Efficient Detection of Early Events of α-Synuclein Aggregation Using a

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Efficient Detection of Early Events of Alpha Synuclein Aggregation using a Cysteine Specific Hybrid Scaffold Satadru Chatterjee, Sumanta Ghosh, Snehasis Mishra, Krishna Das Saha, Biswadip Banerji, and Krishnananda Chattopadhyay Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01161 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Biochemistry

Efficient Detection of Early Events of Alpha Synuclein Aggregation using a Cysteine Specific Hybrid Scaffold Satadru Chatterjee,

†[a]

Sumanta Ghosh,†[b] Snehasis Mishra,

[c]

Krishna Das Saha,[c] Biswadip

Banerji*[a] [d] Krishnananda Chattopadhyay*[b] [d],

†Equal

Contribution

Satadru Chatterjee†a, Biswadip Banerji*a,d aOrganic

& Medicinal Chemistry Division, Indian Institute of Chemical Biology (CSIR-IICB)

dAcademy

of Scientific and Innovative Research, (AcSIR), 4 Raja S. C. Mullick Road, Kolkata, Country. India-700032; Fax: (+) 91 33 24735197, 91 33 24723967; Tel: (+) 91 33 24995709; Email ID: [email protected] Sumanta Ghosh†b, Krishnananda Chattopadhyay*b bStructural

Biology and Bioinformatics Division; Indian Institute of Chemical Biology (CSIRIICB); 4 Raja S. C. Mullick Road, Kolkata, Country. India700032; dAcademy

of Scientific and Innovative Research, (AcSIR), 4 Raja S. C. Mullick Road, Kolkata, Country. India-700032; Fax: (+) 91 33 24735197, 91 33 24723967; Tel: (+) 91 33 24995843 Email ID: [email protected] Snehasis Mishrac, Krishna Das Sahac eCancer

Biology & Inflammatory Disorder Division; Indian Institute of Chemical Biology (CSIR-IICB); 4 Raja S. C. MullickRoad, Kolkata, Country. India700032

Keywords: Cysteine; Fluorescence sensor; Protein conformation; live cell imaging 1 ACS Paragon Plus Environment

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Abstract: In the present study, we have designed and synthesized a new hybrid ligand (SCG) which can selectively detect cysteine in the free and protein bound states within minutes at sub-nanomolar level. The photo induced electron transfer (PET) was responsible for the visible color change as well as a large increase in steady state fluorescence. This detection was validated by using multiple model protein systems with differing cysteine environments and spatial arrangements. SCG was able to monitor the early events of the folding/aggregation kinetics of alpha synuclein (-syn), a protein involved in the pathology of Parkinson’s disease (PD). The early events consisted of conformational fluctuations between different forms of the protein and oligomer formation. SCG was found effective in detecting early isomers of -syn in vitro, and in live cell environments.

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Biochemistry

Introduction: A number of small molecule-based fluorescence probes has been developed recently for sensitive monitoring of different complex cellular events.1, 2 The molecular properties of these probes can be tuned using simple synthetic chemistry approach offering a wide range of optical properties and measurement options for in vitro, live cell and animal experiments. One cellular event is the regulation of redox homeostasis, in which levels of thiol groups (SH) are highly controlled using key protein molecules.3-5 Interestingly, there is only limited reports of the development of small molecule probes to monitor –SH groups in protein environment in a conformational specific manner.6-9 In the present study, we describe a small molecule probe (SCG) for sensitive and rapid detection of cysteine in the free and protein bound states. The target molecule worked based on photo-induced electron transfer (PET) and Michael acceptor properties. In the presence of cysteine, SCG displayed an excellent turn on fluorescence response along with a visual colour change which was absent when other amino acids were employed. Using a number of model protein systems of varying cysteine environments, we found that SCG differentiated between the number (single/multiple), positions (exposed/buried) and redox states (oxidised/reduced) of the SH group in a protein. In addition, SCG offered selective fluorescence response to a cysteine residue present at different regions of a natively unfolded protein alpha synuclein (-syn, SNCA, UniProtKB P37840), which aggregates in Parkinson’s disease (PD) brain.10 This experiment was carried out by employing a number of single cysteine mutants, in which the -SH group was placed at different regions of -syn. Small molecule based fluorophores to monitor the aggregation of -syn has been developed by other groups.11, 12 The results presented in this paper show that although the cysteine mutants of the protein -syn are natively unfolded as that of wild type protein, it can form local structures in which the –SH groups experience region-specific variation in surface exposures. Most importantly, we developed an application of SCG fluorescence to study the early events of -syn aggregation. Although the early stages of -syn aggregation contribute strongly to PD related cytotoxicity,13, 14 these events have been relatively unexplored because of the lack of suitable experimental techniques. The present study show that SCG can offer a simple, convenient and sensitive method to monitor early events of -syn aggregation. Using Confocal microscopy and a neuroblastoma cell line SH-SY5Y, we demonstrated that SCG can also be effective in cellular environments. 3 ACS Paragon Plus Environment

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Materials and Methods: Sample and reagents: BSA (A7638) was obtained from Sigma Chemical Co. (St. Louis, MO). Tris(2 carboxy -ethyl) phosphine hydrochloride solution was also obtained from Sigma Aldrich. Reduction of disulphide bonds in BSA was done by incubating 10µg/ml TCEP solution. Then the solution was applied for overnight dialysis to remove free TCEP in the solution. The expression, purification of WT and mutant proteins have been carried out using published procedure. -syn does not contain any cysteine residue. We introduced a cysteine mutant (Gly132Cys) at the c-terminal end of the protein using standard site-directed mutagenesis procedure. We used G132C mutant, because structurally and functionally it behaves like wild type. All other reagents and solvents used in this present study were purchased from Acros organics, and TCI chemicals respectively. Cell Lines and Chemicals: Human Neuroblastoma cell line (SH-SY5Y) was procured from National Centre for Cell Sciences (NCCS), Pune, India. Cell culture components viz. Dulbecco’s Modified Eagle Medium (DMEM), F-12, Penicillin-Streptomycin-neomycin (PSN) antibiotic cocktail, Fetal bovine serum (FBS), trypsin and ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Grand Island, NY, USA). Other raw and fine chemicals were obtained from Sisco Research Laboratories (SRL), Mumbai, India, Sigma-Aldrich, St. Louis, MO, USA and eBioscience, San Diego, USA. Cell Culture: Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and F-12 medium with the ratio of 1:1 having 10% fetal bovine serum (FBS) and 1% antibiotic cocktail at 37 °C in a humidified condition under constant 5% CO2. After 75−80% confluences, cells were harvested with trypsin (0.25%) and EDTA (0.52 mM) in phosphate buffered saline (PBS) and plated at a necessary density to allow them to re-equilibrate before the experimentation.

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Biochemistry

Confocal Microscopy: To visualize the localization of SCG, SH-SY5Y cells were seeded on the cover slip. After 24 hours, cells were treated with 40 nM of SCG in the absence (control) and presence of 1M cysteine or G132C (α-syn) samples (as our panelling). G132C protein of 200M concentration incubated at 370C for different time intervals (0, 2, 5, 12 and 18h) was taken out, diluted to 1M, and then added extracellularly into SH-SY5Y cells. After incubation for 2 hours, 40nM SCG was added and then the cover slips were washed three times using PBS, which was then fixed by 4% paraformaldehyde using previously published method 15. Before mounting for the microscopy studies, Alexa Fluor 555 Phalloidin was used to visualize the cytoskeleton. An Olympus FV10i confocal microscopy was used for these measurements. MTT assay: MTT assay was performed to evaluate the cell viability. For the initial screening experiment, the HCT116 cells (4×103 cells per well) were seeded in a 96 well plate and kept in an incubator followed by treatment with different concentrations of SCG (0.0–0.5 µM) for 24 h. After 24 h of incubation, cells were washed with PBS, and the MTT solution was added to each well and kept in an incubator for 4 h to form formazan salt. The formazan salt was then solubilized using DMSO and the absorbance was observed at 595 nm using an ELISA reader (Emax, Molecular device, USA). Cell proliferation was determined from the absorption intensity.16 Steady state Fluorescence: We used PTI fluorometer to study SCG as a sensor towards cysteine containing proteins along with free cysteine only by steady state fluorescence. We varied the concentration of cysteine containing systems (free cysteine, wild type -syn, G132C, oxidised BSA and reduced BSA) up to their saturation. We used a 2:1 mixture of 20 mM sodium phosphate and ethanol in each case. Extent of fluorometric response is the indication of SCG’s sensing capacity towards different samples. The steady-state fluorescence emission spectra of SCG was recorded at an excitation wavelength of 365 nm. Reaction of SCG with cysteine gives rise to two peaks at 383 and 400 nm respectively. The peak intensity values at 400 nm were plotted against concentration of all the model systems. 5 ACS Paragon Plus Environment

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The data have been fit using the Hill equation, as follows (eq 1) (1) Where, F and F0 refer to the fluorescence intensity of SCG in the presence and absence of model systems, respectively. Fe denotes the minimum intensity in the presence of a higher concentration of model systems, and K is the equilibrium dissociation coefficient of the SCG-cysteine complex. Here, n is the Hill coefficient, which measures the cooperatively of binding, and x is the concentration of the protein. Table 1: Association and dissociation constant of SCG binding with different model cysteine systems along with LOD Cysteine containing model system

Ka (M-1)

Kd (M)

LOD

Free Cysteine

4.47× 104

2.2 (±0.3)× 10-5

1.2 (± 0.1) nM

Native BSA

11.2× 104

8.9 (±0.9)× 10-6

482 (± 20) pM

Reduced BSA (by TCEP)

33.9× 104

2.9 (±0.5)× 10-6

6.6 (± 0.3) pM

G132C (-syn)

6.76× 104

1.5 (±0.9)× 10-5

77.6 (±4 ) pM

G7C

6.14× 104

1.6 (±0.2)× 10-5

199 (± 9) pM

G86C

4.5× 104

2.2 (±0.3)× 10-5

414 (± 18) pM

The error bars for Kd and LOD were determined using three measurements. Ka was calculated from measured Kd Limit of Detection (LOD): Limit of detection (LOD) in each case was calculated from Intensity vs. Concentration graph.17

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Biochemistry

The formula is:

SD = Measured standard deviation of the blank solution. The value is 0.559. Absorption spectrum: Spectrophotometric assay was done to analyse absorption profile with different cysteine containing system. SCG corresponds to three peaks at 240 nm, 275 nm and 365 nm. Varying concentration of cysteine leads to formation of isobestic points confirmed by absorption profile at 275 nm and 365 nm. Varying concentration of other cysteine containing systems shows other kind of absorption profile in these two maxima. These correspond to different approach of SCG binding towards different cysteine containing system. Fluorescence Correlation Spectroscopy (FCS): We carried out FCS experiment using a home-made setup with 40x water immersion objective. The labelled protein samples were excited using an argon laser of 488 nm (for excitation of Alexa488 labelled -syn) and the signals were collected using two avalanche photodiodes (APD). Correlation functions were calculated from the signal obtained in these APDs. The normalized form of the autocorrelation function of fluorescence intensity I(t) at time t is represented by: ……………… (1) In this expression, is the average of fluorescence signal over time, and δI(t) is the signal fluctuation at time t minus the average: ……………. (2) Correlation for a simple solution containing a single diffusing species represented as: ……………… (3) 7 ACS Paragon Plus Environment

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Where τD is the characteristic diffusion time, N is the average number of particles in the observation volume, and S is the structural parameter. The value of τD obtained from the diffusion coefficient (D) by the following equation: ……………. (4) Size of the observation volume () was calculated using the reference of FCS measurements with Rhodamine 6G, whose value of D has been well established.18 The value of the hydrodynamic radius (rH) can be obtained from D using the Stokes’ Einstein formalism (Equation11). ……………... (5) Where η is the viscosity and k is the Boltzmann constant. It may be noted that Equation 5 assumes that the molecules under investigation are spherical. In order to study conformational change of -syn in the early phase of aggregation, we used 200nM of alexa488 labelled along with 200M unlabeled cysteine mutant of -syn and measured the diffusion time (D), from which we determined hydrodynamic radius (rH) using Stokes’-Einstein equation. MALDI-TOF Analysis: For MALDI-TOF analysis we used 1 µM of SCG solution with 20 µM -syn. 0.5 µl was spotted on a cyano-4-hydroxy-cinnamic acid matrix. Analysis of the sample was done using MALDI TOF by an Applied Bio systems Q10 4800 MALDI TOF/TOF™ analyzer using 4000 series explorer software for acquisition and GPS explorer software, version 3.6 for analysis. 4700calmix (Applied Bio systems) were used to calibrate the instrument under reflector mode. The error limit is 50 ppm. Other Information: 1H

and 13C spectrum of SCG were recorded in a Bruker 600 MHz spectrometer. High resolution

mass spectrum of SCG was recorded with JEOL The Mstation JMS-700 instrument. Singlet (s), 8 ACS Paragon Plus Environment

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Biochemistry

doublet (d), triplet (t), double doublet (dd) & multiplet (m) were designated as 1H NMR multiplicity patterns. All the column chromatographic separations were performed using silica gel of 100-200 mesh with petroleum ether and ethyl acetate as eluent.

Results & Discussion: Synthesis of Pyrene-DEM conjugates fluorescence probe and efficient detection of free cysteine using photo-induced electron transfer mechanism: The ligand SCG was synthesized using a simple two-step procedure involving easily available starting materials (Figure S1). The first step was the classical Suzuki coupling reaction between compound 1 & 2, forming compound 3 which was then subjected to a simple condensation reaction with diethylmalonate (DEM) to obtain the desired compound 4, SCG in 80% yield. The target molecule (SCG) was designed based on the principle of photo induced electron transfer (PET) and fluorescence off-on technique.

Figure 1: A. Proposed mechanism of action of SCG, B. Detection of cysteine( in 2:1 NaP/EtOH) using visual and under UV at 365 nm, C. Comparative fluorescence intensity of different thiol containing amino acids (glutathione, cysteine and Homocysteine) & D. Absorption profile of SCG with varying amount of cysteine.

SCG was fully characterized and all the spectroscopic data are provided in the ESI. Initially, the sensing efficiency of SCG towards cysteine was tested using visual colour change and under UV 9 ACS Paragon Plus Environment

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light at 365 nm in the presence of different amino acids. A distinct colour change (yellow to colourless) (Figure 1B) was observed by visual inspection in presence of cysteine only. Under UV light at 365 nm, a turn on blue fluorescence also characterized the presence of cysteine (Figure 1B). For a control set of experiments, SCG (1M) was tested using other common analytes (different cations and anions, 50 equivalents each) and natural amino acids, in which we noticed no significant changes in SCG intensity (Figure S2). Amino acids with free sulfhydryl group (SH) other than cysteine (like Glutathione & homocysteine) were also tested, which did not yield any significant change in fluorescence thus confirming the specificity of our design towards cysteine only (Figure 1C). Although it is difficult to accurately determine the molecular mechanism responsible for the specificity of SCG, the following two factors may contribute: the first one is the bulky nature of DEM which makes the molecule less approachable by GSH. Secondly, it is well known that in neutral pH, the pKa of cysteine is lower compared to GSH and homocysteine, which would enhance the reactivity of cysteine.19 The UV-absorption profile of SCG with varying concentration of cysteine (0-30 M) clearly showed the presence of isobestic points at 275 nm and 350 nm (Figure 1D), suggesting the presence of the free and cysteine bound SCG in equilibrium. Further, we used 1H NMR titration experiment to confirm Michael type reaction operating here. We observed that in presence of cysteine, the olefin proton (Ha) at ~7.9 ppm disappeared with the appearance of a new peak at ~4.6 ppm for Hb (figure S3). The proposed SCG-cysteine adduct, formed during the reaction was further confirmed by ESI-High Resolution Mass Spectrometry (ESI-HRMS, Figure S4). Rapid and sensitive detection of cysteine in free and protein bound forms: Subsequently, we used fluorescence spectroscopy to detect cysteine in its free and protein bound states. A time-based fluorescence study of SCG with free cysteine showed a large increase in fluorescence, which completed within 3 minutes (Figure S5). We carried out fluorescence titration experiments, in which we used 1µM SCG and varying concentrations of cysteine (up to saturation, 30 µM). In the absence of cysteine, the fluorescence spectrum of SCG was found

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Biochemistry

Figure 2: The variation of steady state fluorescence spectra of SCG with varying concentration (up to 30 M) of A) free cysteine B) BSA C) reduced BSA D) Wild type -syn & E) cysteine mutant syn (G132C) with F) comparative bar graph at the saturation point. In the presence of WT -syn, (Fig. C) no change in the fluorescence intensity was observed. The small change in the intensity for BSA (Fig. B) presumably represents the presence of a free but buried cysteine.

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featureless. In contrast, the presence of cysteine resulted in the appearance of two peaks at 383 nm and 400 nm whose intensity increased as cysteine concentration increased (Figure 2A). For the protein based measurements, we used four model systems. These were I) Bovine Serum Albumin (BSA), a globular protein with 34 oxidized cysteine residues (disulphide linked) and one buried cysteine residue20, 21(Figure 2B) II) TCEP reduced BSA with many exposed reduced cysteine residues (Figure 2C) III) -syn, a natively unfolded protein with no cysteine residue (Figure 2D) and IV) G132C mutant of -syn, which contained a single cysteine residue (Figure 2E). This cysteine residue was incorporated using site-directed mutagenesis.22 We chose -syn as its aggregation has strong implication in the pathology of PD. G132C mutant of -syn has been used routinely for biophysical studies, as its aggregation profile is identical to that of WT -syn.23 Figure 2F plots the variation of the fluorescence intensity of SCG in the presence of saturating concentration (30 µM) of free cysteine, native BSA, reduced BSA and G132C (-syn). We saw a ~1.25 fold increase in fluorescence intensity for the native BSA (one –SH only) compared to the free cysteine. In contrast, a ~25 fold increase in intensity was observed for the reduced BSA (approximately 35 free SH). The protein -syn (no cysteine residue) which was used as a negative control, did not result in any fluorescence change (Figure 2D). However, incorporation of a single cysteine by mutation, G132C resulted in ~4.5 fold increase in fluorescence intensity. Relatively higher fluorescence intensity of the G132C mutant (~4.5 times with respect to free cysteine) compared to the native BSA (~1.25 times) could be explained by the intrinsically disordered nature of the former protein, in which cysteine residue would be more exposed. The binding constant (Ka) of BSA was found to be greater than that of free cysteine (1.12×105 M-1 & 4.47×104 M-1 respectively) (Figure S6, table 1). Ka for G132C was less compared to that of BSA and reduced BSA, but greater than that of free cysteine (Table 1). The increase in Ka for the proteins compared to the free cysteine suggested a possible role of the protein chain in facilitating the binding affinity of SCG. To study the binding mechanism in detail, we carried out UV titration experiments of SCG with G132C and BSA. For G132C, we observed a large increase in the absorbance at around 275 nm both with G132C and BSA (figure S7A and S7C). While in the case of free cysteine, the presence of isobestic points in UV-Vis spectra (Figure 1D) were indicative towards two-state reversible mechanism,24 the absence of isobestic points in the case of -syn may be attributed to the heterogeneity in the SCG bound 12 ACS Paragon Plus Environment

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Biochemistry

syn. Mass spectrometry analysis by Maldi-TOF technique revealed the formation of a new peak maximum at 15023, which corresponded to the complex between SCG and -syn along with an intense peak of -syn at 14575 (Figure S7B). Limit of detection (LOD) of each model system was calculated using previously published method17 and was summarised in Table 1. A significantly low LOD (6.6 pM) was observed with reduced BSA which contains 35 –SH groups (Figure S8C). In addition, G132C (the single cysteine mutant of -syn) also showed a very low LOD of 77 pM (Figure S8D and Table 1). SCG can monitor the early events of -syn Aggregation in a position specific manner: We used three single cysteine mutants, namely G7C (cysteine residue at the N terminal), G86C (cysteine residue located at the NAC region) and G132C (cysteine residue at the C terminal). A schematic representation of the mutation sites are shown in Figure 3A. At zero incubation time (when there was no aggregation), we found that the fluorescence intensity was maximum for G132C, which was followed by G7C. SCG showed minimum intensity for G86C (Figure 3B). At this condition (zero incubation time; no aggregation), LOD values were found to be 77 pM, 199 pM and 414 pM respectively for G132C, G7C and G86C (Table 1, Figure S9). There are several factors, which may be responsible for the difference in the fluorescence intensity observed for these cysteine mutants. One of these may be the local structural elements, which is persistent25 even though -syn is an intrinsically disordered protein. As discussed above with respect to the reactivity of cysteine vs homocysteine and GSH, the difference in site hydrophobicity and pKa may also contribute. To obtain further insights into this, we have determined the residue specific variations of the flexibility (Figure S12A) and hydrophobicity (Figure S12B) of -syn using Expasy ProtScale tool.26 The Kyte & Doolittle scale was used for the hydrophobicity calculation. We also determined the sequence specific variation of pKa values of -syn using Depth algorithm.27 The pKa data are shown in Figure S12C. We found good correlation between the observed fluorescence intensity with sequence pKa values and also with the hydrophobicity and flexibility variations. It may be noted that the N-terminal of -syn has membrane targeted sequence which has 11 KTKEGV repeats with 6 lysine residues. As a result, N-terminal region is more prone to acquire secondary structure with comparatively less flexibility.28 The NAC region of the protein, at which cys 86 is located, is known to be hydrophobic and aggregation prone.29 13 ACS Paragon Plus Environment

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Figure 3: A. Pictorial representation of different region of -syn cysteine mutants, B. Comparative bar graph showing the change in SCG fluorescence with increasing concentration (up to 30 M) of G132C, G7C and G86C. These data were obtained at zero incubation time, when there was no aggregation. C. Kinetics study of G132C and WT -syn using ThT fluorescence D. Aggregation kinetics of G132C detected by SCG and ThT fluorescence. E. Kinetics study of G132C mutant using SCG fluorescence at normal and under reduced condition (in presence of TCEP), 14 ACS Paragon Plus Environment

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Biochemistry

The lowest fluorescence intensity of SCG at Cys 86 possibly indicate that the inserted cysteine has the least solvent accessibility because of the locally formed hydrophobic structure at the NAC region of the protein. The C-terminal of -syn has been reported to be the most flexible region30 and highest pKa value of this region makes it quite accessible as a nucleophile towards the electrophilic centre. We first studied the aggregation kinetics of wild type -syn and cysteine mutants using ThT fluorescence, which showed that the inserted cysteine residue had no significant effect on the overall aggregation kinetics of the protein (Figure 3C shows the data for G132C mutant). The incubation of -syn at 37 oC for several days lead to the generation of amyloid fibrils through a nucleation-dependent pathway. We found that the aggregation kinetics was composed of following events: a) a lag phase in which no change in ThT fluorescence was observed (between 0 and ~20 hours); b) an exponential phase which was characterized by a large increase in fluorescence intensity (between ~20 and ~50 hours); c) a stationary phase, (~60 hours and beyond), in which ThT fluorescence saturates. Figure 3D compares the aggregation kinetics of G132C as monitored by ThT method and by SCG fluorescence measurements. The aggregation kinetics of G132C showed an early increase in the SCG fluorescence (marked by a circle in Figure 3D), which occurred early in the lag phase, during which there was no change in ThT intensity (Figure 3D). This increase in SCG fluorescence was followed by a large decrease in SCG intensity, which also occurred within the initial ten hours of incubation. As we used cysteine mutants in this study, possible formation of disulphide bond formation may quench the SCG fluorescence in early stage after a rise in fluorescence. But, we found that the aggregation kinetics in the absence and presence of TCEP (reducing agent) were super imposable indicating no significant effect of a possible disulphide bond formation during the aggregation (Figure 3E). AFM images of the fibrils (collected at around 30 hours) in the absence and presence of TCEP also suggested that the end products were similar in both conditions (Figure S10). Nevertheless, we added excess TCEP for all aggregation experiments presented here to completely rule out the formation of any disulphide bond, which would complicate the present data analysis. Interestingly, G7C and G86C showed different behaviour for SCG fluorescence. SCG fluorescence for G7C increased slightly at the beginning, which was followed by a small decrease at longer incubation time. In contrast, SCG fluorescence for G86C, which was very less 15 ACS Paragon Plus Environment

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to begin with, did not change with time. From the SCG fluorescence data, we divided the initial lag phase (first 10 hours) of -syn into two events: the event A (an early increase for G132C, slight increase for G7C and no change for G86C) and event B (a decrease in fluorescence for G132C and G7C and no change for G86C) (Figure 4A). To obtain further insights into event A and B, we took help of Fluorescence Correlation Spectroscopy (FCS). FCS is a popular

Figure 4.A.Events at different time frame of -syn mutant aggregation; B. change in hydrodynamic radius; C. change in number of particles with time; D. Pictorial representation of the change in number of particle with conformational fluctuation and aggregation.

technique which can measures the diffusion of a fluorescently labelled molecule (here syn) at single molecule resolution. Using FCS, we measured the diffusion coefficient (D) of Alexa488Maleimide labelled G132C (Alexa488syn), and the value of D was used to determine 16 ACS Paragon Plus Environment

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Biochemistry

hydrodynamic radius (rH). The value of rH decreased during event A (Figure 4B), while the protein remained a monomer. The monomeric nature of the protein during event A was ascertained by the fact that the number of particles (N) obtained from FCS experiments did not change during event A (Figure 4C). In contrast, event B (the decrease in SCG fluorescence, Figure 4A) was accompanied by an increase in rH (Figure 4B) and a decrease in N (Figure 4C), suggesting that the event B corresponded to oligomerization of -syn. Figure 4D showed a pictorial representation of why oligomer formation would result in an increase in rH and decrease in number of N.31 It may be noted that similar change in FCS data was previously used to characterize oligomer formation during the lag phase of -syn aggregation.32 Combining SCG fluorescence and FCS measurements, it could be concluded that the event A corresponded to a conformational alteration in the monomeric form of -syn, while event B is an oligomerization step.

. Scheme 1: Schematic representation of the efficiency of SCG fluorescence over ThT fluorescence and FCS method. 17 ACS Paragon Plus Environment

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Scheme 1 provides a depiction of early events of -syn aggregation based on the presented data Event A corresponds to a conformational change in the monomeric protein. The conformational alteration lead to a large compaction of the protein chain, in which G132C (cysteine mutant at C-terminal) becomes more exposed while the NAC region remains buried. The compaction of -syn at the early stage has been reported at substantiated by several other reports.22,

33, 34

Event B, which corresponds to oligomer formation has been monitored

conveniently using an easy to synthesis small molecule. Similar to other amyloidogenic proteins, oligomers of α-syn are highly diverse in structure; some of them are β-sheet rich, some of them are helical while many others are disordered even in the oligomeric stage. Giehm and coworkers characterized stem-like oligomers of diameter 18 nm, which assembled into amyloid fibrils by inducing membrane disruption.35 Apetri et al. also found the oligomers to be rich in helical content, which can be introduced by of metal ions, small molecules, chaperones, and other posttranslational modifications.36 Change in environmental conditions has shown to generate at least three types of oligomeric components, fibrillar, prefibrillar and annular). SCG detects early folding of G132C using SH-SY5Y cell line: Although there exist few measurements on the early events of aggregation under in vitro conditions, no method exists for such measurements in the cellular environments. To determine whether events A and B could be observed inside neuroblastoma cell line SH-SY5Y, we first incubated the cells with Alexafluor555 Phalloidin (alexa555) to visualise the cytoskeleton. The cytotoxicity of SCG was measured through MTT assay (Figure S11) before microscopy and was found to be very less up to a concentration of 0.1 µM. As per the IC50 of SCG, we have selected the dose of 40 nM for in cell studies. Panel 5A correspond to the control data as follows (i) red fluorescence images of alexa555, (ii) without SCG and (iii) merged. The absence of any fluorescence signal in 5A was indicative towards the presence of SCG only. On the other hand, the blue fluorescence in Figure 5B corresponds to SCG bound cysteine. We subsequently checked if SCG could offer an initial method of detection of the early events of -syn aggregation in the cellular environment. These data are shown in Figure 5C-5G. For these measurements, SH-SY5Y cells were treated with G132C samples incubated at 37oC for different time intervals. When SCG was added from outside, we found enhanced fluorescence even at zero 18 ACS Paragon Plus Environment

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Biochemistry

Figure 5: Confocal microscopy images of A. Control (no SCG) B. SCG with cysteine & SCG with syn at C. 0 hr D. 2 hrs E. 5 hrs. F. 12 hrs and G. 18 hrs respectively in SH-SY5Y cell lines along with Alexafluor555 labelling. 19 ACS Paragon Plus Environment

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incubation time samples (-syn monomers) indicating efficient permeability of SCG and effective binding with G132C monomers within cells (Figure 5C). However, a large enhancement of SCG fluorescence was observed with 2 hours incubation samples (Figure 5D). This concurred well with the in vitro data, which also showed an increase in SCG fluorescence as a result of conformational alteration of the protein after 2 hours (Figure 3D, supported by FCS data of Figure 4B). Figure 5E-G showed subsequent decrease in SCG fluorescence with increasein the aggregation time point (from 5 hours to 18 hours) which is a result of initial oligomer formation and subsequent fibrillation. Subsequently, the intensity vs. distance graph comparing Alexafluor555 and SCG intensities were plotted against each panel from 5A-G. SCG data, both in solution and in cellular environment clearly suggested an increase in SCG fluorescence for early collapsed conformer (at 2 hours), which decreased as the protein aggregated.

Conclusions: In this work, an efficient fluorescent probe (SCG) has been designed which can detect the redox state of a cysteine residue by virtue of photo-induced electron transfer. The detection kinetics for free cysteine was found to complete within three minutes. The reaction was sensitive towards the conformational (exposed/buried) or redox (oxidised/reduced) states of the cysteine residues in a protein. Moreover, the use of SCG fluorescence was found to be a convenient yet sensitive method to monitor early events of -syn aggregation. The data presented in this manuscript clearly supported the notion that the lag phase of -syn aggregation consists of the different events, including rapid conformational fluctuations and oligomerization. While on-pathway oligomers have been shown to be significantly more toxic compared to the late stage fibrils, small molecules induced conversion into minimally toxic oligomers have been shown recently.37 A key advantage of using SCG as a probe comes from its exclusive response at the early stage. As found in Figure 4A, while SCG provides large sensitivity at the early stage of -syn aggregation, it is completely unresponsive in the late stage. Given the unresponsive nature of ThT in the early stage, SCG fluorescence offers an easy method to complement ThT experiments.

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Biochemistry

Single molecules Fluorescence resonance energy transfer (smFRET)38 and FCS39 measurements have been used extensively to study the folding/aggregation landscape of -syn. The advantages of the FRET (and single molecule FRET) studies come from their better sensitivity towards understanding the heterogeneity, which is associated when two landscapes of folding and misfolding overlap. However, a FRET experiment typically requires multiple fluorophore labeling in a single protein, which may lead to conformational/structural change and the alteration in the aggregation pathways. Moreover, site specific labeling of a single protein using two fluorophores is non-trivial, requiring extensive optimization of the purification process. In addition, smFRET measurements often involve surface attachment of the protein molecules, a chemistry which itself can be non-trivial because of its associated protein denaturing effect. Since FCS measures diffusion in dilute solution, it does not require any surface chemistry. Arguably, the most important application of FCS may come from its ability to study nsec-sec dynamics. Unfortunately, FCS suffers from the refractive index effect of urea or guanidinium hydrochloride, which are commonly used in a protein folding experiment. Moreover, FCS is typically not used to study large aggregates (or the late stage of aggregation). The correlation functions for the large particles are generally too distorted to be fit using a reliable diffusion model. The use of SCG, on the other hand, is simple and does not require any complicated chemical or biochemical synthesis and/or purification protocol. It is specific towards cysteine and can be easily extrapolated to systems other than -syn. We have shown preliminary applications of SCG for in cell investigations, which can be further extended using a -syn transfected cell line. However, other model protein systems in more complicated solution conditions (including real patient samples) would need to be studied for a detailed understanding of the application of SCG. In any case, concerted use of ensemble and single molecules methods using smart chemical probes, like SCG, would be essential to comprehend the roles of protein aggregation in different neurodegenerative diseases.

Acknowledgements: The authors thank CSIR for funding this project. SC & SG contributed equally to this work. SC & SG want to thank UGC for a Senior Research Fellowship. The authors thank Mr Sandip

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Kundu, CSIR-IICB, for recording X-ray data, Mr E. Padmanaban and Mr. Tapas Chowdhury for recording the NMR spectra and Mr Sandip Chowdhury for recording the EI mass spectra. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXX. 1H

and

13C

NMR spectra of SCG, other characterization data along with twelve supporting

figures are provided.

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ACCESSION ID: SNCA P37840

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