Paradoxical Effect of Trehalose on the Aggregation of Î±-Synuclein

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Paradoxical effect of trehalose on the aggregation of #synuclein: Expedites onset of aggregation yet reduces fibril load Nidhi Katyal, Manish Agarwal, Raktim Sen, Vinay Kumar, and Shashank Deep ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00056 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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ACS Chemical Neuroscience

Paradoxical effect of trehalose on the aggregation of α-synuclein: Expedites onset of aggregation yet reduces fibril load Nidhi Katyal, Manish Agarwal, Raktim Sen, Vinay Kumar, Shashank Deep* Department of Chemistry, Indian Institute of Technology, Delhi, Hauz-Khas, New Delhi 110016, India. AUTHOR INFORMATION Corresponding Author *Shashank Deep Associate Professor, Department of Chemistry, Indian Institute of Technology, Delhi, Hauz-Khas, New Delhi

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Aggregation of α-synuclein is closely connected to the pathology of Parkinson’s disease. The phenomenon involves multiple steps, commenced by partial misfolding and eventually leading to mature amyloid fibril formation. Trehalose, a widely accepted osmolyte, has been shown previously to inhibit aggregation of various globular proteins owing to its ability to prevent the initial unfolding of protein. In this study, we have examined if it behaves in a similar fashion with intrinsically disordered protein α-synuclein and possesses the potential to act as therapeutic agent against Parkinson’s disease. It was observed experimentally that samples co-incubated with trehalose fibrillate faster compared to the case in its absence. Molecular dynamics simulations suggested that this initial acceleration is manifestation of trehalose’s tendency to perturb the conformational transitions between different conformers of monomeric protein. It stabilizes the aggregation prone “extended” conformer of αsynuclein, by binding to its exposed acidic residues of C terminus. It also favors the β-rich oligomers once formed. Interestingly, the total fibrils formed are still promisingly less since it accelerates the competing pathway towards formation of amorphous aggregates. Keywords: ThT fluorescence, protein aggregation, molecular dynamics simulations, intrinsically disordered proteins, trehalose, conformational equilibrium Introduction: Numerous neurodegenerative diseases are unequivocally correlated to intracellular aggregation of proteins in the form of amyloid fibrils and plaques1–4. However, this fibrillation process goes through cascade of events including partial misfolding followed by formation of oligomers, protofibrils, and eventually fibrils. α-synuclein is one such intrinsically disordered protein which is involved in the pathogenesis of Parkinson’s disease


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(PD) and Lewy body dementia5–7. A central tenet of drug designing is aimed at screening lead compounds that are capable of modulating any or all of the above indicated steps. Naturally occurring osmolytes have been intracellularly accumulated to acclimate to thermal and osmotic stress. Their significance in protein folding and stability have already been accepted and addressed in myriad studies8–11. However, scrutinising their role in modulating protein’s aggregation pathway is still underway and only feebly understood. Amidst the numerous osmolytes, trehalose has received special attention owing to its wide range applications12 and GRAS (Generally Regarded As Safe) status13. Numerous theories have been promulgated in literature to explain its stabilization behaviour14–21. However, its mechanism of stabilization is complex and cannot be ascribed solely to one theory22. In the present study, we have explored its effect on different stages of aggregation of αsynuclein protein. Our work was inspired by previous efforts that have examined trehalose’s potential in intervening with different proteins aggregation pathway. Liu et al23 and others24– 26

have observed that trehalose could inhibit aggregation of Aβ42 and Aβ40 peptides (which

are responsible for Alzheimer’s disease) and is also capable of dissolving preformed aggregates. Tanaka et al.27 have also seen its efficiency in inhibiting poly-glutamine aggregation, making it a promising candidate in treatment of Huntington’s disease28. Trehalose could also inhibit prion aggregation29–31. There exist few reports demonstrating its ability to contribute to PD therapy as well32–34. Zhou and co-workers have shown that trehalose inhibits fibrillation of wild type and A53T mutant α-synuclein in a dose dependent manner32. However, contrary to their findings Ipsita et al.35 have observed acceleration of αsynuclein aggregation in presence of trehalose. The variability in their observations could be due to the difference in aggregation conditions (trehalose concentration etc.). Thus, despite the available literature, the actual scenario is still elusive. Questions such as: how trehalose interacts (directly or indirectly) with monomeric, oligomeric and fibrillar species, can the 3 ACS Paragon Plus Environment


interaction mode be correlated to any physiochemical property of the primary sequence, what is the predominant energy minima in aggregation process in the presence and absence of trehalose, mechanism of inhibition/acceleration of aggregation by trehalose, aggregation dependence on trehalose’s concentration etc., still remain unanswered. Additionally it would be interesting to speculate if trehalose affects intrinsically disordered protein in a similar fashion as globular proteins. Consequently, in our current study, we have employed both computational and experimental tools to gain better and deeper insights into the way trehalose alters each of the steps in αsynuclein aggregation pathway namely monomeric, oligomeric, fibrillar and off-pathway aggregates. Results suggest that trehalose triggers the onset of aggregation, and also stabilizes β-rich oligomers. Yet the total fibril load was observed to be less compared to the case in its absence. The rationale behind the observation was explained by considering the effect of trehalose on different conformers of α-synuclein. The work also focuses on controversial dichotomy between direct and indirect interactions of trehalose with protein, and that on contradictory vulnerability of different conformational states of α-synuclein to fibrillation. In addition, the findings from this study showcases diverse response of trehalose towards intrinsically disordered protein versus the globular protein. We have finally proposed a model describing the events involved in α-synuclein’s aggregation pathway and their perturbation by trehalose. The information obtained from this study might be useful in designing therapeutic drugs against Parkinson’s.


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Results and Discussion: Effect of trehalose on the aggregation kinetic profile of α-synuclein via experimental tools: The effect of trehalose on the fibrillation kinetics of α-synuclein was studied at 37◦C, 1200 rpm, and pH 7.5 by monitoring the time variation of ThT Fluorescence emission intensity at 482 nm, as shown in Figure 1.

Figure 1: Kinetics of fibrillation of α-synuclein alone and in the presence of 0.1 M, 0.3 M, 0.5 M, and 0.7 M trehalose at 37◦C, 1200 rpm, and pH 7.5. Continous lines represent fits of the curve to the equation (4).

The kinetic profile is sigmoidal in shape with three distinct stages- a lag phase, a growth phase and a final plateau regime, consistent with nucleation polymerization model of aggregation36,37. SI Table 1 enlists the parameters obtained on fitting these kinetic traces to the equation 4. It can be seen that trehalose decreases the lag time of aggregation drastically from ~34 hrs in the absence of trehalose to ~22 hrs in the presence of 0.1 M trehalose. Further increment in the concentration of trehalose brings down the lag time to approximately 1-8 hrs. The rate of growth of aggregates is only marginally affected in the presence of trehalose. More importantly, the plateau region denoting the extent of amyloid formation is 5 ACS Paragon Plus Environment


significantly reduced six times with respect to the case in its absence. No prominent trehalose’s dose dependent changes could be observed as all the concentrations from 0.3 M and beyond, arrests the amyloidic assimilation process to the same level. Taken together, trehalose affects all the three phases of aggregation to different extents. To delve deeper into the reason behind the observed profile, molecular dynamics simulations have been performed and the effect of trehalose was monitored on all prominent species in the aggregation pathway namely monomers, oligomers and fibrils. Effect of trehalose on monomeric form of α-synuclein: Intrinsically disordered proteins are known to sample numerous rapidly interconverting conformations with the free energy barrier of just few kJ/mol38,39. Similarly, under in vitro physiological conditions, monomeric α-synuclein populates ensemble of conformations including extended and compact conformers40–49. The conformational transitions between these conformers influences α-synuclein’s self assembly. We have carried out molecular dynamics simulations of both these states in explicit solvent in the absence and presence of different concentrations of trehalose as shown in Table 1. The extended state was probed via two approaches. The first approach involves simulating monomeric form of α-synuclein, starting from the available NMR structure of its human micelle bound form (pdb id: 1XQ8)50. This initial state is labelled as “conf 1”. In the second approach, one strand from the fibrillar structure of α-synuclein (pdb id: 2N0A)51 was taken and simulated with and without trehalose molecules (0.2 M trehalose concentration, which showed maximum changes in the above case, was chosen). Upon equilibration, random coil structure was obtained resembling those obtained in experiments. This state is labelled as “conf 3”.


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Table 1: Conditions for MD simulations of α-synuclein in the presence of different concentrations of trehalose: Initial state of protein

Trehalose concentration (M)

No. of trehalose molecules

No. of water molecules

Box length (nm)

Simulation length (ns)

conf 1

0

0

180066

17.69

200

(pdb id: 1XQ850; human micelle-bound α-synuclein)

~0.0009

3

183984

17.82

200

~0.0029

10

148794

16.61

200

~0.0059

20

148462

16.60

200

~0.05 (conf 1a)

166

144143

16.49

200

~0.2 (conf 1b)

668

173096

17.69

200

conf 2

~0.05 (conf 2a)

166

144889

16.52

200

(structures obtained after simulating conf 1 in the absence of trehalose)

~0.2 (conf 2bi)

668

130088

16.16

200

~0.2 (conf 2bii)

668

129955

16.15

200

conf 3

0

0

134040

16.04

100

(one β-strand from fibrillar structure of α-synuclein, pdb id: 2N0A51)

~0.05 (conf 3a)

166

131352

15.99

100

0

0

131584

16.03

100

~0.06

166

128888

15.99

100

~0.1

247

127565

15.97

100

~0.2

668

120849

15.88

100

Ten molecules of conf 3

0

0

128904

16.04

100

(conf3-10mol)

~0.06

166

126202

16.00

100

Five molecules of conf 2

0

0

131791

16.04

100

(conf2-5mol)

~0.06

166

129091

16.00

100

0

0

132298

16.04

100

~0.04

166

129599

15.99

100

0

0

309216

21.29

100

~0.12

579

241544

19.80

300

Five molecules of conf 3 (conf3-5mol)

(repeated twice) Four β-sheets from fibrillar structure of α-synuclein, pdb id: 2N0A51 (preformed-fibril-4ch) Fibrillar structure of α-synuclein (pdb id: 2N0A51), containing 10 β-sheets (Fibril-10ch)



In all the simulations starting from conf 1, α-synuclein rapidly collapses into the stable compact form, consistent with the observations by Gnanakaran and group43 with OPLS-AA force field. This conformation could mimic the compact conformer of α-synuclein, and is labelled as “conf 2”. Compaction of the monomer is revealed by decrease in radius of gyration of protein (Rg) value from ~4.8 nm (initial value) to ~1.6 ±0.02 nm (averaged over last 50 ns, in the absence of trehalose), and a corresponding decrease in solvent accessible surface area (SASA) from ~149.4 nm2 to ~86.7 ±2.9 nm2 (averaged over last 50 ns, in the absence of trehalose) as depicted in Figure 2. However, this collapse is much less on addition of trehalose in a dose dependent manner.

Figure 2: Time evolution of (a) radius of gyration (Rg) and (b) solvent accessible surface area (SASA) of α-synuclein starting from its human micelle-bound form, conf 1, in the presence of different number of trehalose molecules.

To characterize these collapsed compact states, their secondary and tertiary contacts were monitored. SI Figure 1, and Figure 3 depicts the time evolution of alpha helical content and the residue level contact map respectively.


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Figure 3: Cα residue contact map of α-synuclein starting from its human micelle-bound form, conf 1, in the presence of (a) 0, (b) 166 (0.05 M) and (c) 668 (0.2 M) trehalose molecules, averaged over last 30 ns. Distance scale bar is shown below.

It can be seen that trehalose brings about a lesser decrease in helical content compared to the case in its absence and diminishes the probability of formation of long range contacts. The findings suggest that trehalose stabilizes the protein in an extended state with lesser tertiary contacts and retention of partial helical structure. To eliminate the bias arising from initial starting structure, another extended conformation termed conf 3 was also simulated in the absence and presence of 0.05 M trehalose. During the course of simulation, intra β-sheet formation occurs in the aqueous solution similar to the previous experimental observations41. This β-sheet is formed by residues 3-7, 28-31, 48, 6063, 66, 69-72, 79-81, 86-88, 93, 97-103, 109-115, 123-126. However, the number of residues involved in β-sheet as well as the total β-content is dramatically reduced on addition of trehalose molecules (SI Figure 2). More interestingly, analogous to the previous case, extended conformations are populated in the presence of trehalose molecules. This is quite evident from SI Figure 3, by shifting of average value (averaged over last 50 ns) of Rg from



~1.8 nm (±0.03) in the aqueous solution to ~2.3 nm (±0.04) in trehalose containing solution. Correspondingly, SASA is also higher in trehalose containing solution. The effect of trehalose may also depend on the nature of starting structure population. Thus, to investigate its influence on compact conformer, equilibrated α-synuclein conformations in aqueous solution from different time points were extracted (referred as conf 2) and simulated for further 200 ns in the presence of 0.05 M and 0.2 M trehalose, the details of which are also given in Table 1. These two concentrations were chosen since in above simulations they showed maximum difference in properties from that of without trehalose situation. However, in this case no prominent changes were observed in each of these measured variables (SI Figure 4). This is indicative of differential behaviour of trehalose depending on the initial configuration. The mode of interaction (direct/indirect) of trehalose with all the conformations of protein was analyzed by calculating time averaged normalized ratio of water oxygen atoms (gnow) over last 30 ns. This parameter provides an estimate of extent of protein hydration and is plotted in Figure 4. Value greater than 1 denotes preferential exclusion of trehalose molecules while that smaller than 1 symbolizes preferential binding relative to water molecules. The figure evinces that trehalose molecules bind to all the conformations. However, there occurs relative enrichment of trehalose surrounding the protein conf 1 at a distance of 2-3 Ȧ from the closest atoms. This enhancement increases with trehalose concentration. Thus, compared to conf 2, conf 1 has higher affinity towards trehalose molecules. In other words, trehalose favors the extended conformation to a larger extent compared to the collapsed structure obtained in pure water. This is in contrast to the behaviour of trehalose observed in case of globular proteins wherein it stabilizes the compact state of protein.


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Figure 4: Plot of gnow averaged over last 30 ns versus distance from the peptide in the presence of different number of trehalose molecules for (a) conf 1, (b) different initial conformations of α-synuclein (see Table 1 for definition of conformers). Thus, trehalose molecules preferentially bind to all the conformations of the protein. SI Figure 5 shows that hydrogen bonding is the preferred interaction mode on account of more negative Coulombic hydrogen bonding energy compared to hydrophobic Lennard-Jones interaction energy between protein and trehalose and that the corresponding energies become more favourable on increasing the number of trehalose molecules in a dose dependent manner. The electrostatic hydrogen bond energy was also delineated into mainchain and sidechain, wherein the major contribution is observed to come from sidechain residues. Among the various conformers, extended conformers conf 1 and conf 3 seems to interact with trehalose to a larger extent compared to collapsed conformer conf 2, as shown in Figure 5.



Figure 5: Bar graph representing the Coulombic interaction energy between trehalose and sidechain of different conformers of protein, averaged over last 30 ns for system containing (a) 166 (b) 668 number of trehalose molecules. Putative residues involved in the interaction are depicted in Figure 6. It was observed that trehalose binds non-specifically to the polar residues particularly with their sidechains. It has greater affinity towards the negatively charged C-terminal. This also complies with our previous inference obtained in case of lysozyme protein22.

Figure 6: Human micelle bound α-synuclein is depicted in red color using secondary structure representation, using VMD software52. Putative residues involved in bonding with trehalose are shown in beads representation with green color. These include: 2, 13, 20, 21, 28, 46, 57- 61, 72, 75, 79, 87, 98, 99, 101-105, 110, 114, 115, 119-123, 126, 129-135, 137, 139140. Almost similar residues were involved for all the conformations simulated.


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The above findings were further validated via experimental techniques. The secondary structure of α-synuclein in the presence and absence of trehalose was monitored by accessing the changes in the far UV CD spectrum from 197 to 240 nm. In the absence of trehalose, αsynuclein exhibits a negative band around 199 nm, characteristic of its disordered structure in an aqueous environment as shown in SI Figure 6 (a). The addition of trehalose causes a marginal decrease in the negative value, however, unaltering the shape of the profile. This suggests that trehalose causes a slight decrease in the randomness of α-synuclein without induction of any regular secondary structure (alpha helix or beta strand). To decipher the changes in the protein hydrophobic surfaces on addition of trehalose, ANS dye was added to the sample solutions. It is widely used to identify partially folded/unfolded intermediates. As seen in SI Figure 6 (b), increasing concentration of trehalose causes marginal blue shift in wavelength along with prominent concomitant increase in emission maxima. This is reflective of alteration in the tertiary structure induced by high concentration of trehalose the extent of which increases in a dose dependent manner. We speculate that in the aqueous environment, α-synuclein prefers to adopt compact conformation with no exposed hydrophobic patches and also with acidic C-terminal buried, thus causing weaker binding of ANS molecules. In trehalose solution, extended conformations with exposed hydrophobic patches and acidic residues are favoured which are prone to interact with both trehalose and ANS molecules. To summarize our observations in light of the above findings, we can say that trehalose molecules do not impart any global conformational changes to the natively unfolded state of α-synuclein. However, it brings about changes in local tertiary conformational contacts and stabilizes the extended state of the protein having lesser long range contacts, by binding to this state. Binding of trehalose molecules to the native structure of protein was also confirmed by ITC measurements, SI Figure 7. 13 ACS Paragon Plus Environment


Effect of trehalose on the early oligomeric state: Five copies of the monomeric form of α-synuclein, starting from conf 1, conf 2, and conf 3 was created and randomly distributed in a cubic aqueous box with and without trehalose molecules. Initially, all the protein molecules were in the dispersed form. Their aggregation state was then quantified by monitoring time evolution of the number of clusters, as plotted in Figure 7(a,b).

Figure 7: Time evolution of number of clusters of synuclein conformers: (a) conf 3 (b) conf 2; and number of interchain hydrogen bond formation for (c) conf 3 (b) conf 2, in the presence of different number of trehalose molecules. Data obtained for conf 1 was similar to that obtained for conf 3.


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It was observed that trehalose marginally decreases the rate of sampling of the oligomers, for both the extended conformations- conf 1 (data not shown) and conf 3. Interestingly, irrespective of the addition of trehalose molecules, aggregation profile depicts a marked delay in sequestration of the protein molecules when initial state contains collapsed form (conf 2), compared to the extended alpha helical (conf 1) or extended disordered (conf 3) forms. This finding when correlated with our previous observation of trehalose favouring the extended conformation (section: “Effect of trehalose on monomeric form of α-synuclein”) points to trehalose accelerating the assembly process. Data was also repeated for 10 such synuclein molecules. Similar inference was obtained from them as well (data not shown). Thus, the observed expedition of the aggregation phenomenon in the presence of trehalose, as observed by ThT fluorescence measurements, is solely an outcome of shifting the conformational equilibria from collapsed to the extended state on addition of trehalose. This extended state has fewer long range contacts. Release of long range tertiary interactions potentiating aggregation of α-synuclein, has already been documented in literature48. To conclude, we speculate that the observed decrease in lag from ThT kinetics could be attributed to the enhanced stabilization of the extended conformation with fewer long range contacts in trehalose regime. These disaccharide molecules increase the population of these conformations of α-synuclein which are then more prone to interact with other such structures, thereby initiating the aggregation phenomenon. In the absence of trehalose, compact ensembles of α-synuclein populates with considerable long range interactions that may resist aggregation. The oligomers formed were further characterized by calculating time evolution of number of inter-chain hydrogen bonds. Figure 7 (c,d) evinces that α-synuclein molecules in aqueous trehalose solution have lesser inter-chain hydrogen bonds. Hydrogen bond formation 15 ACS Paragon Plus Environment


propensity is directly correlated to amyloid fibril formation53,54. The comparatively less tendency to form these hydrogen bonds in trehalose regime corresponds to the less fibrillar load (Figure 1). To gain experimental insights into the size of the aggregates, size distribution profiles of αsynuclein (without trehalose) were obtained by dynamic light scattering (DLS) (SI Figure 8). The hydrodynamic radius of the monomer was found to be ~3.4 nm, consistent with the observations by Fink and group55. After 4 hours of incubation (during lag phase), almost all the monomers were consumed. Thus, the intensity of monomer peak was drastically decreased, and the oligomers of size ~260 nm begun to appear. The monomer particles were not detected post lag phase. DLS profiles of α-synuclein were also obtained in the presence of 0.7 M trehalose. The size of the soluble oligomers increased with time as observed without trehalose. However, at all time intervals, larger soluble oligomers were detected in the trehalose containing samples. Broadening of peaks was also observed for samples containing trehalose, suggesting the formation of species of heterogeneous size. These soluble oligomers were further characterized using CD spectroscopy (SI Figure 9). It can be seen that β-rich oligomers get populated with time as observed by increased negative intensity in the wavelength region from 210 nm to 230 nm. Notably, the rate of formation of these oligomers is higher in the presence of trehalose. Effect of trehalose on preformed early oligomers: Oligomers formed during MD simulations in the above case does not develop inter β-sheet contacts under the time scale of our simulations. Thus, β-rich oligomers were mimicked by simulating 4 β-strands from the fibrillar structure of α-synuclein (pdb id: 2N0A). Their secondary structure was monitored over the course of time as shown in Figure 8. It was observed that β-sheets remained perfectly intact in the presence of trehalose. While in its absence, specifically for 4 chains, β-sheets are dissolved and reformed repeatedly. This 16 ACS Paragon Plus Environment

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suggests that β-rich oligomers, once formed are stabilized by trehalose molecules. Similar inference was obtained on simulating 2, and 6 β-sheets as well (data not shown).

Figure 8: Secondary structure evolution of 4 chains of β-sheet in the presence of (a) 0 M and (b) 0.05 M trehalose, as calculated by DSSP. However, by ThT fluorescence intensity measurements, we have observed lower extent of fibrillation in the presence of trehalose. This can be true if either it restricts the aggregation in the intermediate stage itself or drives it towards an off pathway route. The first possibility seems less probable since freezing in this intermediate oligomeric stage would imply that either the rate of forward reaction for formation of higher ordered β-structured oligomers or fibril formation is slow in the presence of trehalose, or the rate of dissociation of β-structured oligomers and/or fibrils is higher. However, this scenario doesn’t hold true as was validated by our simulation data that showed retention/enhancement of the β-content for all βoligomers with size ranging from 2 to 6. The second possibility of trehalose changing the 17 ACS Paragon Plus Environment


aggregation pathway was checked in subsequent section (“Effect of trehalose on off-pathway non-fibrillar aggregates”). Effect of trehalose on fibrillar state of α-synuclein: The fibrillar structure of α-synuclein was recently published by Tuttle MD et al51. This structure was simulated along with ~0.1 M concentration of trehalose. Under all the conditions, the secondary structure of the fibrils remains intact, implying lesser propensity of trehalose to perturb the fibrillar state, under the time scale of our simulations (SI Figure 10). This observation negates the probability of trehalose to dissociate fibrils. Effect of trehalose on off-pathway non-fibrillar aggregates: As discussed above, there arises likelihood of trehalose enhancing the aggregation kinetics of formation of off-pathway aggregates. This was checked as follows. Firstly, the CD spectra of soluble oligomers in the saturation phase at the end of 100 hours were taken, as shown in Figure 9.

Figure 9: Far-UV CD spectra of soluble oligomers in the saturation phase, in the absence and presence of 0.7 M trehalose.


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It can be seen that both trehalose-containing and trehalose-free samples contain oligomers that lack β-content. It was also observed that the amount of maximum scattered intensity at 600 nm in both these solutions remains the same (data not shown), suggesting that trehalose does not change the total amount of protein that gets aggregated. These facts along with the observation of reduced fibrillation in the presence of trehalose as monitored via ThT fluorescence measurements, suggests trehalose populating non-fibrillar aggregates. This was further confirmed by analyzing the morphology of the final aggregates (Figure 10). TEM images of the aggregates formed in the absence of trehalose clearly depict fibrillar structures, while those formed in the presence of trehalose showed non-fibrillar amorphous type morphology. This suggests alteration of the aggregation route by trehalose. Bogdan Barz et al56, very recently, have shown for Aβ peptides that their pathway of aggregation depends on oligomer shape which in turn is dependent on type of conformer(s) populated. They hypothesize that extended oligomers drive the aggregation process while compact oligomers are metastable and are less involved in assembly process. On similar grounds, we speculate that trehalose accelerates formation of the off-pathway amorphous species due to differential type of oligomers being sampled (loose aggregates with less intermolecular hydrogen bonding, section: “Effect of trehalose on the early oligomeric state”) which again, is an outcome of perturbation of conformational equilibria of monomeric αsynuclein by trehalose (section: “Effect of trehalose on monomeric form of α-synuclein”).



Figure 10: TEM images of fibrils formed in the presence of (a) 0 M and (b) 0.7 M trehalose.

Effect of trehalose on the structural and dynamical properties of water molecules: Water molecules play a pertinent role in rendering stabilization to proteins57–60 and in mediating the self assembly process61–65. Particularly in context of trehalose, its interaction with trehalose molecules is believed to be an inevitable factor influencing trehalose’s mechanistic action66,67. Thus, we have delved deeper into analyzing the structural and dynamical properties of water molecules in the presence of trehalose under different stages of protein aggregation. SI Figure 11 plots radial distribution function of water molecules and the backbone of protein molecule. It can be seen that average density of water molecules around α-synuclein molecules decreases as the protein aggregates and transforms from monomeric to oligomeric to fibrillar stage. This is reflected from decrease in heights of first peak (~0.184 nm) and second peak (~0.27 nm), representative of first shell and second hydration shell respectively, with subsequent build up of the oligomers. Thus, expulsion of water molecules occurs during initial stages itself when disordered aggregates are being formed. Frequency distribution of tetrahedral order parameter (q) for hydration layer water molecules was computed at varying concentrations of trehalose under different simulation conditions 20 ACS Paragon Plus Environment

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(SI Figure 12 (a)-(c))). This parameter sheds light onto the structural and orientational behaviour of water molecules, explicitly its extent of deviation from ideal tetrahedral network. High q values closer to 1 denotes perfect tetrahedral arrangement while its value closer to 0 is an indication of random orientation. Irrespective of the system (extended\collapsed), addition of trehalose molecules brings about an increase in the unstructured water configurations leading to an increase in low q values with a corresponding decrease in high q values. The effect varies in a concentration dependent manner. This destructuring effect of trehalose on the tetrahedral network of water is consistent with previous experimental observations via Raman scattering and neutron diffraction techniques68–72. Stronger trehalose-water interaction strength compared to water-water is speculated to account for the observed behaviour66,67. This tetrahedral orientational order parameter was also calculated for bulk water molecules for reference. As shown in SI Figure 12(d), these bulk molecules populate more structured configurations compared to the local, complying with observations by Biswas et al73. The structural content is however less compared to the globular proteins22 . The extent of deviation from tetrahedrality was computed during oligomerization process as well (SI Figure 13). It was observed that regardless of the stage of protein aggregation, similar systems brings about similar perturbation in the tetrahedral arrangement of water molecules. Trehalose-water interaction behaviour was explored by computing site-site radial distribution function of water oxygen atoms with trehalose’s hydroxylic, acetylic and glycosidic oxygen atoms (SI Figure 14). It can be seen that compared to acetylic and glycosidic oxygen atoms, hydroxylic oxygens of trehalose are heavily hydrated, with a sharp intense first solvation peak centered at around 0.26 nm. This non-uniform solvation characteristics on account of geometric constraint, is in line with the previous data74–76. As we move ahead in the pathway towards fibril formation, the heights of peaks in g(r) of all types of oxygen atoms of trehalose 21 ACS Paragon Plus Environment


with water decreases indicating less trehalose-water interaction. This is because intermolecular trehalose-trehalose interaction increases with time. The perturbation brought about by trehalose molecules in the dynamical properties of the water molecules was accessed by analyzing the diffusion coefficient values in the presence and absence of trehalose. As seen from the SI Table 2, diffusion coefficient value varies from 4.51 (±0.02) x 10-5 cm2 s-1 in without trehalose system to 4.13 (±0.02) x 10-5 cm2 s-1 in the presence of highest concentration of trehalose being simulated. The general trend and the numerical values are in good agreement with the previous literature77–82. Thus, trehalose remarkably retards the water dynamics in a dose dependent manner. Deeper insights into the hydration layer water molecules during oligomerization process were carried out by computing mean square displacement of water molecules around 4 Ȧ of protein molecules (conf3-5mol and conf3-10mol) as shown in SI Figure 15. It was observed that water molecules in the solvation layer slow down as we go from monomer to oligomer and subsequent fibril formation. The effect becomes more prominent in trehalose containing systems (in direct proportion with trehalose concentration) (SI Figure 16), while the total water MSD (including both bulk and hydration layer water molecules) remains the same. This implies that during oligomer formation water molecules that are being expelled from protein’s surface, enter into the bulk and gain higher mobility in order to keep total MSD of water molecules unchanged. Thus, entropic contribution of water molecules also plays a significant role, particularly in trehalose scenario. This was also observed in our previous study on GNNQQNY peptides83. The slowing down of trehalose molecules with increasing concentration could also be observed from SI Table 2. This retardation in trehalose dynamics increases remarkably on going from monomeric to oligomeric stage. Diffusion coefficient values of trehalose molecules in the fibrillar stage of protein are almost same as that in the oligomeric stage. To 22 ACS Paragon Plus Environment

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delineate the contribution of hydration and bulk trehalose molecules in this total decrease in diffusion, changes in the mean square displacement of total and hydration layer trehalose molecules are plotted simultaneously during the oligomerization process, as shown in SI Figure 18. It can be seen that at lower trehalose concentrations, comparable changes are observed in the total and hydration layer trehalose molecules on going from monomeric to oligomeric stage. However, with increasing trehalose concentration, decrease in the diffusion of total trehalose molecules observed is not solely an outcome of corresponding decrease in hydration layer. Instead, reduction in diffusion of trehalose seems to occur in the bulk as well. This can be ascribed to the increasing tendency of trehalose’s self association which takes place simultaneously with oligomerization process (SI Figure 17), consistent with earlier experimental and theoretical studies19,84–88. The dynamics of trehalose molecules seems to be affected much more than that of water molecules. Plausible mechanism of aggregation in the presence of trehalose: α-synuclein is a natively unfolded protein. In the aqueous solution, it exists in numerous extended and compact ensembles of conformations with structures such as random coil, a mechanically weak fold, and a “β-like”41 state. However, wealth of theoretical49,89–91 and experimental studies40,43,48,49 suggests that it prefers to adopt collapsed conformations in water. Thus, amidst the pool of varied conformations, majority of the conformers are in the collapsed states. These compact structures encompass numerous long range tertiary contacts between C terminus and NAC region via hydrophobic interactions and that between C terminus and N terminus via electrostatic one. Compact structure is consistent with the smaller hydrodynamic radius of α-synuclein compared to the fully unfolded protein48,92. This ensemble of conformers has been shown to behave as autoinhibitory and resists its self oligomerization and aggregation48.



The structural inter-conversion between these extended and compact states, thus, has a profound effect on its mechanistic route to aggregation42,44–48. Our data in the present study demonstrates that trehalose molecules perturb this heterogeneous conformational equilibrium. It preferably binds to the acidic residues of the C-terminus region and populates structures with extended conformations. These extended conformers have exposed hydrophobic patches of the NAC region along with its N terminus fully extended which have more propensities to aggregate. This way trehalose triggers the initial aggregation process by influencing the conformational dynamics of the monomeric protein (rather than by accelerating the rate of encounter of different monomeric units). However, the total fibril load was observed to be much less in trehalose containing samples. Paradoxically, it stabilizes protofibrillar and fibrillar structures. This can be rationalized by proposing that trehalose accelerates parallel route to formation of off-pathway aggregates whose equilibrium constant is much greater than that for the formation of fibrils. It is consistent with the TEM image. These off-pathway aggregates were observed to be amorphous in nature. Trehalose also modulates the properties of water molecules surrounding it. It distorts the tetrahedral arrangement of water and retards their translational diffusion, due to its strong tendency to interact with them. The interaction mainly occurs via its hydroxylic groups. During oligomerization process, water molecules are removed from the protein domain. These expelled water molecules gain higher mobility compared to the case in trehalose-free scenario. This might play an important role in facilitating aggregation process on entropic grounds. With increase in trehalose concentration, there occurs reduction in trehalose dynamics as well, owing to self association tendency of trehalose.


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Prior studies have highlighted that effective therapeutic strategy to combat diseases is based on identification of compounds that are capable of perturbing the multistep aggregation process93–95. The main aim of such inhibitors is to reduce the formation of oligomers and shorter fibrils, which are observed to be more toxic in the pathway. Monomers and long length fibrils are believed to be benign species in the pathway. Thus, these compounds can act broadly via four modes. First, they can bind to the monomers and inhibit the subsequent fibrillation process. Second, they can accelerate the formation of fibrils thereby reducing the lifetime of oligomers. Third, they can stabilize fibrils and limit fibril fragmentation. Fourth, they can interact with the oligomers but form benign off-pathway aggregates. In light of these arguments, we propose by means of our study that trehalose mainly work via last mode wherein it redirects the amyloid aggregation pathway, along with some contribution from the second pathway. To summarize, we hypothesize that α-synuclein fibrillation process proceeds through pathway depicted in Figure 11. Addition of trehalose shifts the conformational equilibrium towards extended state which then proceeds to form soluble oligomers. Trehalose increases the subsequent forward rate constants such that these oligomers are rapidly converted to fibrils and amorphous aggregates. The formation of amorphous aggregates, however, is favoured to a larger extent than fibrils under trehalose regime. The rate constants are speculated to follow following relationships:

1)

` `

>

` `

(Since fibrillar content is less compared to insoluble amorphous

aggregates, in the presence of trehalose, as observed via TEM images) 2) ` > (Since fluctuation/dissociation of interchain β-sheet contacts in fibrillar and pre-fibrillar aggregates is less in the presence of trehalose, as observed through MD simulations) 25 ACS Paragon Plus Environment


3)

>

(Since large amounts of fibrils are formed in the absence of trehalose, as

observed by ThT fluorescence measurements and TEM image) 4)

<

` `

(Since extended conformers are the one that accelerates aggregation, and

experimentally aggregation was found to be expedited by trehalose) 5)

>

` `

(Since encounter of monomers is delayed by trehalose as observed by

number of MD steps) but

` ` ` `

>

(Since experimentally oligomers are

formed faster in the presence of trehalose, as seen via DLS and ThT measurements) ` ` ` where , , , are forward rate constants in the absence of trehalose and ` , , , are

those in the presence of trehalose for steps 1, 2, 3 and 4 respectively (steps are being labelled ` ` ` ` in Figure 11). Similarly , , , and , , , represents the corresponding

backward rate constants in trehalose free and trehalose containing regimes respectively. It should be noted that these rate constants are just the indicators of number of time steps of simulations and not the actual rate constants. These are qualitative relations derived on the basis of experiments and simulations, with the assumption that they are directly proportional to the number of MD steps.


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Figure 11: Schematic representation of the proposed aggregation pathway of α-synuclein in the presence of trehalose. The length of the arrows is in accordance with the equations proposed on page 25. Conclusion: In this study, we have introspected the way trehalose intervenes into the aggregation pathway of α-synuclein. Trehalose is a widely known osmolyte that is conventionally viewed as protein stabilizer14–17,20,21,96–98. Its effect on the protein’s self assembly route is still under investigation23,99–102. In most of the cases, an additive that stabilizes the native folded state of a protein is proposed to act as an aggregation inhibitor, since aggregation is presumed to be initiated by partial misfolding103,104. On similar lines, for intrinsically disordered protein, wherein early stage in fibrillation constitutes partial folding104, such an additive is assumed to enhance aggregation. Here, we have presented a case in which analogous scenario doesn’t 27 ACS Paragon Plus Environment


hold true. Our investigation suggests that trehalose modulates the structural transitions of αsynuclein and accelerate the onset of aggregation by stabilizing its extended conformation. This conformation is more susceptible to interact with other such species, adopt β-sheet structure and subsequently fibrillate. Aggregation process is accompanied by clustering of trehalose and removal of water molecules. These expelled water molecules have larger translational movement, advocating entropy factor to also play a role in favouring the assembly process. Yet, surprisingly, the total extent of fibrillation is reduced in trehalose containing solutions. This can be accounted for by considering the fact that trehalose drives the aggregation route towards formation of off-pathway amorphous aggregates. Unfortunately, it does not destabilize on-pathway species. The findings from this work showcases diverse response of trehalose towards the protein. It calls for relooking into the concept of osmolyte-induced structural perturbation of IDP’s, and to re-explore ameliorative capabilities of trehalose. Trehalose can be used to decrease potentially toxic fibrillar load by diverting it into amorphous aggregates. Our study also demonstrated that the extended conformers of α-synuclein are potential candidates in promoting fibrillation. Thus, the reinforcement of compact structures with considerable long range interactions could serve as a probable therapeutic strategy to impede its aggregation in Parkinson’s disease.

Methods: Computational details: It is well known that results of the simulations are highly dependent on the choice of parameters such as water model and force field used to describe the potential energy surface of the system. These force fields have been calibrated against model compounds and peptides and effectively reproduce the folded conformations of globular proteins. However, their 28 ACS Paragon Plus Environment

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validation for intrinsically disordered proteins (IDPs) is still a challenging task. Alexander D MacKerell Jr and group have shown CHARMM 36m to have improved accuracy in generating conformational ensembles105. Derreumaux et al106 have compared the equilibrium ensembles of Aβ1-42 dimers generated from four different force fields namely AMBERsb14, CHARMM22, OPLS-AA and AMBER96sb-ildn, and observed that there are marked differences in the secondary and tertiary structural properties among these force fields with AMBERsb14, and CHARMM22 slightly overestimating the secondary structure contents. Nonetheless they all agree on few properties such as cross-collision sections, hydrodynamics properties, and small-angle X-Ray scattering profiles. Marie Skepo and group have compared AMBER and GROMOS force fields107. They have shown that with small variations, all standard force fields sample conformations that are slightly compact than those observed experimentally.

With

respect

to

α-synuclein,

previous

studies

have

employed

GROMOS108,109, OPLS-AA42,43,89,110, AMBER90 or CHARMM49,111 force fields. Study by Sangeeta Kundu have shown that GROMOS96 53a6 successfully captures the conformational dynamics of α-synuclein β-hairpin fragment112. Additionally, we have also shown earlier that it could work well for both globular proteins22 and disordered peptides31 along with trehalose. Thus, all atom molecular dynamics (MD) simulations of α-synuclein molecules were carried out in water and in aqueous trehalose solutions using Gromacs 5.1.1113 and Gromos 96 53a6 force field. Automated topology builder software114, which is parameterised with this force field, was used to generate pdb and topology files of trehalose. For α-synuclein, NMR structure of its human miscelle bound form, pdb id 1XQ850, was used as a starting structure for simulations of its monomeric state. This form (labelled as “conf 1”) represents an extended conformer of the protein which contains secondary structure in the form of alpha helix. Another variant of extended form labelled as “conf 3” was created by taking one strand from the fibrillar structure of α-synuclein, pdb id 2N0A51. This strand looses all its secondary



structure upon equilibration and would closely mimic experimentally observed extended state of intrinsically disordered form of α-synuclein. Both these states after production run collapses into the compact form. This collapsed conformer was labelled as “conf 2”. Two different conformations were chosen from this ensemble (labelled as conf 2bi and conf 2bii) and were used for MD simulations. The suffix “a” used with conf 1/conf 2/conf 3 denotes the corresponding system with 0.05 M trehalose while suffix “b” represents the analogous system with 0.2 M trehalose. All these conformers were solvated in a cubical box with SPC water model115. Simulations carried out in the presence of different concentration of trehalose involves random placement of appropriate number of trehalose molecules. Neutralization of the system was done by adding NA+ ions. Steepest descent algorithm with a maximum gradient of 1000 kJ mol-1 nm-1 was used for energy minimization. Equilibration was carried out in two steps similar to our previous studies22,83: a 100 ps NVT equilibration using Velocity rescale temperature coupling algorithm116 with a coupling constant of 0.1 ps, followed by 100 ps of NPT equilibration using 2 ps of coupling constant and Parrinello-Rahman barostat117. Bonds were constrained using LINCS algorithm118. Finally, production MD was run in NPT ensemble at 300 K, with a time step of 2 fs. Long range electrostatic interactions were calculated using Particle Mesh Ewald119 (PME) method with Lennard-Jones and coulomb cutoff of 1.0 nm each. The equations of motion were integrated using leapfrog algorithm120. Similar procedure was repeated for simulating oligomeric and fibrillar structure of αsynuclein. Oligomers of the protein were generated by creating 5 and 10 copies of different conformers (as described above, viz conf 1, 2 and 3) and randomly placing them in a box, while coordinates of fibrillar structure were taken from pdb id 2N0A51.


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Table 1 enlists the details of the systems being simulated, and different initial states of the protein are shown in SI Figure 19. Analysis: The analysis was done on data collected after every 50 ps. Root mean square deviation (RMSD) was calculated by fitting backbone atoms of the protein at different time frames with respect to the first time frame as the reference structure. Radius of gyration (Rg) and Solvent accessible surface area (SASA), evaluated using “gmx gyrate” and “gmx sasa” respectively, were used as measures of protein compaction. The secondary structural components namely alpha helix and β-sheet etc. were defined using DSSP algorithm121. Contact maps were created by calculating distance matrices consisting of the smallest distances between Cα atoms of all residue pairs. A cut-off distance of 1.5 nm was given so that it can account for any long range intramolecular interactions. Hydrogen bonds are considered to be formed between donar(s) and acceptor(s) if the distance between them (D-A) is less than or equal to 0.35 nm and the A-D-H angle is less than or equal to 30◦. Molecules are assumed to constitute part of a cluster if it lies within 0.45 nm from any another molecule of that cluster. “gmx energy” tool of gromacs was employed to delineate interaction energy into Lennard-Jones and Coulombic contributions, and “gmx rdf” was used to calculate pair radial distribution function. Hydration water molecules are defined as those that are within 0.4 nm from the protein. Local distribution of trehalose molecules: The distribution of trehalose molecules around protein in hydration shells of different radius, relative to water molecules was evaluated in a way similar to Liu et al.122 using:

(

) )

= (

equation (1)



Page 32 of 65

where and represents the average number of water oxygen atoms and hydroxyl group oxygen atoms of trehalose molecules, respectively, during the last 30 ns, located at a certain distance from protein.

and

denotes the total number of water oxygen atoms

and hydroxyl oxygen atoms of trehalose, respectively, in the simulation box. Tetrahedral order parameter of water molecules: It is a measure of the extent to which a given water molecule and its four nearest neighbours adopt a tetrahedral arrangement. It was calculated using123:

$

! = 1 − ∑*- ∑ +-* (cos )*+ + )

equation (2)

Where )*+ is the angle subtended between the central oxygen atom of water and its ith and jth neighbours. Average value of q was plotted against its normalized frequency distribution f(q), where normalization was done by setting the area under the curve to unity. The bin width was frequency data collection was set to 0.1. Diffusion Coefficients of water and trehalose molecules: It was calculated for oxygen atoms of water/trehalose, using Einstein equation124 :

. = lim2→ < |5(6) − 5(0)| > /66

equation (3)

where r(t) and r(0) are, respectively, the position vectors of the center of mass of molecules at time t and 0. Experimental details: Materials: Thioflavin T (ThT), 1-anilinonaphthalene-8-sulphonate (ANS) were purchased from Sigma-Aldrich. Tris buffer, and Sodium Chloride were obtained from Merck, and Trehalose, IPTG, PMSF, streptomycin sulphate, and sodium azide from Sisco Research Laboratory, India. All solutions were prepared in 20 mM Tris, 0.1 M NaCl, pH 7.5. 32 ACS Paragon Plus Environment

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Expression and purification of α-synuclein: The protocol followed was similar to that used by Lansbury and group125 and others126,127. Transformation of the plasmid expressing αsynuclein was carried out in competent cells of E.Coli strain BL21 (DE3). Expression of the protein was induced by the addition of 1 mM IPTG (final concentration) once the optical density of the bacterial culture reached 0.6 at 600 nm. The culture was further grown under similar conditions for 4 hours. Thereafter, the cells were harvested and resuspended in 50 mM Tris-HCl, pH 7.5, 2 µL DNase I (2000 U/mL) , 10 mM PMSF and 0.5 M NaCl. Lysis of the cells was then carried out by sonication. The solution was boiled in a water bath for about 5 min. Streptomycin sulphate (136 µL mL-1 of supernatant) and glacial acetic acid (228 µL mL-1 of supernatant) were added dropwise and with continous stirring, to the supernatant obtained. The solution was again centrifuged and the supernatant was precipitated with ammonium sulfate (saturated ammonium sulfate at 4 °C was used 1:1, v/v, with supernatant). The protein was collected as a precipitate by centrifugation and resuspended in 20 mM TrisHCl, pH 7.5, 0.1 M NaCl and thereafter extensively dialyzed against Milli-Q water. The protein was stored lyophilized. The purity of the recombinant wild type α-synuclein was checked by SDS-PAGE. UV visible spectroscopy: The concentration of the protein was determined by measuring absorbance on Varian Cary Eclipse UV visible spectrophotometer using a molar extinction coefficient of 5600 M-1cm-1 at 275 nm. Stock solutions of ANS and ThT were prepared in Milli Q water and concentration was determined using a molar extinction coefficient of 5000 M−1cm−1 at 350nm and 35000 M−1cm−1 at 412 nm for the two dyes, respectively. Preparation of samples for aggregation studies: The protein solution of 180 µM concentration in 20 mM Tris, 0.1 M NaCl and pH 7.5 along with 0.05 % NaN3 was incubated at 37 °C in Eppendorf Thermomixer at 1200 rpm, alone, and in the presence of different



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concentrations of trehalose (0.1 M, 0.3 M, 0.5 M and 0.7 M). The aliquots were withdrawn at different time intervals and analyzed by various techniques. Fluorescence assays: Varian Cary Eclipse fluorimeter was used for recording fluorescence spectra of different samples. The effective concentration of ThT and ANS dyes were kept as 20 µM and 50 µM respectively. ANS was excited at 350 nm, and the emission spectra were collected between 400 to 600 nm. ThT was excited at 450 nm, and the emission was collected at 482 nm. Briefly, 10 µL of the incubated sample was added into 490 µL ThT solution (40 µM ThT in 20 mM Tris buffer, 0.1 M NaCl, pH 7.5) in a quartz cuvette of 1 cm path length. All fluorescence experiments were repeated twice for three batches of protein samples. The kinetic data obtained for fibrillation using ThT binding measurements were fitted using following equation128 :

: = :; +

< ( =>?(

@@ / A

equation (4) ))

Where y is the fluorescence intensity at any time t, :; is the corresponding intensity at time t=0, a represents the maximum fluorescence intensity, 6 / denotes the time at which the intensity is half of its maximum value. Apparent rate constant (kapp) was calculated using

Paradoxical Effect of Trehalose on the Aggregation of Î±-Synuclein

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