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On-off transition and ultrafast decay of amino acid luminescence driven by modulation of supramolecular packing


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Biomolecule luminescence in the visible range of the spectrum has been experimentally observed upon aggregation, contrary to their monomeric state. However, the physical basis for this phenomenon is still elusive. Here, we systematically examine all coded amino acids to provide non-biased empirical insights. Several amino acids, including non-aromatic, show intense visible luminescence. While lysine crystals display the highest signal, the very chemically similar non-coded ornithine does not, implying a role for molecular packing rather than the chemical characteristics. Furthermore, cysteine show luminescence that is indeed crystal-packing-dependent as repeated rearrangements between two crystal structures result in a reversible on-off optical transition. Additionally, ultrafast lifetime decay is experimentally validated, corroborating a recently raised hypothesis regarding the governing role of nπ* states in the emission formation. Collectively, our study supports that electronic interactions between non-fluorescent, non-absorbing molecules at the monomeric state may result in reversible optically-active states by the formation of supramolecular fluorophores.
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On-off transition and ultrafast decay of amino acid
luminescence driven by modulation of
supramolecular packing
Zohar A. Arnon,
Topaz Kreiser,
Boris Yakimov, ...,
Evgeny Shirshin,
Davide Levy, Ehud
characterization of all
Lysine, a non-aromatic
amino acid, exhibited the
highest fluorescent signal
Revirsible transition
between cysteine crystals
determines on-off
Ultrafast lifetime decay
shown experimentally,
confirming governing role
in emission
Arnon et al., iScience 24,
July 23, 2021 ª2021
On-off transition and ultrafast decay
of amino acid luminescence driven
by modulation of supramolecular packing
Zohar A. Arnon,
Topaz Kreiser,
Boris Yakimov,
Noam Brown,
Ruth Aizen,
Shira Shaham-Niv,
Pandeeswar Makam,
Muhammad Nawaz Qaisrani,
Emiliano Poli,
Antonella Ruggiero,
Inna Slutsky,
Ali Hassanali,
Evgeny Shirshin,
Davide Levy,
and Ehud Gazit
Luminescence of biomolecules in the visible range of the spectrum has been
experimentally observed upon aggregation, contrary to their monomeric state.
However, the physical basis for this phenomenon is still elusive. Here, we system-
atically examine all coded amino acids to provide non-biased empirical insights.
Several amino acids, including non-aromatic, show intense visible luminescence.
Lysine crystals display the highest signal, whereas the very chemically similar
non-coded ornithine does not, implying a role for molecular packing rather than
the chemical characteristics. Furthermore, cysteine shows luminescence that is
indeed crystal packing dependent as repeated rearrangements between two
crystal structures result in a reversible on-off optical transition. In addition, ultra-
fast lifetime decay is experimentally validated, corroborating a recently raised
hypothesis regarding the governing role of np* states in the emission formation.
Collectively, our study supports that electronic interactions between non-fluores-
cent, non-absorbing molecules at the monomeric state may result in reversible
optically active states by the formation of supramolecular fluorophores.
In nature, proteins and peptides offer a broad range of characteristics and attributes, which provide an
extensive variety of mechanical, electrical, and optical properties. These are often derived from the phys-
icochemical properties of the amino acid building blocks that constitute the proteinaceous polymer. In
addition to the amino acid content, the arrangement of the protein in its environment, i.e., secondary, ter-
tiary, and quaternary structure, can dramatically influence the resulting physical properties. This arrange-
ment is affected by factors such as the solvent, temperature, and ionic strength. Recently, several studies
described the intrinsic luminescence in the visible range of protein assemblies, whereas the monomeric
protein in solution is not luminescent (Bhattacharya et al., 2017;Pinotsi et al., 2016). Consistently, it is
now evident that some amyloids, once assembled, exhibit fluorescence that is not demonstrated in the
monomeric state in solution (Ardona et al., 2017;Chan et al., 2013;Del Mercato et al., 2007;Pinotsi
et al., 2013).
Monomeric amino acids were subjected to extensive research over the years. Recently, a great emphasis
was given to the ability of amino acids to serve as building blocks for the formation of various supramolec-
ular assemblies with attractive features (Guerin et al., 2018;Ji et al., 2019;Makam et al., 2019). It was shown
that single amino acids in their aggregated form can also produce a fluorescent signal, which is not evident
in the monomeric state (Babar and Sarkar, 2017;Banerjee et al., 2020;Niyangoda et al., 2017;Shaham-Niv
et al., 2018). Numerous observations suggest that the optical phenomenon of aggregation-induced
intrinsic fluorescence is broader than originally speculated, as it also applies to many other metabolites
and nucleic acids (Arnon et al., 2019;Berger et al., 2015;Chen et al., 2018;Lakowicz et al., 2001;Stephens
et al., 2020;Zou et al., 2002). Circumstantial evidence and argumentation suggested that supramolecular
packing is important. It was suggested that the hydrogen bonds and, in specific cases, the aromatic stack-
ing, can underlie these optical properties (Jong et al., 2019;Pinotsi et al., 2016). Yet, other views have sug-
gested impurities and oxidation as alternative mechanisms, ascribing no significant role to the molecular
Department of Molecular
Microbiology and
Biotechnology, George S.
Wise Faculty of Life Sciences,
Tel Aviv University, Tel Aviv
6997801, Israel
Faculty of Physics, M.V.
Lomonosov Moscow State
University, Moscow 119991
Drug Discovery, Metabolite
Medicine Division, Tel Aviv
University, Tel Aviv 6997801,
International Centre for
Theoretical Physics, Strada
Costiera, 11, 34151 Trieste,
Department of Physiology
and Pharmacology, Sackler
Faculty of Medicine, Tel Aviv
University, Tel Aviv University,
6997801 Tel Aviv, Israel
Institute for Regenerative
Medicine, I.M. Sechenov
Moscow State Medical
University, 119991 Moscow,
X-Ray Diffraction Lab,
Wolfson Applied Materials
Research Centre, Tel Aviv
University, Tel Aviv 6997801,
Department of Materials
Science and Engineering Iby
and Aladar Fleischman
Faculty of Engineering, Tel
Aviv University, Tel Aviv
6997801, Israel
These authors contributed
Lead contact
iScience 24, 102695, July 23, 2021 ª2021
This is an open access article under the CC BY-NC-ND license (
Figure 1. Fluorescence of the 20 coded amino acids
Images of all 20 amino acids, both brightfield (left) and confocal fluorescence at excitation wavelength of 405 nm (right). Amino acid samples as originally
obtained from Sigma-Aldrich are displayed in rows marked with OS (Original Sigma). Amino acids dissolved, reassembled, and lyophilized are displayed in
2iScience 24, 102695, July 23, 2021
arrangement (Tikhonova et al., 2018). Indeed, oxidation of aromatic moieties is known to result in the for-
mation of fluorescent products. However, this cannot readily explain the emergence of intrinsic fluores-
cence upon aggregation of non-aromatic species (Del Mercato et al., 2007;Jong et al., 2019;Stephens
et al., 2020). Overall, the key evidence to distinguish between these options and elucidate the mechanism
of intrinsic fluorescence upon aggregation was so far missing. We believe that the reversible transitions be-
tween the luminescent and non-luminescent states upon controllable change of aggregate structure is
such an evidence. In this work, we aimed at designing and performing such an experiment.
The phenomenon of aggregation-dependent luminescence of proteins is a rapidly evolving field (Ardona
et al., 2017;Arnon et al., 2019;Babar and Sarkar, 2017;Banerjee et al., 2020;Berger et al., 2015;Bhatta-
charya et al., 2017;Chan et al., 2013;Kong et al., 2019;Niyangoda et al., 2017;Pansieri et al., 2019;Pinotsi
et al, 2013,2016;Prasad et al., 2017;Shaham-Niv et al., 2018). Protein aggregates were shown to absorb
light at wavelengths above 300 nm and to exhibit a structure-specific fluorescence in the visible range,
even in the absence of aromatic amino acids (Del Mercato et al., 2007). A plausible explanation for this phe-
nomenon is the formation of structure-specific supramolecular fluorophores that are permissive to proton
transfer across hydrogen bonds (Grisanti et al., 2017;Pinotsi et al., 2016;Yamaguchi et al., 2009). Other hy-
potheses postulate that the fluorescence is associated with electron-hole recombination due to charge
transfer between charged amino acids (Ardona et al., 2017;Kumar et al., 2020;Prasad et al., 2017)or
that deep-blue autofluorescence stems from carbonyl double bonds of the protein backbone (Niyangoda
et al., 2017). Following these studies, we have decided to look into the intrinsic optical properties of single
amino acid and metabolite assemblies. Indeed, we have shown that adenine, phenylalanine, tyrosine, and
tryptophan exhibit autofluorescence in the visible range upon self-assembly (Shaham-Niv et al., 2018). This
allows the detection of metabolite assemblies within living cells, without any use of external dyes that could
interfere with the ability to accurately model a given sample (Buell et al., 2010). Other derivatives of tyrosine
were also shown to exhibit luminescence upon aggregation (Ren et al., 2019). Yet, the underlying mecha-
nism of the autofluorescence of these assemblies is controversial, as in all cases it can be attributed to the
aromatic moiety. In addition, the explanation of impurities as the source of fluorescence is still a plausible
option. Here, we have endeavored to study the optical properties of all 20 coded amino acids in a system-
atic, non-biased approach. Each amino acid was optically characterized in two states: the original powder
form, as obtained from the commercial supplier, and after being dissolved in heated water and allowed to
cool down for recrystallization. Aiming to simplify the process of assembly and i n order to eliminate as many
factors as possible, we avoided altering solvents, salt concentrations, changing pH, etc. The systematic
evaluation of all amino acids, before and after recrystallization, provides mechanistic insights into the
broad phenomenon of assembly-dependent intrinsic fluorescence.
First, we examined the optical properties of the crystalline powders of all 20 coded amino acids in the dried
powder form. In addition, each powder was dissolved in water at a high concentration (Table S1). In order to
completely dissolve the powders, the aqueous solution was heated to 90 C and vortexed until a clear,
transparent solution was achieved. The amino acid solutions were then cooled down gradually to room
temperature and incubated for several days to allow the formation of assemblies within the solution.
The samples were then lyophilized to attain a dried pow der of the amino acid assemblies. The optical prop-
erties of these powders were examined as well.
The brightfield and confocal fluorescence images obtained under excitation of 405 nm for all 20 amino
acids, as originally received (OS, original Sigma) and after dissolution and reassembly (DR, dissolved
and reassembled), as well as quantification of the fluorescence signals, are displayed in Figure 1 (see Fig-
ure S1 for spectra). For all samples, similar excitation and detection parameters were used. Based on the
current measurements there is no systematic trend between the optical properties and the polarity of the
amino acids. However, it does appear as though the charged amino acids tend to be characterized by
stronger fluorescence, whereas those amino acids with hydrocarbon side chains with methyl groups
tend to exhibit very weak optical activity.
Figure 1. Continued
rows marked with DR (Dissolved and Reassembled). At the bottom of each amino acid panel are the normalized fluorescence signals of the OS sample
(light blue) and the DR sample (dark blue).
See also Tables S1 and S2 and Figure S1.
iScience 24, 102695, July 23, 2021 3
We then explored the intense fluorescence of L-lysine, which exhibited the brightest signal of all 20 amino
acids. We first inquired whether the length of the amine residue chain of lysine, which has four carbons, plays
a role in the optical properties of the crystal. For this purpose, we explored the fluorescence of three additional
molecules derived from lysine: L-ornithine, L-2,4-diaminobutyric acid, and DL-2,3-diaminopropionic acid,
which comprise 3, 2, and 1 side chain carbons, respectively (Figure 2A). The rationale of this experiment was
to alter the crystal packing by shortening the amine residue, thereby modulating the electronic interactions
by varying the distance between molecules, and to examine any effect on the resulting optical properties.
All four samples (including lysine) comprised small crystalline powders (Figure 2B). The fluorescent signal
from each sample was obtained using confocal microscopy, similar to the amino acids shown in Figure 1.
Figure 2. Fluorescence of lysine and its derivatives
(A) The chemical scheme of lysine and its derivatives comprising shorter carbon chains.
(B and C) Microscopy images showing (B) brightfield and (C) fluorescence (excitation at 405 nm) analyses of the four powders. Pixel color represents the
intensity; black, non; blue, dim; white, strong.
(D) Crystalline structures of lysine and its derivatives as determined using PXRD.
See also Data S1 for additional crystallographic information.
4iScience 24, 102695, July 23, 2021
Intense fluorescent signals were prominent in the molecules comprising 1-, 2-, and 4-carbon chains,
whereas in the 3-carbon chain molecule, ornithine, no visible signal was detected (Figure 2C). The powder
X-ray diffraction (PXRD) patterns of the samples were further examined in order to understand the molec-
ular arrangement of the crystals, which might provide insight into the favorable interactions allowing the
crystal fluorescence (Figure 2D). The crystal structures of lysine, ornithine, and diaminopropionic acid
were previously published (Chiba et al., 1967;Mar
´et al., 2019;Williams et al., 2016). The cry stal struc-
ture of diaminobutyric acid was determined by thePXRD pattern obtained in this study CCDC Deposition #
1990651 (see STAR Methods). The results revealed the crystal packing of all four samples, allowing molec-
ular inspection of the optical phenomenon. A possible explanation lies in the type of hydrogen bond
network that is formed within the supramolecular structure. The NH
group of lysine hemihydrate and dia-
minobutyric acid, and the NH
of diaminopropionic acid, may interact with a water or chloride ions and
donate hydrogen bonds to the molecule complexed in the supramolecular structure. On the other
hand, in the case of ornithine, the NH
groups do not form hydrogen bonds that are donated from the
amine group to either a chloride ion or a water molecule in the complex. Thus, the differences in the supra-
molecular packing involving the lysine side chains, chloride ions, and water molecules can lead to
differences in the optical properties. We believe that the packing of the crystal is an important factor in
determining its optical behavior, much like the properties of microenvironments such as different solvents,
ionic strength, polarity, or molecular concentration could be significant for structure-function attributes.
Following this line of evidence, we focused on possible interconnections between the optical and structural
properties of amino acid powders.
Next, each of the 20 amino acid samples was examined using PXRD to determine the crystalline structure.
The results are summarized in Table S2. Most amino acid crystals were composed of the same crystal struc-
ture in both the OS and the DR samples. Of the 20 amino acids, only cysteine and serine displayed different
powder diffraction between their respective OS and DR samples, with a single crystal packing observed in
each sample. However, both serine samples exhibited some level of fluorescence, whereas the cysteine DR
sample did not show any detectable fluorescence (Figure 1), making the differentiation between the
cysteine samples much more straightforward. In addition, the DR serine sample was found to comprise
a hemihydrate crystal packing (Table S2), adding another factor of complexity,whilebothcysteinecrystals
showed different arrangements of cysteine alone, with four molecules in both unit cells. Hence, we further
investigated the OS and DR samples of cysteine (Figures 3A and 3B), which showed a clear difference in the
fluorescent signal (Figure 3C) and exhibited an orthorhombic and monoclinic packi ng, respectively (Figures
3Dand3E)(Harding and Long, 1968;Kerr and Ashmore, 1973). Since the process of attaining the OS pow-
der is unknown to us, we have decided to crystallize an orthorhombic cysteine crystal after dissolving the
amino acid in order to control the entire procedure. We were able to crystallize both crystal packings,
orthorhombic and monoclinic, depending on the crystallization temperature, 4 Cand25C, respectively,
as the only difference in the crystallization conditions. This allowed a reversible crystallization of both crys-
tal packings, exemplifying the significance of the crystal packing to the optical properties of the crystal.
Thus, the fluorescent OS powder (Figure 3F) was dissolved and recrystallized at 25 C to obtain the non-
fluorescent monoclinic DR sample (Figure 3G) and then re-dissolved and recrystallized at 4 Ctoform
yet again the fluorescent orthorhombic crystals (Figure 3H). To confirm that the fluorescence does not
stem from impurities, the non-fluorescent DR sample was washed several times to remove any impurities
that may reside in the supernatant, before recrystallizing as orthorhombic crystals. Although the fluores-
cent signal of cysteine is relatively low, a difference in the fluorescent signal between the two crystal pack-
ings is clearly evident. A comparison of the two structures showsthat there is a subtle difference in the prox-
imity of the S-H bonds relative to each other. In the orthorhombic structure, the sulfide groups are packed
closer to each other and may facilitate charge transfer, as seen in previous studies (Del Mercato et al., 2007;
Pinotsi et al., 2016;Prasad et al., 2017). It is important to note that the overall macroscopic morphology is
dictated by other factors in addition to the packing, as evidenced by d ifferent morphologies for an identical
packing (compare Figures 3F–3H), and vice versa (compare Figures 3G–3H). For this reason, the crystal
packing was determined using PXRD for each sample and for every iteration. Morphologically, the recrys-
tallized orthorhombic structures (Figure 3H) are similar to the non-fluorescent monoclinic structures (Fig-
ure 3G) and are dissimilar to the relatively large OS crystals (Figure 3F); hence, this affirms that the differ-
ence in fluorescence is not derived from any morphological discrepancy. As stated above, the crucial
experiment to prove the influence of crystal packing on intrinsic fluorescent formation could be an obser-
vation of reversible transitions between the luminescent and non-luminescent states upon controllable
iScience 24, 102695, July 23, 2021 5
change of the crystal structure. We believe that the observed on-off switching of cysteine fluorescence
upon changing its crystal packing (Figure 3) represents such an experiment.
In an attempt to explain the underlying mechanism of the self-assembly-induced fluorescence of amino
acids, we tried to find a common thread in the aggregation-induced fluorescence related literature. First
principles quantum chemistry calculations of self-assembly-induced fluorescence mainly deal with absorp-
tion spectra (Grisanti et al., 2017;Pinotsi et al., 2016;Stephens et al., 2020). Without novel absorption bands,
no novel fluorescence bands may appear, and the key to the explanation of the presence of new emission
properties is the understanding of long-wavelength absorption formation. This paradigm was extended in
the recent work of Grisanti et al., where, using ab initio calculations, fluorescence properties of model pep-
tides aggregates were assessed (Grisanti et al., 2020). Besides the prediction of the emission in the visible
spectral range, a conclusion was made on the time-resolved behavior of the aggregation-induced fluores-
cence. Namely, the presence of an ultrafast fluorescence decay and accompanying spectral diffusion, i.e., a
gradual fluorescence emission redshift in time, on a sub-picosecond timescale, was described theoretically.
This result prompted us to search for experimental indication of an ultrafast component in a self-assembly-
induced fluorescence system by means of the ultrafast spectroscopy. Taking into account the universal char-
acter of this emission, i.e., similarity of its spectral properties for a broad range of systems, we chose to work
with a known model system—fibrillar structures that are formed as a result of phenylalanine self-assembly.
The fluorescence of phenylalanine was previously characterized, and showed high fluorescent signal, much
higher than the unfortunately too weak signal of cysteine, which was insufficient for these measurements
(Shaham-Niv et al., 2018). Thus, by the example of the fibrils made of phenylalanine, we aim at corroborating
the presence of an ultrafast fluorescence decay of the self-assembly-induced fluorescence.
Figure 3. Fluorescence of cysteine
For a Figure360 author presentation of Figure 3, see
(A and B) Confocal microscopy images of the cysteine (A) OS sample and (B) DR sample.
(C) The normalized fluorescence intensity of cysteine samples as a function of the emission wavelength (excitation at 405 nm).
(D and E) Crystalline structures of cysteine based on PXRD analysis showing (D) the orthorhombic packing of the OS sample and (E) the monoclinic packingof
the DR sample.
(F–H) Confocal imaging of the cysteine (F) OS sample, (G) DR sample, and (H) DR sample that was re-dissolved and recrystallized at different conditionsto
regain an orthorhombic packing. For confocal images, pixel color represents the intensity; black, non; blue, dim; white, strong.
See also Table S3 and Figure S2.
6iScience 24, 102695, July 23, 2021
The self-assembly of phenylalanine in aqueous solution was initiated by cooling a supersaturated solution
of phenylalanine (40 mg/ml) at 90 Cto20C, similar to previous work (Shaham-Niv et al., 2018). This leads
to the formation of elongated fibrillary aggregates, as shown by bright-field microscopy (Figure 4A). The
phenylalanine fibrils exhibited relatively high fluorescence emission in the visible spectral range, which is
absent in the monomeric state of phenylalanine, and were characterized by the wavelength-dependent
Stokes shift, i.e., the spectra measured at longer excitation wavelength exhibited red-shifted emission (Fig-
ure 4B). Such a behavior of the fluorescence emission band is known as the red-edge excitation shift effect
and is characteristic for the self-assembly-induced fluorescence of amino acids, peptides, and proteins (Ar-
non et al., 2019;Berger et al., 2015;Kumar et al., 2020). The presence of elongated fibrillar structures was
detectable by fluorescence lifetime imaging, without the use of any external dyes, only by using the auto-
fluorescence signal of the sample (Figure 4C). The autofluorescence decay curve obtained for the phenyl-
alanine fibrils revealed the presence of 0.35- and 2.53-ns decay times after fitting to a biexponential model
(Figure 4D). However, the temporal resolution, or the width of the instrument response function (IRF) of the
fluorescence lifetime imaging setup, which employs the standard time-correlated single photon counting
technique, is 50 ps and does not allow for detecting any ultrafast decay components, which will be
completely masked by the 50-ps IRF (Rovnyagina et al, 2019,2020). Hence, we have utilized the fluores-
cence up-conversion technique, which provides for sub-picosecond resolution of fluorescence decay (for
more information, see STAR Methods).
Using fluorescence up-conversion, it was observed that the phenylalanine fibrils exhibited ultrafast decay
lifetimecomponentasfastas0.67ps(Figure 4E). The second decay component was characterized by a 12.6
ps decay. The presence of the sub-picosecond fluorescence decay is in agreement with the calculation of
the recent published work (Grisanti et al., 2020), where the spectral diffusion and relaxation was observed
on a few hundred picoseconds timescale.
In the work of Grisanti et al., the ab initio nonadiabatic dynamics simulations were used to reveal charac-
teristic properties of the excited electronic states of model amyloid-like peptides. It was shown that a
visible (blue-green) fluorescence could originate from the np* states localized on the amide groups. The
specific structure of amyloids gives rise to stabilization of some of these states, thus lowering the energy
gap between the ground and minimum np* state leading to the shift of absorption and fluorescence to
the near UV-visible range. Grisanti and co-workers observed that, after excitation in the manifold of np*
states in 10–15 fs all trajectories from different np* states reached the lowest excited S
(or np*
, accord-
ing to Grisanti et al.) state. In the range of 15–40 fs after excitation UV fluorescence from S
state is observed
andispeakedat3.3eV(370 nm). After that, on the timescale of 100–200 fs, internal vibration redistri-
bution occurs that leads to additional redshift of the emission spectrum, down to 2.0 eV (620 nm)
(Figure 4F).
This evolution occurs on the timescale of 100 fs and is accompanied by the gradual shift of the fluores-
cence emission from the UV to the blue-green region (Figure 4G). By setting the registration emission
wavelength to the blue-green region (in our case, to 500 nm [2.5 eV]), the ultrafast relaxation along the
potential curve shown in Figure 4F can be detected. This process is illustrated in the inset of Figure 4G
and corresponds to the fluorescence decay curve presented in Figure 4E. The ultrafast relaxation occurs
on the timescale of 100 fs, corresponding to the non-radiative decay rate of 10
ever, lower than in the case of monomeric proteins, where the lack of stabilization of the np* states results in
the absence of near-UV and visible absorption and blue-green fluorescence (Grisanti et al., 2020). More-
over, the presence of the ultrafast decay may result in low fluorescence quantum yield of the aggrega-
tion-induced emission of proteins (0.01) (Niyangoda et al., 2017). Further relaxation from the lowest
energy np*
state may then occur on a longer timescale owing to the stabilization of the geometry of
the aggregate, and this relaxation corresponds to the 1 to 2-ns fluorescence lifetime, which was observed
for the self-assembly-induced fluorescence in this paper (Figure 4D) and other previous works (Tikhonova
et al., 2018). Hence, by detecting the ultrafast decay of the fibrillar structures formed as the result of phenyl-
alanine self-assembly, the model of Grisanti et al. can be extended frompeptides to amino acids, therefore
elucidating the origin of their enigmatic fluorescence emission upon packing (Grisanti et al., 2020).
To conclude, in thiswork we have studiedthe optical properties of all20 coded amino acid powders. Eachpowder
was examinedas commercially obtained and also afterdissolution, reassembly, and lyophilization. It is surprising
iScience 24, 102695, July 23, 2021 7
that lysine exhibited the most intense fluorescence, even though it has no aromatic moieties. Other charged
amino acidsshowed little to no fluorescence. In addition, short-chain derivatives of lysinedisplayed no correlation
betweenthe chain length and their intrinsicfluorescence. This indicates that, in somecases, not only the chemical
identity ofthe monomeric moleculedictates the optical properties of the assembly, but alsothe supramolecular
arrangement within the assembly. To substantiate this notion, PXRD analysis together with confocal imaging re-
vealed that the orthorhombic crystal of cysteine is fluorescent, whereas the monoclinic crystal is not. The recrys-
tallization of the non-fluorescent monoclinic crystals into fluorescent orthorhombic crystals confirms that, in this
case, changing the crystal packing is sufficient for conferring optical properties, and that the fluorescence does
not stemfrom contaminations, impurities, or oxidation.It is important to note that we donot imply that impurities,
Figure 4. Ultrafast decay
(A) Chemical scheme and fibrils of phenylalanine (macroscopic and microscopic photos).
(B) Fluorescence emission of phenylalanine assemblies at excitations between 370 and 420 nm.
(C) Autofluorescence lifetime imaging of phenylalanine fibrils. Excitation was performed in a two-photon regime at
700 nm.
(D) Fluorescence decay curve of the phenylalanine fibrils measured with (sub)nanosecond resolution.
(E) Fluorescence decay curve of phenylalanine fibrils measured using fluorescence up-conversion technique. Excitation
and emission were set to 380 and 450 nm, respectively. The inset shows the parameters of fluorescence decay obtained
using the biexponential decay model (D and E).
(F) Schematic decrease in the energy gap between the excited np* and ground state of the model fibrils.
(G) Scheme of the shift of the aggregation-induced fluorescence spectrum with time, demonstrating the presence of the
ultrafast decay and spectral migration.
8iScience 24, 102695, July 23, 2021
oxidation, or aromatic interactions are not valid mechanisms that couldexplain the fluorescence in some cases.
However,in addition to those,supramolecularinteractions mayalso affect the opticalproperties of otherbiomol-
ecular assemblies, as unambiguously presented here, thus playing a key role in a phenomenon that could be ex-
plained byother known mechanisms. In the literature it was proposed that peptide aggregation-induced fluores-
cence in the blue-green spectral range is due to (1) delocalization of electrons over a network of hydrogen bonds
(Shukla et al.,2004), (2) hydrogenbond-mediated interactionsbetween the amidegroups (Ye et al., 2017), (3) pro-
ton transfer across hydrogen bonds(Pinotsi et al., 2016),and (4) a decreasein the energy gap between the excited
and groundstates caused by the influence of the hydrogenbonds on the amide groupgeometry (Ye et al., 2017).
The importance and role of the structure for fluorescenceformation in aggregates of non-aromatic peptideshas
been recently addressed using ab initio nonadiabatic dynamics simulations of the excited electronic states (Ye
et al., 2017). In addition, previous work has shown that low-energy optical excitations and subsequent fluores-
cence can be induced by charge-transfer excitations (Jong et al., 2019;Mandal et al., 2019;Prasad et al., 2017).
Specifically, charge transfer excitations involving sulfuratoms of methionine and thepositively charged N termini
of amyloid aggregates were presented (Jong et al., 2019). In the case of cysteine demonstrated in this work, the
difference in the molecular packing within the crystal alters the distances between the sulfur atom and the N
termini of the neighboring molecule, which could affect the charge transfer potential. Indeed, these distances
are shorter for the orthorhombic crystal in comparison with the monoclinic packing (Figure S2). Overall, on the
basis of previous research, it can be summarized thatthe chromophore responsible forthe aggregation-induced
absorption and fluorescence in the visible range is structure specific and can be formed either in the absence or
presence of aromatic moieties in peptides. Our results demonstrate that even the simplest and most thoroughly
investigated molecular systems, such as amino acids, can still serve as the basis for new, intriguing, and unknown
phenomena. The reversibility of cysteine fluorescence serves as a strong evidence that the molecular arrange-
ment has a crucial role in the observed optical properties.
The prediction of ultrafast decay by Grisanti et al. was experimentally confirmed in an aggregation-induced
fluorescence model system of phenylalanine fibrils. Specifically, the presence of the ultrafast decay of the
blue-green autofluorescence is in agreement with the hypothesis regarding the governing role of amyloid
structure-stabilized np* states in the emission formation. Although the experimental data do not exclude
other hypotheses, its relevance to the theoretical calculations can be a step toward understanding the
origin of the blue-green emission in amyloids and other systems that recently attracts increased interest,
which appear as a result of peptides and amino acid self-assembly. Further understanding of the underlying
mechanisms could aid in harnessing the intrinsic properties of supramolecular polymers self-assembled by
simple and cost-effective building blocks to develop smart optoelectronic materials.
Limitations of the study
The current study aims to systematically explore, in a non-bias manner, the requirements for autofluores-
cence upon aggregation. Nevertheless, it is limited only to the 20 coded amino acids. Although we
observed similar optical behavior for other small molecules, such as metabolites and nucleotides, further
research, beyond the scope of this study, is necessary to understand the fundamental principles of this phe-
nomenon. In addition, there are numerous other questions that could be asked and researched for
regarding these systems, such as the effect of racemic amino acids, different solvents, or ionic strength.
Questions like what is the ‘‘proper packing’’ for fluorescence? What are the requirements? This is the
main enigma. Once we know the answer, we can potentially pre-design the optical properties of a material.
Although we did not find a clear-cut answer to this question, we believe that this article is a big step in the
right direction.
Detailed methods are provided in the online version of this paper and include the following:
BLead contact
BMaterials availability
BData and code availability
BAmino acid reassembly
BConfocal imaging
iScience 24, 102695, July 23, 2021 9
BImage analysis
BPowder X-Ray diffraction
BCysteine crystallization
BPhenylalanine fibrils sample preparation
BSteady-state fluorescence measurements of phenylalanine fibrils
BFluorescence lifetime microscopy (FLIM)
BSub-picosecond fluorescence lifetime measurements
Supplemental information can be found online at
Z.A.A. and T.K. contributed equally to this work. We thank our lab members for the fruitful discussions. This
work was supported by the Israeli National Nanotechnology Initiative and Helmsley Charitable Trust (E.G.),
ner Institute (Z.A.A.). The work was supported by the Ministry of Science and Higher Education of the
Russian Federation within the framework of state support for the creation and development of World-Class
Research Centers "Digital biodesign and personalized healthcare" No075-15-2020-926 (E.S.). We thank
members of the Gazit group for the helpful discussions.
Z.A.A., T.K., S.S.-N., and E.G. conceived and designed the experiments. Z.A.A., T.K., N.B., R.A., S.S.-N.,
P.M., M.N.Q., E.P., A.R., I.S., A.H., E.S., and D.L. planned and performed the experiments. Z.A.A., T.K.,
and E.G. wrote the manuscript. D.L. performed PXRD experiments and analysis. All authors discussed
the results, provided intellectual input and critical feedback, and commented on the manuscript.
The authors declare no competing interests.
Revised: May 17, 2021
Accepted: June 4, 2021
Published: July 23, 2021
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12 iScience 24, 102695, July 23, 2021
Lead contact
Further information and requests for resources and materials should be directed to and will be fulfilled by
Materials availability
Direct material requests to lead contact, Ehud Gazit.
Data and code availability
The published article includes all datasets generated or analyzed during this study. Crystallographic Infor-
mation File (CIF) for the crystal structure of L-2,4-diaminobutyric acid can be found in the CCDC database
(CCDC Deposition # 1990651).
Chemicals, peptides, and recombinant proteins
L-Alanine Sigma-Aldrich A7627
L-Arginine monohydrochloride Sigma-Aldrich 11039
L-Aspargine Sigma-Aldrich A-0884
L-Aspartic acid Sigma-Aldrich A9256
L-Cysteine Sigma-Aldrich 168179
L-Glutamine Sigma-Aldrich G-3126
L-Glutamic acid Sigma-Aldrich G1251
L-Glycine Sigma-Aldrich G7126
L-Histidine monohydrochloride monohydrate Sigma-Aldrich H8125
L-Isoleucine Sigma-Aldrich I2752
L-Leucine Sigma-Aldrich L8000
L-Lysine Sigma-Aldrich L5501
L-Methionine Sigma-Aldrich M9625
L-Phenylalanine Sigma-Aldrich P2126
L-Proline Sigma-Aldrich P-0380
L-Serine Sigma-Aldrich S4311
L-Threonine Sigma-Aldrich T8625
L-Tryptophan Sigma-Aldrich T0254
L-Tyrosine Sigma-Aldrich T3754
L-Valine Sigma-Aldrich V0500
Deposited data
Crystal structure of L-2,4-diaminobutyric acid This Paper; Cambridge Crystallographic Data
CCDC: 1990651
Software and algorithms
GraphPad Prism 9.0.1 GraphPad
ImageJ 1.52 NIH
Inkscape 0.92
Mercury 4.1.3 Cambridge Crystallographic Data Centre
ChemBioDraw Ultra 14.0 PerkinElmer
iScience 24, 102695, July 23, 2021 13
Amino acid reassembly
The original samples from Sigma-Aldrich were dissolved in double distilled water, at a relatively high
concentration, depending on the water solubility, as detailed in Table S1. The samples were then heated
to 90 C and vortexed to obtain a clear, transparent solution. The solutions were then incubated at room
temperature for a week to allow self-assembly and crystallization. The samples were then lyophilized to re-
move the water and attain a dry crystalline powder.
Confocal imaging
All confocal images were taken using a Leica SP8 Lightning confocal microscope with a Leica Application
Suite X (LAS X) software. The samples were excited using a 405 nm laser, with laser intensity set to 50% and
the gain set to 500 for all images. The emission range was set between 415 nm to 600 nm. All images were
taken at a magnification of 5X.
Image analysis
Images were analyzed using Image J. Since we use confocal microscopy, we can assume that the depth (Z)
is very small in comparison to the width and height (X and Y). Thus, in these images we can consider the
areas rather than the volumes. For each image, we divided the number of ‘‘fluorescent’’ pixels by the num-
ber of ‘‘black’’ pixels in the corresponding brightfield image. The threshold for ‘‘fluorescent’’ and ‘‘black’’
pixels were identical for all the images in each experimental set.
Powder X-Ray diffraction
X-ray diffraction was collected using a Bruker D8 Discover diffractometer with LYNXEYE EX linear position
detector. The diffraction pattern to analyze the amino acid structure was performed in a classical w-wBragg-
Brentano setup. The diffraction patterns were corroborated based on the reported phases in the PDF-4-
organics-2019 database. As the crystal structure of L-2,4-diaminobutyric acid was not reported in the
literature, a full crystal structure determination was performed (CCDC Deposition # 1990651; Data S1).
The crystalized powder was placed in a 0.7 mm quartz capillary and a full diffraction pattern was collected
between 2 and 50 ,step0.02A
˚. The capillary setup was employed: Go
¨bels mirrors to obtain a parallel
beam, rotating capillary holder. The crystallographic structure determination was performed using the
EXPO2014 software (Altomare et al., 2013). These EXPO2104 features used were cell indexing (N-TREOR09
algorithm) and the Simulated Annealing Method.
The solution with the lowest cost function was used as model to perform further crystal refinement on the
structure using the GSASII software (Toby and Von Dreele, 2013). The final error indexes were: wR=10.9%
and GoF=3.77.
Cysteine crystallization
Both cysteine crystal packings were crystallized in double distilled water, at an amino acid concentration of
200 mg/ml. Monoclinic packing was attained as described in the ‘‘Amino Acid Reassembly’’ section above.
Orthorhombic packing was attained by incubating the solution over night at 4 C.
Phenylalanine fibrils sample preparation
The sample preparation protocol was analogous to the procedures previously used to create self-assem-
bled phenylalanine -aggregates as previously described (Shaham-Niv et al., 2018) using the heat-cool tech-
nique. Briefly, L-phenylalanine (PanreacApplichem, CAS 63-91-2, no additional purification) in a concentra-
tion of 40 mg/ml was dissolved in distilled water (Millipore-Q) at 90 C (temperature was controlled by the
thermostat Qpod 2e, Quantum Northwest, USA) and was stirred for 1 hour using magnetic stirring for com-
plete dissolving. To obtain self-assembled aggregates, the heated phenylalanine solution was cooled to a
room temperature of 23-25 C under normal conditions in cuvettes or on glass slides, depending on the
type of the measurement being performed.
Steady-state fluorescence measurements of phenylalanine fibrils
Steady-state fluorescence measurements were performed using the FluoroMax-4 spectrofluorometer
(HORIBA Jobin Yvon, Japan). Excitation-emission matrices were measured in the 400-750 nm emission
14 iScience 24, 102695, July 23, 2021
range with 1 nm step; the excitation wavelength was varied in the 370-420 nm range with a 10 nm step. The
spectral widths of the excitation and emission slits were set to 5 nm. The measurements were carried out in
a quartz cuvette with an optical path of 1 cm. The measurements were carried out for samples cooled to
room temperature.
Fluorescence lifetime microscopy (FLIM)
Fluorescence lifetime imaging microscopy (FLIM) with multiphoton excitation was performed using a
custom-build multiphoton multimodal microscopy setup. Femtosecond optical parametric oscillator
TOPOL-1050-C (Avesta, Russia), providing excitation by signal wave in the 680-1000 nm range, was used
as an excitation source. The pulse width of exciting radiation was 150 fs, with frequency of 80 MHz,
average power at the excitation wavelength on the sample was 1 mW. Scanning overthesamplewasper-
formed using DSC-120 scan head (Becker&Hickl, Germany). Imaging was performed using oil immersion
Plan Apochromat 603objective with NA=1.4 (CFI Plan Apochromat Lambda 603,Nikon,Japan).Fluores-
cence decay curves were detected using hybrid GaAsP detector HPM-100-40 (Becker&Hickl, Germany) with
sensitivity in 250-720 nm range and instrument response function characteristic time-width of 120 ps. To cut
off the excitation, a 680 nm short-pass dielectric filter was used. Both autofluorescence of phenylalanine
-fibers and the thioflavin T fluorescence signals were excited at 730 nm.
Fluorescence decay curves were fitted using SPCImage 8.3 software (Becker&Hickl, Germany) after spatial
binning (bin size was equal to 5 for ThT-fluorescence measurements and 10 for autofluorescence measure-
ments) by bi-exponential decay law with respect to instrument response function. Average fluorescence
lifetime was calculated as t
), where a
are the amplitudes and lifetimes
obtained from fit.
Sub-picosecond fluorescence lifetime measurements
Time-resolved fluorescence emission measurementsin the sub-picosecond time range were carried out us-
ing a commercially available femtosecond fluorescence spectrometry system FOG100 (CDP Systems,
Russia). The samples were excited by 100 fs pulses at 380 nm with a frequency of 80 MHz (second harmonic
of Ti:Sapphire oscillator Mai-Tai, Spectra Physics, USA). The fluorescence signal from the sample was
focused on a 0.5 mm b-barium borate crystal alongside the fundamental beam (80 fs, 760 nm), acting as
a gate pulse for the frequency up-conversion. The gate pulse was delayed by an automatically controlled
delay stage. The upconverted light was focused onto the entrance slit of the double monochromator (spec-
tral resolution < 1.5 nm) and was detected by a photomultiplier tube. The reproducibility of the measure-
ment was checked by 10 times, measuring decay trace. Special rotation cuvette unit was used to avoid pho-
todegradation of the sample.
Similar to steady-state fluorescence and microscopy measurements, a phenylalanine sample in a liquid
state, heated to a temperature of 90 C, was poured into a cuvette and was cooled down to room temper-
ature for 15-20 minutes. The measurements were carried out when the sample was turbid. Fluorescence
decay curves were fitted by biexponential decay law with respect to instrument response function fitted
as Gaussian function. Data analysis on the sub-picosecond decay curves were performed using custom-
built Python scripts using LmFit, Matplotlib, Numpy, Pandas libraries.
[CCDC: 1990651 contains the supplementary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data Centre via
iScience 24, 102695, July 23, 2021 15
... Interestingly, cysteine displays a crystal structure dependent fluorescence, as different arrangements between two crystal structures result in a reversible on−off optical transition. 34 Thus, the supramolecular assembly of the amino acids clearly has a significant impact on their optical properties. ...
... 19 It is clear, however, that the nature of the electronic transitions underlying the role of the carbonyl groups will be highly sensitive to specific environmental interactions. For example, it would be interesting to investigate in the future the effects of the supramolecular assemblies in lysine or cysteine crystals 34 in the electronic states associated with the carbonyl. ...
Full-text available
While in the vast majority of cases fluorescence in biological matter has been attributed to aromatic or conjugated groups, peptides associated with neurodegenerative diseases, such as Alzheimer's, Parkinson's, or Huntington's, have been recently shown to display an intrinsic visible fluorescence even in the absence of aromatic residues. This has called the attention of researchers from many different fields, trying to understand the origin of this peculiar behavior and, at the same time, motivating the search for novel strategies to control the optical properties of new biophotonic materials. Today, after nearly 15 years of its discovery, there is a growing consensus about the mechanism underlying this phenomenon, namely, that electronic interactions between non-optically active molecules can result in supramolecular assemblies that are fluorescent. Despite this progress, many aspects of this phenomenon remain uncharted territory. In this Perspective, we lay down the state-of-the-art in the field highlighting the open questions from both experimental and theoretical fronts in this fascinating emerging area of non-aromatic fluorescence.
... The mechanism of dbAF phenomenon is not yet fully understood and it may not be straightforward as dbAF, in addition to proteins, was documented also for other polymers and simple organic compounds [49,50]. The dbAF of proteins seems to occur mainly due to the emission of excited electrons of peptide bonds in the hyperstable β-sheet structure [51,52]. ...
... The dbAF of proteins seems to occur mainly due to the emission of excited electrons of peptide bonds in the hyperstable β-sheet structure [51,52]. Recent studies have shown that other structure-related factors such as non-covalent π-π stacking between tyrosine residues, chirality of n→π* transitions or supramolecular packing contribute to the spectroscopic properties of the dbAF fluorophores [49,50,52]. The ordered arrangement of atoms and the organization of hydrogen bonds cause a lowering of the energy barrier of electron transitions of the fluorophore, resulting in the emission of light in the visible range [51][52][53]. ...
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Human transthyretin (TTR) is a tetrameric protein transporting thyroid hormones and retinol. TTR is a neuroprotective factor and sensor of oxidative stress which stability is diminished due to mutations and aging, leading to amyloid deposition. Adverse environmental conditions, such as redox and metal ion imbalances, induce destabilization of the TTR structure. We have previously shown that the stability of TTR was disturbed by Ca²⁺ and other factors, including DTT, and led to the formation of an intrinsic fluorophore(s) emitting blue light, termed deep blue autofluorescence (dbAF). Here, we show that the redox state of TTR affects the formation dynamics and properties of dbAF. Free thiols lead to highly unstable subpopulations of TTR and the occurrence of frequent dbAF. Oxidative conditions counteracted the destabilizing effects of free thiols to some extent. However, strong oxidative conditions led to modifications of TTR, which altered the stability of TTR and resulted in unique dbAF spectra. Riboflavin and/or riboflavin photoproducts bound to TTR and crosslinked TTR subunits. Riboflavin-sensitized photooxidation increased TTR unfolding, while photooxidation, either in the absence or presence of riboflavin, increased proteolysis and resulted in multiple oxidative modifications and dityrosine formation in TTR molecules. Therefore, oxidation can switch the role of TTR from a protective to pathogenic factor.
... Concentrationinduced Lys aggregation can also be observed in histones by nonlinear absorbance at 325 nm, and it is hypothesized to involve the α-amino group in Lys-Lys pair coupling [10]. Furthermore, experiments on crystallized Lys and its derivates have demonstrated the important role of supramolecular arrangement in optical emission [11]. ...
L-lysine is an essential amino acid whose peculiar optical properties in aqueous solutions are still in search of a comprehensive explanation. In crystalline form L-lysine does not emit, but when in an aqueous solution, as the concentration increases, emits in the blue. The origin of such fluorescence is not yet clear. In the present article, we have combined quantum mechanics and classical simulations with experimental techniques to demonstrate that optical absorption and excitation-dependent fluorescence are directly correlated with the formation of aggregates, their dimensions and intermolecular interactions. The nature of the aggregates has been studied as a function of the pH and concentration of the amino acid. At low concentrations, fluorescence intensity increases linearly with molarity, while at high concentrations a new condition is established in which emitting and non-emitting molecular species coexist. The L-lysine aggregation and the formation of intermolecular H-bonding are at the ground of the emission in the blue range.
... 14,15 Moreover, the self-assembling properties of the compound also influence its photophysical characteristics [16][17][18][19] which include its conductivity, 20,21 optoelectronic properties, 22 and dispersion capability, 23,24 as well as the lattice packing of the molecules. 25,26 The selfassembling properties of the material are also affected by various parameters such as concentration, 27 temperature, 11,28,29 pH, 30,31 solvent, 32,33 and lattice arrangement. [34][35][36] The importance of the self-assembly/aggregation on the photophysical properties of the material can be broadly understood by two terminologies aggregation-induced emission [37][38][39] and aggregation caused quenching. ...
We report the synthesis and characterization of the self-assembled structures formed by 4-chloro-2(3H)-benzothiazolone (CBT) to panchromatic fibres and its application in cellular imaging. The aggregation properties of the synthesized compound were studied extensively under different solvents and concentrations and their morphologies examined at a supramolecular level by various microscopic techniques such as atomic force microscopy (AFM), fluorescence microscopy, and optical microscopy. Interestingly, the self-assembled structures formed by CBT reveal panchromatic emission properties and show blue, green, and red fluorescence under different excitation wavelengths. The mechanism of the structure formation of the self-assemblies were studied through different techniques such as concentration-dependent 1H-NMR, ATR-FTIR, UV-visible spectroscopy, and fluorescence spectroscopy. Finally, the utility of CBT for cell imaging applications was demonstrated and it can be noted that CBT was efficiently up taken by mammalian cells and the cells revealed panchromatic emission under blue, green, and red channels. The intensities of the fluorescence observed were blue>green>red and the dye interestingly does not exhibit any fluorescence quenching.
Metabolites are the set of substances produced or utilized in the biochemical process of metabolism known to perform diverse physiological functions in every living organism. As very simple molecules, metabolites can self‐assemble into functional materials for biomedical and nanotechnology applications. Simple amino acid‐based crystals exhibit interesting physicochemical properties of piezoelectricity, fluorescence and optical waveguiding. Combinations of metal‐coordinated metabolites display catalytic properties mimicking natural enzymes for chemical reactions and environmental remediation. Furthermore, excessive accumulation of metabolites spontaneously forms toxic assemblies implicated in the pathogenesis of metabolic and neurodegenerative diseases. Herein, we mainly review the progress of recent three years on the assembly of minimalistic metabolite‐based building blocks into bionanomaterials and their potential applications in energy harvesting, optical waveguiding, enzymatic catalysis, and biomedicine. We hope this review can promote the understanding and development of metabolite materials to meet functional requirements.
Metabolites are immensely important for the routine function of every cell and take part in numerous physiological processes. Yet, in excess amounts, metabolites can self‐assemble, giving rise to cytotoxic amyloid‐like structures. Such structures may underlie some of the pathological effects associated with inborn error of metabolism disorders characterized by metabolite accumulation due to genetic mutations. Furthermore, such assemblies may have a role in neurodegenerative disorders due to abnormal accumulation. On the other hand, metabolites were shown to form functional assemblies in various organisms. Interestingly, various proteins and peptides showing amyloidal properties also have physiological roles and have been used for the fabrication of functional nanomaterials. Following this notion, metabolite self‐assembly could also be utilized due to several advantages including exceptional biocompatibility, inexpensive production, facile modeling and biodegradability in vivo. Co‐assembly of metabolites resulting in high rigidity can be further used in different biomedical and nanotechnological applications. Metal‐coordinated metabolite assemblies can be used as electrocatalysts in energy harvesting applications. Consequently, the study of metabolite self‐assemblies is not only crucial in order to understand their role in normal physiology and in pathology, but can also uncover a new route in exploring the fabrication of organic, biocompatible structures for material sciences and various technological applications.
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Amyloids have unique structural, chemical and optical properties. Although much theoretical effort has been directed toward understanding amyloid nucleation, the understanding of their optical properties has remained rather limited. In particular, the photophysical mechanisms leading to near UV excitation and characteristic blue-green luminescence in amyloid systems devoid of aromatic amino acids have not been resolved. We use ab initio static calculations and nonadiabatic dynamics simulations to study the excited electronic states of model amyloid-like peptides. We show that their photophysics is essentially governed by the multitude of nπ* states with excitation localized on the amide groups. The strong stabilization of the nπ* states with respect to the amide group deplanarization and the concomitant increase of the oscillator strength makes excitation in the near UV possible. With respect to emission, our dynamics simulations revealed that the amyloid cross β arrangement effectively hinders the nonradiative relaxation channels operative in functional proteins. Finally, we show that after relaxation of the photoexcited peptides toward the minimum of the different nπ* states, fluorescence takes place in the visible (green) part of the electromagnetic spectrum.
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Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.
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We had earlier reported on the presence of broad UV-Vis electronic absorption (250—800 nm) in a monomeric protein rich in charged, but lacking aromatic amino acids, referred as Protein Charge Transfer Spectra (ProCharTS). Specifically, it was shown that the cationic amino/anionic carboxylate headgroups of Lys/Glu sidechains act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other. In this work, we show that such excitations produce weak intrinsic luminescence in proteins originating from charge recombination. We investigated aqueous solutions of proteins with varying abundance of charged amino acids like human serum albumin (HuSA), hen lysozyme and intrinsically disordered proteins like PEST fragment of human c-Myc protein, α-synuclein and dehydrin. The absorbance and luminescence in all protein samples were a linear function of concentration (0—50 μM) employed, confirming its origin from a monomeric species. The slope of luminescence/[protein] plot directly correlated with the fraction of charged amino acids present in protein. Specifically, the higher slope in protein like HuSA was chiefly accounted by large molar extinction coefficient rather than the quantum yield. This coefficient directly correlates with population of charged side chain headgroups lying in close spatial proximity in the protein, contributed by the 3D fold of the polypeptide. ProCharTS luminescence parameters appear conserved across proteins. These include, overlapping excitation/emission spectra; large Stokes shifts (14000—3000 cm—1) that decrease with increasing excitation wavelength; low quantum yields (0.002—0.026) indicating poor radiative recombination efficiency and multiexponential decays (mean lifetimes = 0.4—2.9 ns).
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Fluorescence in biological systems is usually associated with the presence of aromatic groups. Here, we show that specific hydrogen bonding networks can significantly affect fluorescence employing a combined experimental and computational approach. In particular, we reveal that the single amino acid L-glutamine, by undergoing a chemical transformation leading to the formation of a short hydrogen bond, displays optical properties that are significantly enhanced compared to L-glutamine itself. Ab initio molecular dynamics simulations highlight that these short hydrogen bonds prevent the appearance of a conical intersection between the excited and the ground states and thereby significantly decrease non-radiative transition probabilities. Our findings open the doors for the design of new photoactive materials with biophotonic applications. Significance statement Intrinsic fluorescence of non-aromatic amino acids is a puzzling phenomenon with an enormous potential for technological and biomedical applications. The physical origins of this effect, however, remain elusive. Herein, we demonstrate how specific hydrogen bond networks can modulate fluorescence. We highlight the key role played by short hydrogen bonds in the networks on the ensuing fluorescence and we provide a detailed molecular mechanism to explain this unusual non-aromatic optical properties. Our findings should benefit the design of novel optically active biomaterials for applications in biosensing and imaging.
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Enzymatic activity is crucial for various technological applications, yet the complex structures and limited stability of enzymes often hinder their use. Hence, de novo design of robust biocatalysts that are much simpler than their natural counterparts and possess enhanced catalytic activity has long been a goal in biotechnology. Here, we present evidence for the ability of a single amino acid to self-assemble into a potent and stable catalytic structural entity. Spontaneously, phenylalanine (F) molecules coordinate with zinc ions to form a robust, layered, supramolecular amyloid-like ordered architecture (F–Zn(ii)) and exhibit remarkable carbonic anhydrase-like catalytic activity. Notably, amongst the reported artificial biomolecular hydrolases, F–Zn(ii) displays the lowest molecular mass and highest catalytic efficiency, in addition to reusability, thermal stability, substrate specificity, stereoselectivity and rapid catalytic CO2 hydration ability. Thus, this report provides a rational path towards future de novo design of minimalistic biocatalysts for biotechnological and industrial applications. Simple enzyme mimetics are highly desired for industrial settings providing high catalyst stability and low production costs. This work reports that phenylalanine and zinc ions can self-assemble into a supramolecular structure showing catalytic properties similar to those of the natural enzyme carbonic anhydrase.
The “Mother Nature” utilizes molecular self-assembly as an efficient tool to design several fascinating supramolecular architectures from simple building blocks like amino acids, peptides and nucleobases. The self-assembling behavior of various biologically important molecules, morphological outcomes, molecular mechanism of association and finally their applications in real world draw broad interest in chemical and biological point of views. In this present feature article, the amyloid hypothesis is extended to include non-proteinaceous single metabolites that invoke a new paradigm for the pathology of inborn metabolic disorders. In this scenario, we dedicate this article to understand the morphological consequences and mechanistic insight of the self-assembly of some important amino acids (eg. L-phenylalanine, L-tyrosine, glycine etc.) and nucleobases (adenine and eight uracil moiety derivatives). Using proper spectroscopic and microscopic tools, distinct assembling mechanisms of different amino acids and nucleobases have been established. Again, lanthanides, polyphenolic compounds such as crown ethers and a worldwide drink, beer, are elegantly employed as inhibitors of the resulting fibrillar aggregated structures. As a consequence, this study will cover literally a vast region in the self-assembling outcomes of single biologically important molecules and therefore, we expect a detailed understanding of such morphological outcomes using spectroscopic and microscopic approaches may open a new paradigm in this burgeoning field.
Recent reports of distinctive UV-vis absorption profiles for monomeric proteins rich in charged amino acids which span 250-800 nm have opened up a new label-free optical spectral window for probing biomolecular structure and interactions. Combined experimental-computational studies have revealed that such broad absorption profiles in these proteins arise from photoexcited charge transfer (CT) transitions in spatially proximal charged amino acids such as lysine (Lys) and glutamate (Glu). Here, using Time-dependent Density Functional Theory (TDDFT) with an optimally tuned CAM-B3LYP functional, we refine the computed UV-Vis spectra for Lys-Glu dimers within protein folds and quantify the percentage CT character of the constituent transitions. The optimally tuned functionals are derived through a careful analysis of the CAM-B3LYP parameter space for Lys-Glu dimers as a function of amino-acid conformation and sidechain separations. Our studies reveal that the tuned Lys-Glu dimer spectrum spans 150-650 nm and possesses 5 specific types of CT excitations with diverse and large spatial charge separation lengthscales of 2-10 Å. These include inter-/intra-residue peptide backbone to peptide backbone (BB-CT) excitations spanning 160-210 nm, inter-/intra-residue peptide backbone to sidechain (BS-CT) excitations spanning 160-260 nm, and sidechain to sidechain (SS-CT) excitations which show the broadest absorption range spanning 250-650 nm.
There is a growing body of experimental work showing that protein aggregates associated with amyloid fibrils feature an intrinsic fluorescence. In order to understand the microscopic origin of this behavior observed in non-aromatic aggregates of peptides and proteins, we conducted a combined experimental and computational study on the optical properties of amyloid-derived oligopeptides in the near-UV region. We have focused on a few model systems having charged termini (zwitterionic) or acetylated termini. For the zwitterionic system, we were able to simulate the longer tail absorption in the near UV (250-350 nm), supporting the experimental results in term of excitation spectra. We analyzed optical excitations responsible for the low-energy absorption and found a large role played by charge-transfer states around the termini. These charge-transfer excitations are very sensitive to the conformation of the peptide and in the realistic fibrils may involve inter and intra chain charge reorganization.
The self‐assembly of simple units into well‐ordered supramolecular polymeric structures may give rise to a broad range of desirable attributes. In the field of bionanotechnology, there are two main classes commonly used as building blocks for self‐assembly—amino acids and nucleobases. While protein, peptide, and amino acid building blocks propose broad chemical versatility, the specific Watson–Crick base pairings of nucleic acids allow rational design and precise control over the assembly process. Specifically, artificially synthesized peptide nucleic acids (PNA), short DNA mimics that have an amide backbone, can present a unique set of properties while preserving the specific base pairing of the nucleic acids. The use of complementary building blocks allows the study of supramolecular polymer coassemblies. Here, the coassembly of two paired di‐PNA building blocks provides a new layer of control, as both building blocks are required for the spontaneous assembly to occur. Utilizing a microfluidic system, the growth of the well‐ordered structure can be regulated and monitored. The crystal structure of the formed assemblies display Watson–Crick base pairing with similar distances as those found in typical DNA double helices. Moreover, the crystals exhibit intriguing optical properties that may be implemented in various materials science and nanotechnological applications. The specific Watson–Crick base pairing of peptide nucleic acids (PNA) governs their assembly into ordered structures. The use of complementary building blocks allows the study of supramolecular polymer coassemblies. Here, the controlled assembly of two paired di‐PNA building blocks is demonstrated using a microfluidics system to generate crystalline fluorescent structures.