Investigation of Fibril Forming Mechanisms of l-Phenylalanine and l

Jan 25, 2017 - Phenylketonuria and tyrosinemia type II, the two metabolic disorders, are originated due to the complications in metabolism of phenylal...
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Investigation of Fibril Forming Mechanisms of L‑Phenylalanine and L‑Tyrosine: Microscopic Insight toward Phenylketonuria and Tyrosinemia Type II Debasis Banik, Sangita Kundu, Pavel Banerjee, Rupam Dutta, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India S Supporting Information *

ABSTRACT: Phenylketonuria and tyrosinemia type II, the two metabolic disorders, are originated due to the complications in metabolism of phenylalanine (Phe) and tyrosine (Tyr), respectively. Several neurological injuries, involving microcephaly, mental retardation, epilepsy, motor disease, and skin problems etc., are the symptoms of these two diseases. It has been reported that toxic amyloid fibrils are formed at high concentrations of Phe and Tyr. Our study indicates that the fibril forming mechanisms of Phe and Tyr are completely different. In the case of Phe, −NH3+ and −COO− groups of neighboring molecules interact via hydrogen bonding and polar interactions. On the other hand, there is no role of − NH3+ group in the fibril forming mechanism of Tyr. In Tyr fibril, the two hydrogen bonding partners are −OH and −COO− groups. In addition, we have also investigated the effect of three lanthanide cations on the fibrillar assemblies of Phe. It has been observed that the efficiencies of three lanthanides to inhibit the fibrillar assemblies of Phe follow the order Tb3+< Sm3+< Eu3+.



problems etc.8 In a recent study, Gazit and co-workers have reported the formation of fibrillar assemblies of Phe and Tyr and these have similar biophysical, biochemical, and cytotoxic properties like other amyloids.9,10 On the other hand, Perween et al. suggested that far-UV absorption in the CD spectra of these two amino acids is due to n−π* and π−π* transitions involving carbonyl group and benzene ring, respectively. These CD spectra do not reflect the secondary structure characteristics of the amino acids like other amyloids.11 Amyloid fibrils are associated with several complications like Alzheimer’s disease, prion disorders, and type II diabetes in the lifestyle of millions of worldwide people.12,13 Therefore, by considering the neurological complexity due to amyloid formation, it is very interesting to focus the fibril forming mechanisms of these two amino acids. It has been proposed that the self-assembly processes of L-Phe and L-Tyr are guided by the hydrogen bonding and electrostatic interactions between neighboring molecules.11,12 In addition, aromatic interactions (π−π stacking) assist the amyloid formation.14,15 Apart from this, amyloid formation depends on several factors, such as chirality of the molecule, pH of the medium, variation of the solvent, sequence of amino acid within the peptide, etc.16−19 Inhibition of amyloid formation by polyphenols, β-cyclodextrin, molecular tweezers, and cucurbit[7]uril (CB[7]) have already been reported.20−24 Singh et al. and Luigi et al. have shown the inhibition of Phe fibril formation by 25,26 D-phenylalanine and doxycycline, respectively. Tian et al.

INTRODUCTION Phenylalanine (Phe), one of the essential amino acids for human, is naturally converted into tyrosine through an enzyme phenylalanine hydroxylase (PAH).1−4 This tyrosine (Tyr) is degraded into small molecules (either released through kidney or used in the citric acid cycle) via the pathway of five enzymatic reactions.4 The first step of this reaction involves an enzyme called tyrosine transaminase (TT). Genetic mutation of these enzymes (PAH and TT) inhibits the degradation of Phe and Tyr; as a result, the concentrations of these two amino acids (Phe and Tyr) in blood increase abnormally. This causes several genetic disorders, such as phenylketonuria (PKU) and tyrosinemia type II etc.1−5 Kaufman has mentioned that in the case of hyperphenylalaninemia patients the blood phenylalanine concentration exceeds 1.2 mM.3 This concentration is 20 times greater than normal levels (0.055−0.060 mM). Francis et al. reported that an 11 year old tyrosinemia type II patient has plasma tyrosine concentration of 1.4 mM.5 In case of mink, the tyrosine concentration reaches above 2 mM.6 The biosynthetic preparation of neurotransmitter (i.e., dopamine) requires the availability of tyrosine and the presence of a standard concentration of Phe within the brain. However, this condition is not maintained in PKU patients, as the excess Phe in blood saturates the transporter of amino acids through blood-brain-barrier and reduces the ratio of Phe/ tyrosine in brain. This causes a deficiency of the neurotransmitter dopamine, reduction in protein synthesis, and demyelation. The early and late manifestations of PKU are microcephaly, severe mental retardation, epilepsy, and progression of a motor disease, respectively.7 On the other hand, tyrosinemia causes several skin problems, photophobia, excessive tearing, neurological © 2017 American Chemical Society

Received: December 5, 2016 Revised: January 24, 2017 Published: January 25, 2017 1533

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The Journal of Physical Chemistry B Scheme 1. Chemical Structures of the Materials Used in Our Study

of our knowledge, this is the first approach to differentiate the fibril forming mechanism of L-Phe and L-Tyr along with the effect of lanthanides on the fibrillar assembly of Phe.

have shown retarded amyloid-β formation using 12-crown-4 (12C4).27 Very recently, we have shown that the efficiency of 18-crown-6 (18C6) is greater than 15-crown-5 (15C5) to arrest the L-Phe fibril.28 Bera et al. have shown that Tyr substituted analogues of diphenylalanine do not form fibrillar assemblies.29 However, until now, there is no comparative information about the fibril forming mechanisms of L-Phe and L-Tyr. In this article, we have addressed two things: (i) Is there any difference in fibril forming mechanism between L-Phe and L-Tyr (Tyr is para-hydroxy Phe)? (ii) In our previous study, we have observed that crown ethers inhibit the fibril formation of L-Phe by forming host−guest complex with the −NH3+ group.28 What will happen if we target the carboxylate group of L-Phe? Therefore, to address the first point, we have employed 18C6 to capture the −NH3+ group of Tyr and studied the kinetics up to 48 h. Second, we have deprotonated the hydroxyl group of Tyr by increasing pH of the medium and observed the effect. Lastly, it is reported that the lanthanide ions interact with the amino acids via carboxylate group.30 Therefore, we have investigated the effect of lanthanide ion (Eu3+) on the Tyr fibril. By combining the results of these three experiments, we have observed that fibril forming mechanism of L-Tyr is completely different from the + − L-Phe (−NH3 and −COO groups of neighboring molecules interact via hydrogen bonding and polar interactions28). Fibrillar assemblies of L-Tyr are due to formation of hydrogen bonds between − OH and −COO−groups of neighboring molecules. To elucidate our second issue, we have employed three different lanthanide ions (Tb3+, Sm3+, and Eu3+) to study the interaction with L-Phe fibril. We have observed that less than 50 μM concentrations of Sm3+ and Eu3+ are able to inhibit the fibrillar assembly of Phe. In presence of Tb3+, inhibition process has been retarded. Metal complexes are found to be inhibitors of amyloid fibrils.31−47 We have chosen lanthanide ions due to their potential use as cancer biomarkers, apoptotic agents, in vivo neuroimaging agents, as therapeutic molecules in heart disease, myocardial infractions, etc.48−54 In both cases, we have monitored the kinetics using field emission scanning electron microscopy (FESEM) and fluorescence lifetime imaging microscopy (FLIM) techniques. Fluorescence correlation spectroscopy (FCS) studies have been used to corroborate our results. To the best



MATERIALS AND METHODS Chemicals. L-Phenylalanine (L-Phe), L-tyrosine (L-Tyr), 18-crown-6 (18C6), terbium(III) nitrate hexahydrate (Tb(NO3)3·6H2O), samarium(III) nitrate hexahydrate (Sm(NO3)3· 6H2O), and europium(III) nitrate pentahydrate (Eu(NO3)3· 5H2O) were bought from Sigma-Aldrich. 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) dye was supplied by Exciton. We have used double-distilled Milli-Q (ddH2O) water in our experimental work. Scheme 1 represents the chemical structures of all the materials. Preparation of Fibrillar Samples. At first, we dissolved solid L-Phe and L-Tyr in double distilled H2O to prepare 1 mM concentration. We kept the solution for 24 h at room temperature for the preparation of fibrillar assemblies. After that, we added 0.5 (Tyr:18C6 = 1:0.5), 1.0 (1:1), 1.5 (1:1.5), and 2.0 (1:2) mM 18C6 to 1 mM Tyr and studied the kinetics. Three lanthanide salts [Tb(III), Sm(III), and Eu(III)] have been used to inhibit the fibrillar assembly of Phe. Transmission electron microscopy (TEM) measurements of L-Phe and L-Tyr fibrillar aggregates were performed at 80 kV voltage using JEM 2010 transmission electron microscope. Other instrumental techniques have been discussed in details in our earlier publication (sections 2.3 to 2.7 of ref 28).



RESULTS AND DISCUSSION First, the experimental results have been shown to differentiate the fibril forming mechanism between L-Phe and L-Tyr. For this purpose, we have taken the help of our previous publication and most of the experiments using L-Phe are not performed again.28 At first, we monitored the formation of Tyr fibril by measuring the fluorescence intensity of DCM compared to neat water. Enhancement in fluorescence intensity has been observed in the presence of Tyr (Figure S1, Supporting Information). Similar observation is also reported by Cheng et al. in the presence of cerebral amyloid fibrils.55 This provides an indication about the formation of Tyr fibril. As L-Tyr is a chiral molecule, we have 1534

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Figure 1. FESEM and TEM images of Phe and Tyr fibrils [(a,b): FESEM (L-Phe); (c,d, and e): TEM (L-Phe); (a-,b-): FESEM (L-Tyr); (c-,d-, and e-): TEM (L-Tyr)].

Tyr:18C6 = 1:2 at 48 h (Figure 4). FESEM images also support the flakes type morphology of Phe in the presence of 18C6, whereas, no morphological change is observed in Tyr. As the −NH3+ group of Phe plays an important role in the fibril formation, inhibition has been observed due to formation of 18C6:NH3+ host−guest complex. Therefore, our experimental results indicate that there is no role of −NH3+ group in the fibril forming mechanism of Tyr. In addition, we have observed the appearance of very tiny fibrils in the presence of 18C6 (Figure S3, Supporting Information). With increasing 18C6 concentration and time, tiny fibrils are agglomerated to mature fibrils, which are morphologically different from the neat Tyr fibril (Figure 2). This indicates the occurrence of some favorable interaction in the presence of 18C6. FLIM technique also provides information about the lifetime of the dye within fibrillar environment. Lifetime distributions of DCM in Phe and Tyr fibrils and in the presence of 18C6 are shown in the bottom portion of every lifetime image (Figures 2 and 3). We have observed that the lifetime distributions of DCM in Phe and Tyr fibrils are in between 1650 and 2100 ps and

performed circular dichroism (CD) measurement. We have observed a 222 nm absorption peak at 1 mM concentration (Figure S2, Supporting Information).11 Fluorescence and CD measurements of L-phe are provided in ref 28. For microscopic evidence, we have taken the FESEM, TEM, and FLIM images of L-Phe and L-Tyr fibrils at 1 mM concentrations (Figures 1 and 2). Fibrous morphologies of L-Phe and L-Tyr are confirmed from these images. After confirming the fibril formation by these two amino acids, we have investigated the effect of 18C6 on the Tyr fibril. It is reported that crown ether recognizes the ammonium ions of amino acids.56,57 We have maintained the same ratio of Tyr:18C6 like our previous report and studied the kinetics up to 48 h (please see the Materials and Methods Section for details).28 FLIM images of Tyr fibril in the presence of different concentrations of 18C6 at different times are shown in Figure 3. From Figure 3, we have observed that fibrillar morphology of Tyr remains intact in the presence of Tyr:18C6 = 1:2 up to 48 h. On the contrary, in case of Phe, inhibition starts at Phe:18C6 = 1:0.5 from 20 h.28 We have taken the FESEM images of Phe:18C6 and 1535

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We have also measured the diffusion of DCM dye in Tyr fibril and in the presence of 18C6. In water, DCM diffuses at a rate of 300 μm2 s−1.58 The autocorrelation traces of DCM in Tyr fibril and in the presence of 18C6 are nicely fitted using single component diffusion models (Figure 5). Within Try fibril, the diffusion of DCM dye slows down to 198 μm2 s−1. In presence of Try:18C6 = 1:2, we have observed similar diffusion coefficient of DCM (190 μm2 s−1). Therefore, the combined results of microscopy and diffusion indicate that there is no effect of 18C6 on the fibrillar morphology of Tyr. Except for the −NH3+ group, Tyr contains two polar moieties (−OH and −COO−) at neutral pH. Therefore, we should discuss individually the role of −OH and −COO− groups on the fibril forming mechanism of Tyr. For this purpose, deprotonation of −OH group has been done by increasing the pH of the medium. The pKa of side chain hydroxyl group of Tyr is 10.07. Therefore, we have investigated the effect of pH 12 on the preformed L-Tyr fibril using FESEM (Figure 6). We have not found any trace of fibril from these images. Therefore, the deprotonation of −OH group inhibits the formation of Tyr fibril. Singh et al. have observed the formation of Phe fibril at pH 2 and 9 along with the neutral aqueous solution.25 Bera et al. found the involvement of −OH group in the higher order aggregation of a protected dityrosine peptide.29 It is possible that the −OH group of Tyr forms strong hydrogen bonds with the −COO−, deprotonation causes the repulsion between two negatively charged groups. As a result, we have observed inhibition of Tyr fibril. Adler-Abramovich et al. performed molecular dynamics simulation to investigate the effect of high pH on the L-Phe fibril.9 At high pH, the overall charge of L-Phe becomes negative and

Figure 2. Fluorescence intensity [(a) L-Phe (c) L-Tyr] and lifetime images [(b) L-Phe (d) L-Tyr] of Phe and Tyr fibrils.

1100−2300 ps, respectively. Similar lifetime distribution in Tyr is found in the presence of 18C6. However, we have reported the change in lifetime distribution of DCM on going from Phe fibril to Phe+18C6 system.28 This corroborates our FESEM and FLIM results of Phe and Tyr in 18C6.

Figure 3. Effect of 18C6 on the Tyr fibril. Kinetic measurements have been performed using FLIM technique. 1536

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Figure 4. FESEM images of (a) Phe and (b) Tyr assemblies in the presence of 2 mM 18C6.

lanthanide ion (Eu3+). We have employed 40 μM Eu3+ to observe the effect on Tyr fibril. FESEM and FLIM images are taken at 24 h after the addition of Eu3+(Figure 7). Complete inhibition of Tyr fibril is observed from these two images. Therefore, our experimental results clearly indicate that −OH and −COO− groups are the two hydrogen bonding partners of Tyr fibril. It is interesting to predict the very different aggregation mechanism of two similar amino acids. L-Phe and L-Tyr have very similar chemical structures. However, the solubility of these two compounds in water is significantly different. Although, Tyr contains an additional hydroxyl (−OH) group, the water solubility decreases on going from Phe to Tyr. This indicates the involvement of −OH group of Tyr with the other group (−COO−) and this decreases the water solubility. In other words, if Tyr has to form fibril in aqueous solution, its −OH group should not interact with the water molecules. On the other hand, Phe has two polar groups (−NH3+ and −COO−) and within the selfassembly these two groups are connected via hydrogen bonding. Therefore, we feel that this is the probable approach to differentiate the fibril forming mechanism of these two amino acids. Now, we would like to focus on the effect of three lanthanide ions (Tb3+, Sm3+, and Eu3+) on the L-Phe fibril. For this purpose, we have employed two different concentrations of three lanthanides (20 and 40 μM of Tb3+, Sm3+, and Eu3+). Initially, we have monitored the kinetics at 6 and 24 h after the addition of

Figure 5. Autocorrelation traces of DCM in Tyr fibril and in the presence of 2 mM 18C6.

they have observed filamentous aggregates. Within this aggregates, they have predicted strong packing between benzene rings. At pH 12, we have also observed similar morphology of L-Tyr fibril as predicted by Adler-Abramovich et al.9 Therefore, we feel that these assemblies are due to strong π−π stacking interactions between L-Tyr molecules. To clarify the interaction of −OH and −COO−groups of Tyr, we have engaged the −COO− group by interacting with a

Figure 6. FESEM images of L-Tyr assemblies at pH 12.

Figure 7. (a) FESEM and (b) FLIM images of Tyr assemblies in the presence of 40 μM Eu3+. 1537

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Figure 8. Kinetics of Phe fibril inhibition by different concentrations of (a) Tb3+; (b) Sm3+; (c)Eu3+. (FESEM images were taken at 6 and 24 h after the addition of lanthanides.)

are probably due to the disassembly of L-Phe fibril in the presence of Sm3+ and look like flakes. Singh et al. have also observed similar morphology of L-Phe fibril in the presence of 25 D-phenylalanine. Simultaneous existence of two types of LPhe aggregates (flakes and Palmyra leaf) in the presence of 40 μM Sm3+ is clearly observed from the FLIM images. In case of Eu3+, the “Palmyra leaf” type morphology is only observed at 20 μM from 6 to 24 h. After this concentration flakes type aggregates have been observed. In comparison, the fibril inhibiting efficiency of Tb3+ is the lowest than those of the other two lanthanide ions.

lanthanides using FESEM technique (Figure 8). After that, the detailed kinetics (at 6, 12, and 24 h) have been studied by FLIM technique (Figure 9). FESEM and FLIM results indicate that 20 μM Tb3+ cannot inhibit the fibril formation of Phe within 24 h. In presence of 40 μM Tb3+ from 12 h, we have observed different morphology other than fibril. This morphology looks like “Palmyra leaf”. Similar morphology is observed at 20 μM Sm3+ from 6 to 24 h. However, in the presence of 40 μM Sm3+, we have observed the generation of a new morphology along with the “Palmyra leaf” type aggregates. These newly appeared aggregates 1538

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Figure 9. Kinetics of Phe fibril inhibition by different concentrations of (a) Tb3+; (b) Sm3+; (c) Eu3+. (FILM images were taken at 6, 12, and 24 h after the addition of lanthanides.) (Scale bar is 100 μm.)

of L-Phe arises due to its fibrillar morphology.9 Luigi et al. have reported that the doxycycline hinders the fibril formation of L-Phe. They have also performed the cell viability experiments in absence and presence of doxycycline.26 According to their observation,

In between Sm3+ and Eu3+, complete inhibition is observed in case of Eu3+ at 40 μM concentration. Therefore, the inhibition efficiency of three lanthanide ions follows the trend Eu3+> Sm3+> Tb3+. Adler-Abramovich et al. have reported that the cytotoxicity 1539

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Figure 10. Lifetime distribution of DCM in (a) Phe fibril and (b) in the presence of 40 μM of different lanthanides at 6 h.

cell viability (SH-SY5Y cell line) increases from 58 to 70% in the presence of 50 μM doxycycline. However, doxycycline cannot neutralize the toxic effect of L-Phe under their experimental conditions. Similar to our study, they have observed the generation of smaller aggregates in the presence of doxycycline. Singh et al. mentioned that as the fibrillar morphology of L-Phe is responsible for the cell toxicity, the inhibition strategy of fibril formation is a good approach for amyloid disease.25 Therefore, we feel that newly appeared aggregates in the presence of lanthanides are less toxic compared with the normal fibril. We have plotted the lifetime distribution of DCM in Phe fibril and in the presence of lanthanides. Plots are shown in Figure 10. As discussed earlier, we have observed that the lifetime distribution of DCM remains in between 1650 to 2100 ps with a maximum at around 2000 ps in neat Phe fibril (Figure 10a). Ongoing from Tb3+ → Sm3+ → Eu3+, we have observed the decrease in the lifetime distribution of DCM (Figure 10b). Similar observation is also found in our previous study with increasing crown ether concentration.28 In presence of Tb3+, single distribution is observed which is similar to the neat fibril. Sm3+ gives dual bands in the lifetime distribution. These dual bands correspond to the “Palmyra leaf” and flakes type aggregates. On the other hand, in the presence of Eu3+ a single lifetime distribution band is observed at a lower lifetime region than neat fibril. This band is due to the flakes type morphology appeared from the inhibition of fibrillar assembly. In summary, the overall lifetime distribution analysis indicates that the inhibition efficiency of three lanthanides follows the order Tb3+< Sm3+< Eu3+ and the result is completely supported by FESEM and FLIM techniques. FCS is a sensitive technique to measure the molecular diffusion. We have collected autocorrelation traces of DCM in L-Phe fibril and in the presence of different concentrations of lanthanides at 12 h. Fitted FCS traces are shown in Figure 11 (depending upon the fitting residuals and R2 values single or double component diffusion models were used to fit the data). We have observed that diffusion of DCM retards to ∼248 μm2s−1 and ∼198 μm2s−1 within L-Phe and L-Tyr fibrils, respectively (Table 1). From FESEM and TEM images, it can be stated that the size of L-Tyr fibrils are larger compared to L-Phe (Figure 1). As a hydrophobic probe, when DCM enters into this large aggregates significant retardation in diffusion coefficient has been observed compared to neat water and in L-Phe. Therefore, from retarded diffusion dynamics of DCM one can predict the size and hydrophobic interactions within other amyloids.

Figure 11. FCS traces of DCM in neat fibril and in the presence of 40 μM different lanthanides at 12 h.

Table 1. Diffusion Coefficients of DCM in L-Phe Fibril and in the Presence of Lanthanides systems

α1

DCM in L-Phe fibril 20 Tb3+ (μM) 40 20 Sm3+ (μM) 40 20 Eu3+ (μM) 40

1 1 0.90 0.74 0.65 0.61

α2

0.10 0.26 0.35 0.39 1

D1 (μm2/s) 248 ± 3.2 251 ± 3.6 242 ± 4.5 254 ± 5.2 241 ± 4.1 252 ± 2.3

D2 (μm2/s)

152 ± 5.2 138 ± 3.7 155 ± 6.6 145 ± 5.6 146 ± 4.5

In presence of 20 μM Tb3+, the diffusion coefficient of DCM is similar to the neat fibril. The similarity in diffusion coefficient indicates that there is no inhibition at this concentration which is ascertained from the FESEM and FLIM images. Interestingly, in the presence of 40 μM Tb3+ we have observed the generation of a slow diffusing component ∼150 μm2s−1 having the contribution 10%. Therefore, one can predict the percentage of inhibition from the relative contribution of this slow diffusing component. In presence of 20 and 40 μM Sm3+, the relative contribution of slow diffusing component is 26 and 35%, respectively. Therefore, one-third portion of fibril is disrupted in the presence of 40 μM of Sm3+ at 12 h. In case of Eu3+, we have observed the presence of a single diffusing species at 40 μM. It is reported that L-Phe interacts with Eu(III) and Tb(III) via carboxylate group.30 Using CHN analysis, it has been observed that three L-Phe molecules are connected to a single Eu(III) or Tb(III). Multiple coordination 1540

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The Journal of Physical Chemistry B number of lanthanide complexes is also suggested by Frederick S. Richardson.59 Therefore, in the presence of lanthanide ions [especially Eu(III)], intermolecular interaction between neighboring L-Phe molecules disappears, along with the generation of distinct aggregates (dye diffusion coefficient ∼150 μm2s−1) where multiple monomers are attached with lanthanide cations (flakes type aggregates). This is the probable reason for getting a distinct dye diffusion coefficient (∼150 μm2s−1) other than the monomeric one (∼300 μm2s−1). In summary, the appearance of the slow diffusion coefficient is an indication of fibril inhibition process and when this component is predominantly present in the autocorrelation traces complete inhibition takes place. Therefore, we have verified the inhibition of L-Phe fibril by lanthanides and their inhibition efficiencies using FCS. We have also investigated the effect of 18C6 and lanthanides by incubating these compounds with L-Phe and L-Tyr from the very beginning. As reported earlier, we have maintained the same concentrations of two amino acids, 18C6 and lanthanides. The detailed kinetics has been monitored using FLIM experiments (Figures S4, S5, and S6). We have observed that up to 1:1 concentration of L-Phe:18C6, the fibrillar morphology remains intact (up to 48 h). At 1:1.5 of L-Phe:18C6 at 3 h, we have observed the simultaneous presence of fibrils and arrested fibrils. As reported earlier, flakes type morphology has been observed at 1:1.5 (at 20 and 48 h) and 1:2 (at 3, 20, and 48 h) of 28 L-Phe:18C6. On the other hand, in case of L-Tyr, we have not found any changes in the morphology up to 1:2 concentration of 3+ L-Tyr:18C6 (up to 48 h). In case of lanthanide addition, Tb cannot inhibit the fibril formation at all. We have observed “Palmyra leaf” type morphology in the presence of Sm3+. In presence of 40 μM Eu3+, we have observed the simultaneous presence of “Palmyra leaf” and “flakes” type aggregates. Therefore, the retardation of fibril inhibition kinetics has been observed in these cases (incubation of stimuli from the very beginning) compared to the data already present in our article (stimuli addition after the formation of fibril). If two L-Phe molecules are connected via hydrogen bonding after the formation of fibrillar network, disruption can be achieved by capturing one of the −NH3+ groups by 18C6. This is the probable reason for L-Phe fibril inhibition at a ratio of 1:0.5 (fibril:18C6). However, in case of 18C6 (fibril:18C6 = 1:0.5) incubation from the very beginning, 50% L-Phe molecules lose their effectiveness and the remaining 50% forms fibril. This is how the retardation occurs.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Phone: +91-3222283332; Fax: 91-3222-255303. ORCID

Nilmoni Sarkar: 0000-0002-8714-0000 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS N.S. is thankful to SERB, Department of Science and Technology (DST), Government of India, for generous research grants. D.B. is thankful to IIT Kharagpur for the research fellowship. S.K., P.B., and R.D. are thankful to CSIR for their research fellowships.



REFERENCES

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CONCLUSION In conclusion, fibril forming mechanisms of L-Phe and L-Tyr have been investigated. It has been observed that hydrogen bonding interactions between −NH3+ and −COO− groups of neighboring Phe molecules are responsible for the fibril formation. On the other hand, in the case of Tyr, −NH3+ group has no role in the fibril formation. The −OH and −COO− groups of Tyr are the two hydrogen bonding partners. We have also shown that lanthanides can inhibit the fibril formation of L-Phe and follow the trend Tb3+< Sm3+< Eu3+.



fibrils in Tyr+18C6 system, and the effects of 18C6 and three lanthanides during the preparation of L-Phe and LTyr fibrils (PDF)

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b12220. Steady-state emission spectra of DCM in water and in Tyr fibril, CD spectrum of Tyr in water, generation of tiny 1541

DOI: 10.1021/acs.jpcb.6b12220 J. Phys. Chem. B 2017, 121, 1533−1543

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