides insights into the multifaceted roles
artificial chaperones may play in a
dosage-dependent manner, and
enriches the toolkit for efficient and controllable construction of
complex assembly systems.
1. Introduction
The pathway complexity of supramolecular polymerization, along with its
high sensitivity to solvents, additives, impurities, and environmental
conditions, poeses significant challenges for thermodynamic and kinetic
regulations. [1-13] In recent years, strategies
have been developed to trigger supramolecular polymerization, select
polymerization pathways, and control polymer structures. These are
represented by (seeded) living supramolecular polymerization,[14-18] fuel-driven supramolecular polymerization,[19, 20] supramolecular interfacial
polymerization, [21] controlled release monomers
from macromolecules, [22] and using external
stimuli.
Assembly assisted by molecular chaperones has been recognized as an
efficient strategy in biological systems, however, less explored in
supramolecular polymerizations. Chaperones are well known to assist in
the correct folding and assembly of proteins. ATP-independent chaperones
often act as ‘holdases’, [23] they bind to
misfolded proteins to prevent their further aggregation. Examples of
artificial chaperones based on similar mechanisms have been developed
for regulating protein folding. [24] Recently,
this strategy was employed in controlling the supramolecular
polymerization of squaraine dye monomers, in which the monomer was
kinetically trapped by a macrocycle chaperone, and the living
supramolecular polymerization was then triggered by adding an initiator
to remove the chaperone. [25]
Some ATP-independent molecular chaperones can play the role of
‘foldases’, assisting in the folding of substrate proteins after
binding. [23, 26-29] Achieving this process
requires a loose binding between the molecular chaperone and the
protein, allowing the protein enough flexibility to explore its
conformation space. [23] Once the folded protein
is formed, the binding affinity between the protein and the molecular
chaperone decreases, resulting in the regeneration of the chaperone.[27, 30] Such a chaperone-mediated
folding-while-bound mechanism was employed in artificial chaperone
systems, which realizes the renaturation of proteins.[31-34] Inspired by these catalytic molecular
chaperones as well as the catalytic chemistry, a concept of catassembly[35, 36] had been proposed as an efficient
assisted assembly strategy. As a prove of concept, polyhedral molecular
cages were demonstrated to serve as chaperones to enantioselective
catalyze the supramolecular polymerization of a porphyrin monomer.[37] The molecular cages eventually dissociate
from the supramolecular polymer through phase separation.
As mentioned above, the content of additives can significantly influence
assembly pathways, highlighting the intricate complexity in regulating
assembly kinetics. This has propelled us to explore the impact of
stoichiometry of chaperones as additives on the kinetics of
supramolecular polymerizations. Here, we report an artificial chaperone
serves a dual role in regulating peptide assembly in a dosage-dependent
manner, i.e. catalyzing assembly at low dosages while inhibiting
assembly at high dosages. As shown in scheme 1, carboxymethyl cellulose
(CMD) was employed as a molecular chaperone to regulate the assembly of
Aβ16-22 peptide (KLVFFAE, abbreviated as KE).
CMD initially enriched the
peptide monomers and formed coacervate droplets through liquid-liquid
phase separation (LLPS). Subsequently, at different CMD dosages, CMD
played two opposite roles within the coacervates. When the dosage of CMD
was low, coacervates facilitated the nucleation of peptides, and further
fibrillation by desolvation and releasing CMD through phase separation.
In contrast, CMD with higher dosage trapped the system in the coacervate
state, slowed down and even inhibited the peptide fibrillation. We
believe this phenomenon where a molecular chaperone plays opposite roles
at different dosages is likely widespread. Investigating the underlying
mechanism can help us gain a deeper understanding in chaperone-assisted
molecular assembly and develop new assembly strategies, e.g.
catassembly.
2 . Results and
Discussion
2.1.Low dosage of CMD promoted the peptide assembly
The KE monomer was initially trapped into a random coil conformation in
the aqueous solution at pH 4.25 (Figure S1). The addition of low dosage
of CMD (< 1 mol% of KE,
calculated in moles of glucose units) triggered the conformational
transition of KE from α -helix to β -strand and subsequently
the assembly into fibrils (Figure 1). The conformational transition of
the peptide was demonstrated by the circular dichroism (CD) spectra, in
which the peak at 215 nm gradually diminished, and the spectra
eventually became a single peak with only the β -strand
conformation (Figure 1B). [38] Meanwhile, the
supramolecular polymerization of β -strand KE was evidenced by a
gradual increase in the emission at 482 nm using a ThT fluorescence
probe [39] (Figure 1C), and also the images of
transmission electron microscopy (TEM) (Figure S2) sampled at different
time points. Some droplet-like coacervates in the early-stage samples
were observed under TEM, suggesting that KE may complex with CMD in
solution and undergo LLPS (Figure 1A). The phenomenon of LLPS often
originates from the complexation of oppositely charged polyelectrolytes,
such as nucleic acids and proteins, in aqueous solution, which leads to
phase separation and the formation of coacervate droplets. Since CMD
molecules were negatively charged, while KE molecules were positively
charged under the experimental conditions (Figure S3A), it is plausible
that they formed coacervate droplets through electrostatic interactions
and facilitate KE fibrillation. In contrast, a neutral polysaccharide,
dextran, which has the same backbone structure as CMD but without
carboxyl groups (Figure S3B), had no effect on KE assembly (Figure S4).
Confocal laser scanning microscopy (CLSM) provided further insight into
the LLPS and fibrillization process (Figure 2, Movie S1).
The CMD and KE were labelled by
fluorescein isothiocyanate (FITC, emitting green fluorescence) and
rhodamine B (RhoB, emitting red fluorescence), respectively. In the
initial stages, yellowish-green spherical coacervates were observed, and
fluorescence imaging analyses [40, 41] indicated
that these coacervates were enriched in CMD and KE (Figure 2A).
Subsequently, protofibrils generated within these droplets, and then
elongated into fibrils that interconnect multiple droplets (Figure
2B-D).
As
the droplet diminished over time, a large number of mature fibrils were
eventually obtained (Figure S5). [42] These
results indicate that CMD and KE can form coacervates through LLPS, and
these metastable coacervates then function as nucleation centers to
promote the fibrillation of the
peptide.
2.2. Highdosage of CMD inhibited
the peptide assembly
However, the formation of coacervates did not necessarily promote
peptide fibrillation. In contrast, large amounts of CMD inhibited KE
fibrillation, despite forming coacervates with KE (Figure 3). By adding
5 equivalents of CMD, a notable inhibition effect on KE fibrillation was
observed both visually and from the fluorescence kinetic data (Figures
3A and 3B). Upon the introduction of 50 equivalents of CMD, the solution
remained consistently transparent and devoid of fibril formation.
Although coacervates were still found, these entities remained
stabilized and refrained from transitioning into fibrils (Figure 3C). CD
spectra showed that the peptides maintained the initial random coil
conformation instead of transitioning into the β -strand
conformation (Figure S6). At this point, CMD plays a role similar to
that of an inhibitor in molecular chaperones. These results indicate
that although CMD tends to form coacervates with KE, whether the CMD
acts as a promotor or inhibitor inside the coacervates is closely
related to its molar ratio. Therefore, the intricate kinetic mechanisms
behind this phenomenon necessitate a comprehensive investigation.
2.3. Low dosage of CMD
catalyzed the assembly of KE through multi-step phase separation
To explore the mechanism of peptide fibrillation promoted by low dosage
of CMD, a quantitative fluorescence analysis of selected regions using
CLSM was carried out. As the fibrillation progressed, the red emission
from the peptides gradually enhanced, whereas the green emission from
CMD attenuated (Figure 4A-C and Movie S1). This phenomenon implies that
while KE monomers were stacking into fibrils, CMD molecules were
progressively dissociating from KE monomers, eventually expelled from
the coacervates. Furthermore, correlation analysis showed a low
correlation between green and red signals at the end of assembly (Figure
4C). This finding signifies a tendency for the KE fibrils and CMD to
undergo phase separation.
To gain deeper insights into the evolution of interaction between CMD
and KE, time-dependent Förster resonance energy transfer (FRET)
measurements were performed. CMD
was labeled with FAM, while KE was labeled with Rhodamine B. When
excited at 380 nm, the maximum emission of CMD-FAM and KE-RhoB peaked at
512 and 575 nm, respectively. After mixing the CMD-FAM and KE-RhoB
solutions, the emission at 575 nm quickly increased in 15-20 s,
accompanied by the emission at 512 nm declined (Figure 4D and 4F). In a
control experiment, the excitation of dye molecules FAM could not induce
the enhancement of the emission of KE-RhoB, verifying the FRET effect
between CMD-FAM and KE-RhoB (Figure S7). This result indicated a fast
association between CMD and peptides upon mixing, which triggered the
FRET effect after the proximity of the two fluorescent groups.
Subsequently, the emission at 575 nm started to attenuate, while the
emission at 512 nm recovered in around 2 min (Figure 4E and 4F). We
speculated that this corresponded to the process of KE monomers
dissociating from CMD and assembling into fibrils. Due to the
consumption of KE monomers and the phase separation of fibrils from CMD,
the FRET signal gradually attenuated. This observation is in line with
the findings from CLSM analysis.
The self-assembly of peptides often follows a non-classical
nucleation-growth mechanism, where peptides first undergo LLPS to form
coacervates, and then nucleate inside the coacervates and eventually
generate fibrils. [42] During this process, the
stacking of peptide molecules involves a desolvation process, i.e.
expelling the water molecules that interacting with the peptides.[42, 43] In this research, since the participation
of the polyelectrolyte CMD, the phase separation underwent at least two
stages and was not just a desolvation process. First, CMD and KE formed
coacervates mainly through electrostatic interactions, corresponding to
the first stage of phase separation. Afterwards, KE molecules stacked
with each other, while further expelling solvent water and hydrophilic
CMD from the coacervates, representing the second stage of phase
separation. The isothermal titration calorimetry (ITC) measurements
(Figures S8 and S9) demonstrated that the assembly of KE chaperoned by
CMD is entropy-driven, which supported this phase separation-mediated
assembly process. [44] Phase separation phenomena
were also observed in other chaperone-assisted assembly systems, such as
the departure of molecular cages through phase separation after inducing
chiral supramolecular polymerization of porphyrins.[37] Therefore, the CMD acted as an enzyme
analogous that can catalyze the assembly of peptides.
Figure 5A illustrates the process of CMD-catalyzed KE assembly. When
mixing, negatively charged CMD rapidly enriches positively charged KE
molecules and forms coacervates through LLPS. These concentrated
compartments facilitate KE molecules to adopt β -strand
conformation and stack into nuclei, which further induces more KE
monomers to transform into β -strand conformation and participate
in assembly, eventually forming fibrils. The stacking of KE molecules
drives their dissociation from CMD, prompting their assembly into
fibrils and the consequent release of CMD. Reflecting on this, we have
outlined the assembly equation of KE catalyzed by CMD (Figure 5B). This
process is analogous to protein assembly catalyzed by non-ATP
independent chaperones within biological systems. As we mentioned above,
it requires a loose binding between the chaperone and the substrate,
while the dissociation constant (KD) between them should
be at the millimolar level. [23] In our research
system, the KD of KE and CMD is 1.30 mM as measured by
the surface plasmon resonance technique (SPR) (Figure S10), which just
meets the above requirements. The relatively high KDvalue results in a continuous interplay of association and dissociation
between CMD and KE peptides, facilitating the peptides to adjust
conformation and assembly. In biological system, the binding affinity
between molecular chaperones and products is often weaker than that with
substrates, therefore prone to dissociation due to competitive binding.[23] Here, the release of CMD should be driven by
the competitive stacking of KE monomers that finally expel the CMD
through phase separation.
2.4.
Kinetic analysis of peptide assembly inhibited by high dosage of CMD
Based on the assembly equation in Figure 5B, we inferred that increasing
the dosage of CMD, while facilitating the nucleation of KE, may
subsequently inhibit the growth of nucleus. To validate this hypothesis,
we investigated the effect of CMD dosage on KE assembly kinetics by
collecting kinetic data (Figure 6A) and fitting the data to a
theoretical model (Details in the SI, Theoretical Methods
section ). Initially, the addition of CMD increased both the apparent
nucleation and elongation rate constants. The lag time for the assembly
of KE was significantly shortened. When 0.01 equivalent (1 μ M) of
CMD was added, the lag time was almost undetectable. However, as the CMD
content continued to increase, a distinct decline manifested in the
apparent elongation rate constant
(\(k_{E}\)). Therefore, an optimal CMD amount at around 1 mol% (Figure
6B, Supporting Information Table S1) resulted the highest apparent
polymerization rate constant (Figure S12). A similar result was obtained
from kinetic data collected by CD spectroscopy (Figure S13). At high CMD
content, KE monomers and nuclei associated with CMD at a higher
probability. On one hand, this reduced the concentration of free KE
monomers in the solution, and on the other, competitive binding from
high dosage of CMD prevented the association of KE monomer and nuclei.
This complex interplay ultimately resulted in a decrease of fibril
growth rate constant.
The
competitive binding from high dosage of CMD that inhibit the
fibrillation was further verified by
time-dependent FRET experiments
(Figure S14). The addition of varying molar ratios of CMD-FAM to the
KE-RhoB solution leads to a rapid decrease in the emission at 512 nm.
The extent of the decrease in luminescence intensity corresponds to the
amount of peptide monomers adsorbed by CMD-FAM. As the dosage of CMD-FAM
increased, the extent of emission
reduction correspondingly increased, until reaching a plateau due to the
completely adsorption of the peptide (Figure 7A). As the fibrillation
progressed, KE-RhoB monomers disassociated from CMD-FAM and assembled
into fibrils, resulting in a gradual recovery of the emission at 512 nm
and an attenuation of FRET signal. However, the extent of CMD-FAM
dissociation from complexes correlated with the dosage of CMD-FAM
(Figure 7B). Low dosage of CMD-FAM generated small number of nuclei,
resulting in slow monomer consumption, and correspondingly slow recovery
of emission at 512 nm. For high dosage of CMD-FAM that adsorbed the
peptide completely, a less recovery of the emission at 512 nm was
observed, indicating that the competitive binding of CMD-FAM hindered
the assembly of peptide and the release of CMD-FAM.
As
we have demonstrated in this work, artificial molecular chaperones can
exert complex effects on the kinetics of supramolecular polymerization
due to their dynamic and reversible interactions with substrates,
intermediates, and products. According to the assembly equation we
proposed (Figure 5B), excessive molecular chaperones may cause the
assembly to be trapped at intermediate states instead of transforming
into the product, therefore inhibiting the assembly. Consequently, the
quantity of molecular chaperones does not follow the intuition that
‘more is better’ for catalyzing assembly, but instead has an optimal
molar ratio. Similar phenomena have been reported in biological systems.
For example, low content of liposome vesicles can induce α-synuclein
aggregation, while high molar ratios inhibit its aggregation[45]. In the case of RNA-binding proteins like FUS
and TDP43, low content of RNA can promote the phase separation of
protein and further aggregation, whereas high ratios prevent droplet
formation, keeping the proteins dissolved [46]. It
was found that low concentrations of Prostaglandin E2 can promote
dendritic cell migration, while high concentrations have the opposite
effect [47]. Therefore, we believe that such
dosage-dependent role switching phenomena may be ubiquitous in assembly
systems, yet they have not received adequate attention. For instance,
extensive studies have demonstrated that liquid-liquid phase separation
is closely related to the assembly of proteins [42,
46, 48]. However, the formation of droplets may either facilitate or
inhibit [46, 49] protein assembly. Studies argue
that protein aggregation and the complex coacervation are independent
processes [50]. Therefore, how LLPS affects
protein assembly remains elusive. We suggest that for systems involving
multicomponent in LLPS, the impact of their dosage on peptide assembly
should not be disregarded. Additionally, for drugs that inhibit amyloid
aggregation of proteins, the potential risks brought by concentration
changes after metabolism should be evaluated. Insufficient inhibitors
may keep protein aggregation in the highly toxic oligomeric stage, and
residual small amounts of inhibitors may promote aggregation of protein
monomers [51].
3 .