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Impact of #-Synuclein Initial Ensemble Structure on Fibrillation Pathways and Kinetics Jia Bai, Kai Cheng, Maili Liu, and Conggang Li J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b01225 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 8, 2016

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The Journal of Physical Chemistry

Impact

of

α-Synuclein

Initial

Ensemble

Structure

on

Fibrillation Pathways and Kinetics Jia Bai,

†

†,‡

Kai Cheng,

†,‡

†

Maili Liu, and Conggang Li *,†

Key Laboratory of Magnetic Resonance in Biological Systems, State Key

Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center of Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P.R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

1

ACS Paragon Plus Environment


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ABSTRACT: The presence of intracellular filamentous α-synuclein (αS) aggregates is a common feature in Parkinson’s disease. Recombinant expressed and purified human αS is also capable of forming fibrils in vitro. Many studies have shown that solution conditions heavily influence αS fibrillation kinetics, fibril structure and morphology that exhibit differential biological effects. Nevertheless, αS ensemble structure in various solution conditions is not well characterized, furthermore, how the initial solution ensemble structures impact αS assembly kinetics and pathways which result in diverse fibril structure and morphology remains elusive. Here, we mainly employed NMR spectroscopy to characterize the initial ensemble structure of αS in the presence or absence of 150 mM sodium chloride (NaCl) solution, where two polymorphs of αS were demonstrated in previous studies. Our data show that αS exhibits distinct conformations and fibrillation kinetics in these two solutions. αS adopts a more compact and rigid ensemble structure that has faster fibrillation kinetics in the absence of NaCl. Based on the ensemble structure and dynamics, we proposed a possible molecular mechanism in which αS forms different polymorphs under these two conditions. Our results provide novel insights into how initial conformation impacts on fibrillation pathways and kinetics, suggesting that micro-environment can be used to regulate intrinsically disordered proteins assembly.

2


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INTRODUCTION α-synuclein (αS) is a 140-residue intrinsically disordered protein that was normally found in both soluble and membrane-associated fractions of the neuron cells in the brain. Its filamentous aggregates are the major component of Lewy bodies that is the pathological hallmark of Parkinson’s disease (PD), one of the major neurodegenerative diseases.1-2 αS is comprised of three distinct regions: the positively charged amphipathic N-terminus (residue 1-60); the hydrophobic non-amyloid-component (NAC) region (residues 61-95) and the highly negatively charged C-terminus (96-140), that may possess different functions.3 Although αS shows no apparent ordered secondary structure in dilute solution, it possessed long-range transient interaction between the N-terminus and the C-terminus,4-7 which is regarded as a major factor to affect αS fibrillation. The physiological function of αS remains unknown, but the aggregation of αS into fibrils is thought to play important role in the pathogenesis of PD.8 Recombinant expressed and purified human αS were used to understand why αS forms into β-sheet riched amyloid like fibrils in cells, many studies have shown that recombinant αS can form fibrils and the fibrillation kinetics, fibril structure and cellular toxicity are highly dependent on the solution in which fibrils assemble.9-12 For example, αS concentration, solution pH, temperature, the cosolutes of osmolytes, macromolecular crowding agents dramatically alter the kinetics of aggregation and the morphology of the fibrils.9,

12-13

Nevertheless, high resolution structural

characterization of αS in various solutions is rarely investigated, how the solution 3



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conditions affect αS self-assembly rate and pathways is not known. A recent study shown that αS could assemble into different high-molecular weight structures: a cylindrical assemblies, referred as fibrils, and a twisting assemblies, referred as ribbons, are formed in the presence and absence of 150 mM sodium chloride (NaCl), respectively. Both forms, ribbon and fibrils have β-sheet elements, but their number, length and distribution differs greatly, thus the biophysical properties and cellular responses are also different.14 NMR spectroscopy can provide detailed residue-level structural and dynamic information and it is a suitable technique for intrinsically disordered proteins ensemble structure characterization. With the advantages of free background and high sensitivity,

19

F NMR has become a useful technique to monitor conformational

changes and fibrillation kinetics of amyloid protein or petides, such as αS, IAPP and amyloid-beta 1-40.15-18,21 Here, we used NMR spectroscopy to characterize the initial ensemble structures that eventually assemble into two αS polymorphs in the presence or absence of 150 mM sodium chloride. Our NMR and fluorometry data show that αS exhibits distinct conformations and fibrillation kinetics in these two solutions. In the absence of NaCl, αS is a more compact and rigid ensemble structure that has faster fibrillation kinetics. Based on the ensemble structure and dynamics, we proposed a possible molecular mechanism in which αS forms different polymorphs under different salt concentrations.

METHODS 4


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Experimental Section. Plasmids contained the genes of human recombinant α-synuclein were transformed into E.coli BL21 (DE3) competent cells. Expression and purification of 15N, 19F and 13C/15N labeled αS and its variants were followed by the described protocols,

respectively. The purified αS cysteine mutants were

19-21

dialyzed to spin-labeling reaction buffer, including 50 mM PBS, 100 mM NaCl of pH 7.2,

and

then

added

a

5-fold

molar

excess

of

the

MTSL

(1-Oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) to react at 4 ℃ for at least 16 hours by agitation. Unreacted spin label was removed by using desalting columns. Complete labeling with the nitroxide radical was verified by Mass Spectrometry. Chemical Reagents. MTSL was purchased from Toronto Research Chemicals Inc. All other chemicals were of analytical grade from Sinopharm Chemical Reagent Co. Ltd. NMR Experiments. The triple-resonance experiments, relaxation experiments, 1D

15

N transverse

F spectra, T1 and T2 relaxation experiments of αS

19

were performed on Bruker 600 MHz and 700 MHz spectrometers equipped with

19

F/1H/13C/15N or

1

H/13C/15N cryoprobe, respectively. NMR buffer

contained 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) with or without 150 mM NaCl (pH 6.0). Protein was dissolved in 90% NMR buffer and 10% D2O. C/15N labelled αS (0.3 mM) in absence or presence of 150mM NaCl was used to

13

acquire triple-resonance experiments for αS backbone assignment. The 3D spectra were obtained using standard heteronuclear correlation experiments including HNCA, 5



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HN(CO)CA, and CBCA(CO)NH. 2D 1H-15N HSQC spectra were used to check the stability of αS before and after acquiring the 3D spectra. The relaxation delays t=17, 34, 51, 68, 85, 136, 170, 187, 204, 254 ms were used for 15N R2 experiments and the relaxation delays t=10, 20, 40, 80, 160, 320, 640, 1280, 1800 ms were used for the

15

N R1 experiments. The recycle delays for relaxation

measurements were 3 s. Relaxation times were determined by fitting the decay peak intensities to an exponential function. The steady-state heteronuclear NOE measurements were collected with and without proton saturation during the relaxation delay of 5 s. 1D

19

F R1 was measured by inversion recovery. R2 was measured with a CPMG

sequence. 1D 19F spectra were recorded with a 11.3 kHz sweep width, 16 K complex points and a recycle delay of 3 s. Proton decoupling was applied to all 1D 19F NMR spectra. For

19

F exchange spectra, the mixing time was set to 250 ms, 500 ms, 1000

ms, respectively. 19F Chemical shifts are referenced to trifluorotoluene at -63.72 ppm. The inter-molecular and intra-molecular PRE experiments were performed on a Bruker 800 MHz spectrometer. The PRE data were measured from the cross-peak intensity ratios between two 2D 1H-15N HSQC NMR spectra obtained in the presence and absence of MTSL (the nitroxide radical). For the intra-molecular PRE measurements, sample comprised of 0.1 mM MTSL-labeled 15N αS were used for the experiments (low concentration of αS 0.1 mM is used to avoid inter-molecular PREs). For the inter-molecular PRE measurements, sample comprised of 0.05 mM MTSL-labelled

14

N-αS and 0.05 mM wild-type

15

N-αS mixtures were used for the 6


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experiments. A 10-fold molar excess of L-ascorbic acids were added to MTSL-labeled sample for generating the diamagnetic sample. For the 1H pulsed-field gradient NMR experiments, we used 1, 4-dioxane as an internal radius standard (2.12 Å) and viscosity probe. Diffusion spectra were acquired using the stimulated echo experiment with 3-9-19 pulse module for water suppression (stebpgp1s19). The peak in 0.81-0.60 ppm region was used to calculate αS hydrodynamic radius (RhαS) according to the equation, RhαS= (Ddioxane / DαS)*Rhdioxane.

22

All NMR data were processed by

NMRPipe 23 and analysed by Sparky. 24 Fibrillation Experiments. The incubation reactions contained 200 uM αS in 20 mM MES with or without 150 mM sodium chloride (pH 6.0), 1 mM ethylenediaminetetraacetic acid (EDTA) and 500 uM phenylmethanesulfonyl fluoride (PMSF). Fibril formation was induced at 37 ℃ by agitation with 220 rpm in the shaker. Sample (10 ul) were removed and mixed with 1 mL 25 uM ThT, and fluorescence intensities were assed from the emission spectra at 482 nm on a HORIBA Fluoromax-4 spectrometer. All the aggregation data was fitted to equation (1),25-26 where α[t] is the parameter measured by ThT fluorescence experiments, t is the incubation time, kapp and t1/2 are the values related to the fibrillation reaction.

α [t ] = (1 - e

- k app t

) / (1 + e

- kapp ( t -t1/ 2 )

)

(1)

Structural Constraints. The PRE restraints were determined from the intensity ratios between two HSQC spectra acquired in the presence and absence of the nitroxide radical. The paramagnetic relaxation enhancement rate (R2sp) was calculated

7



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from: 27 red sp I ox R 2 exp ( − R2 t INEPT ) = I red R2red + R sp 2

(2)

Where Iox and Ired are the peak intensities of oxidized and reduced resonances, respectively. R2red, the intrinsic transverse relaxation rate, was estimated from the peak line-width in the reduced spectra. tINEPT is the total INEPT evolution time of the HSQC experiment (~11 ms). The distance between the nucleus and the spin label, r, was calculated from: 1/6

K  3τ c   r =  sp  4τ c +  1 + ωh2τ c2    R2 

(3)

Where K is a constant comprising the nuclear spin gyromagnetic ratio, the electronic g factor, and the Bohr magneton. The constant K is 1.23╳10-32 cm6s-2 for a nitroxide radical, ωh is the Larmor frequency of the proton nuclear spin. The correlation time for the electron-nuclear interaction τc was set to 4 ns, in agreement with previous studies.28

RESULTS & DISCUSSION 19

F NMR Provides Multiple Conformations Information in the Absence of

Sodium Chloride. To investigate why different polymorphs of αS were formed in different salt concentration, we first compared the 1H-15N spectra of αS and the chemical shift perturbations in two different solutions with or without NaCl are small (< 0.1 ppm)(Figure 1 A). The narrow chemical shift range (7.8-8.3 ppm) of amide 1H suggested that αS is intrinsically disordered in both solutions. In the absence of 8


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sodium chloride, some cross peaks of residues showed chemical shift perturbations, and residues with larger chemical shift change were labelled in the Figure ( The assignments were according to Biological Magnetic Resonance Data Bank (BMRB entry number is 16543 for α-synuclein)). The residues that showed larger chemical shift perturbations were mainly distributed in N-terminus (Figure 1 B). 19

F NMR chemical shift is more sensitive than 1H-15N HSQC to conformational

change, partly due to its larger chemical shift dispersion. We then recorded the

19

F

spectra of 3-19F tyrosine labelled αS in the presence and absence of 150 mM NaCl. αS has four tyrosines (Y39, Y125, Y133, Y136), the

19

F spectra of αS in 150 mM

sodium chloride solution has four resonances as expected. In contrast to one set of peak in

19

F spectrum in 150 mM NaCl solution, we observed more than four

resonances in the absence of NaCl, which suggested more than one conformational state existed in solution without NaCl and the conformational exchange is slow at 19F NMR chemical shift time scale. To assign the multiple

19

F resonances, mutagenesis

approach was employed and only one tyrosine was kept and the others are changed to phenylalanine, then 3F-tyrosine labelled only one tyrosine residue at each time. For variant labelled only Y125 or Y133 or Y136, only one resonance was observed in the presence of 150 mM NaCl (Figure 1 C), but in the absence of NaCl, they all exhibited two sets of peaks, a major peak and a minor peak (Figure 1 D, asterisk ). For variant labelled only Y39, it just exhibited one peak regardless of the presence or absence of NaCl. To assess the conformational properties of major and minor species present in 9



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both solutions, we used solvent isotope shifts to study the extent of solvent exposure of the labeled sites. The solvent isotope shift could be determined from the difference of the 19F chemical shifts in the 10% and 100% D2O solvent and the 19F NMR spectra for Y39, Y125, Y133, Y136 sites in the presence and absence of 150mM NaCl with 10 or 90% D2O were shown in Figure S1 (A-F). Free 3F-tyrosine is completely solvent exposed, and its frequency changes induced by solvent isotopic effects were 132 Hz,. solvent isotopic effects on

19

F chemical shifts of Y39, Y125, Y133, Y136

labeled sites were smaller than that of free 3F-tyrosine, suggesting these sites are not completely solvent exposed. The

19

F data indicated that αS adopt at least two

conformations in the absence of NaCl, the major conformation showed a lower degree of solvent exposure, and the minor conformation possessed a higher degree of solvent exposure, similar as those in the presence of 150 mM NaCl. Dynamic properties of αS. To further probe the change of αS dynamics induced by salts, we measured

19

F longitudinal and transverse relaxation rates, respectively

(Table S1-2). The major peak of Y125, Y133, Y136 variants all showed larger transverse relaxation rates (R2) than their minor peaks in the absence of NaCl. In the presence of 150 mM NaCl, all resonances showed similar transverse relaxation rates (R2) as those minor peak in the absence of NaCl. No large change of R1 was observed for all variants in the presence or absence of NaCl. These data indicated that αS adopt at least two conformations with different dynamics in the absence of NaCl, the major peaks represent a compact conformation, and the minor peaks represent an extended conformation similar as that in the presence of 150 mM NaCl. To assess the 10


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dynamical exchange timescale of the different conformations in the absence of NaCl, we acquired the 19F exchange spectra of Y133 and Y136 variants at 250 ms, 500 ms, 1000 ms, respectively (Figure S2). No cross-peaks were observed in the 19F exchange spectra during the above mixing time, suggested that multiple conformations of αS are in dynamical exchange at least above 1s timescale in the absence of NaCl. The

19

F longitudinal and transverse relaxation rates only supplied dynamic

information of limited sites. In order to gain dynamic information of more residues, backbone 15N NMR longitudinal, transverse relaxation, and steady state heteronuclear 1

H-15N NOE were measured in both solutions, respectively. The measured values as a

function of αS residue number were shown in Figure 2. Although the overall pattern is similar, the transverse relaxation rates (R2) are different in two solutions, indicated that αS may exhibit different local dynamics. The R2 values of residues in the NAC region were relatively smaller, indicating that this region was the most flexible section of the protein. The residues in the N-terminus and C-terminus show larger differences, the R2 values in the absence of sodium chloride are larger than those in the presence of 150 mM sodium chloride, suggesting that the N-terminus and C-terminus of αS were experiencing restricted motion in the absence of sodium chloride, consistent with

19

F results. Such rigidity of N-terminus was also reported in the ribbons.29 No

large change of R1 was observed in the presence or absence of NaCl. The

15

N

steady-state heteronuclear NOE is sensitive to local dynamics and the values in the absence of NaCl were larger than those in the presence of NaCl, indicating a reduced flexibility in the absence of NaCl, consistent with R2 data. 11



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Salts Effects on the Secondary Structure of αS. 13C backbone chemical shift are sensitive to the change of secondary structure, and Cα, Cβ chemical shifts are usually used to determine secondary structure propensities. Here we used 13Cα, 13Cβ chemical shifts to assess the secondary structure content of αS in the present or absent of 150 mM NaCl (Figure 3). Site-specific secondary structure propensities were determined based on the observed 13Cαand 13Cβ chemical shifts using SSP program. Residues in well formed α-helix were expected to yield SSP score close to 1, while residues in fully developed β-sheet structures were expected to yield SSP score of -1. According to the SSP scores reported in our study, it was obvious that αS showed almost the same SSP scores in the presence and absence of 150 mM NaCl, suggesting that the absence of NaCl did not cause significant secondary structure change of αS. Salts Effects on the Tertiary Structure of αS. Paramagnetic relaxation enhancement (PRE) method that can yield long-range distance information between a paramagnetic center and nuclei were employed to probe salt effects on α-synuclein tertiary structure. The distance up to 25 Å for MTSL ( a paramagnetic radical used in our study) could be obtained from intensity ratios of cross peaks in 1H-15N HSQC spectra acquired in the presence and absence of the radical (Iox/Ired). We mutated A19, A90, G132 to cysteine, respectively, which is used as attachment points for nitroxide radical MTSL. We performed intramolecular PRE experiments at three positions, A19C, A90C, G132C, in the presence and absence of 150 mM NaCl, respectively. The resulting Iox/Ired intensity ratios are shown in Figure S3. Figure S3 showed the only intra-molecular PRE values, and the corresponding 12


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inter-molecular PRE values in such low concentration of αS were negligible (Figure S4). From the PRE data in Figure S3, we calculate the distance between each residue and MTSL labeled sites (Figure 4). In 150 mM NaCl, the distance between spin label at A19C and residues 110-140 in C-terminus is shorter than 25 Å, suggesting long-range interactions between N and C-terminus. The distance from other two label sites also shows that there were many short-range contacts near the spin label location sits, as well as transient long-range contacts between N-terminal, NAC and C-terminal regions, which is consistent with previous study4. In the absence of NaCl, no matter the spin label located at A19C, A90C, or G132C sites, all distance between spin labels and residues is shorter than 20 Å. While some medium and long range PREs in αS are seen in the presence of 100 mM salt, larger PRE effects are observed in the absence of salt, suggesting that αS samples more closely compact conformations in the absence of NaCl than that in 150 mM NaCl. Pulsed-field gradient NMR (PFG-NMR) translational diffusion experiments were also used to estimate the hydrodynamic radius (Rh) of αS in the presence or absence of 150 mM NaCl (Figure S5). The Rh values in the absence of NaCl (Rh=27.9 Å) is smaller than that in the presence of 150 mM NaCl (Rh=30.4 Å), in agreement with the PRE and relaxation measurements. Comparing A30P, E46K and A53T Variants with Wild-Type of αS. Missense mutations in αS, including A30P, E46K, A53T, have been identified in autosomal-dominantly inherited early-onset PD.30-32 We wonder sodium chloride salts 13



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would have similar effects on αS variants (A30P, E46K, A53T) structures and dynamic properties. 2D 1H-15N HSQC and 1D 19F spectra of αS variants (Figure S6-7) showed very similar spectral characteristics with those of wild-type αS. For 1D

19

F

spectra, we also observed more than four resonances of these three variants in the absence of NaCl, which suggested that more than one conformation can be observed by

19

F NMR. We compared R2 values of αS and its variants in the corresponding

solution conditions, and the overall patterns of the R2 values are similar (Figure S8), indicating that NAC region was the most flexible section of the protein, and the N-terminus, C-terminus of such variants were experiencing restricted motion and were more rigid in the absence of sodium chloride. It has been pointed that the interaction between N- and C-terminus is regulated by electrostatic interaction. For E46K variant, glutamic acid was mutated to lysine residue, altering the local electric charge distribution, so the salts have larger effects on E46K variant structures and dynamics properties, which induced larger chemical shifts and transverse relaxation rates changes of the N- and C-terminus. We also measured the hydrodynamic radius (Rh) of αS variants in the presence or absence of 150 mM NaCl (Figure S5), and the variants (A30P, E46K, A53T) are like wild type, adopting a more compact conformation in the absence of NaCl. Comparing ThT Fluorescence Data with and

19

F NMR Data. We used fluorometry

19

F NMR spectroscopy to monitor the fibrillation of αS in the presence and

absence of 150 mM NaCl. Figure 5A-B showed the time dependence of the

19

F

spectra of αS acquired in the presence and absence of 150 mM NaCl, respectively. 14


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The four resonances of αS in the presence of 150 mM NaCl disappeared gradually with increasing time, which suggested that monomer and small aggregates formed large fibrils and their resonances were too broad to detect. The resonances of αS in the absence of NaCl showed very different characteristics, and only the four major peaks decreased with increasing time, and the intensity of minor peaks did not decrease. To further confirm the results, we also performed the

19

F spectra of Y133

variants as a function of incubation time in the presence and absence of 150 mM NaCl, respectively (Figure 5 C-D). For the resonance of Y133 variant in the presence of 150 mM NaCl, the resonance disappeared with the increasing time. For the resonance of Y133 variant in the absence of NaCl, only the major peak disappeared with increasing time, and the intensity of minor peak did not decrease, which were consistent with those observation of wild-type αS. For the whole process, no obvious chemical shift changes were observed, suggesting that low molecular weight intermediates either are absent or cannot be detected. Figure 5E-F showed the ThT fluorescence intensities and corresponding integrated

19

F signals as a function of time. Samples for

fluorometry and 19F NMR analysis were taken out for the measurements at the same time. The

19

F NMR spectra were consistent with these observations of ThT

fluorescence experiments, αS fibrillated faster in the absence of NaCl. The little different fibrillation rates of wild-type αS and its variant Y133 is possibly due to the C-terminal mutation of Y133 variant. Mechanism for Accelerating αS Aggregation. αS has shown different fibrillation kinetics and fibril structure in the presence and absence of NaCl, but the exact 15



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Page 16 of 30

mechanism remains elusive. Our results provide a possible clue from initial structure from which the fibril formed. The secondary structural propensities of αS in the presence and absence of NaCl were quite similar, suggesting that secondary structure was not a main reason. From

19

F spectra, αS and the variants adopt at least two

conformations in the absence of NaCl, the major state showed a compact conformation, and the minor state possessed a extended conformation which showed the similar structural properties as that in the presence of 150 mM NaCl. We did not observe transitions of αS between the compact conformation and extended conformation during the fibrillation, and the peak intensities of the compact conformation decreased more quickly than those of extended conformation, suggesting that compact conformation is critical to aggregation. Disorder protein can adopt a variety of conformational states and interconvert between them on a wide range of timescales. Fluctuations within the unfolded ensemble states in the presence or absence of NaCl are likely to be important in determining the fibrillation pathway. We speculated that αS aggregation pathway in the absence of NaCl is different from that in the presence of 150 mM NaCl. Based on our results, a schematic illustration of αS aggregation in the presence and absence of NaCl was shown in Figure 6. The structural transformation to the intermediate and the formation of the nucleus are the two key steps to fibrillation kinetics. αS in the absence of NaCl is a more compact conformation, which stands for an aggregation prone state. The compact conformation may be closer or easier converting to the intermediate state, resulting in more quickly assembly process. However, the initial 16


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structure of αS in the presence of NaCl adopts a more extended conformation that may be greatly different from the intermediate state, and the initial structure need more time to convert to intermediate state, which results in slow fibrillation. Notably, we did not observe 19F chemical shift changes during fibrillation, suggesting that the rate for intermediate convert to nucleus may be very fast, or the population is beyond NMR detection limits. Interestingly, fibrils and ribbons both formed in-register parallel β-sheets aggregates, but they possess greatly different secondary structures and dynamics, residue 1-42 in the ribbons seems to be more rigid than the corresponding residues in the fibrils,29 consistent with our R2 experiments. The results suggested that the initial ensemble structure may be potentially related to αS self-assembly.

CONCLUSIONS In conclusion, we show that αS adopts a more compact and rigid ensemble structure that has faster fibrillation kinetics in the absence of NaCl. For the first time, we observed conformational selection during αS assembly by

19

F NMR. Our results

imply that solution conditions affect aS initial ensemble structure and eventually affect fibrillation kinetics and final fibril structure. Our study also suggests that cellular environment may influence intrinsically disordered proteins assembly and the effects of physiological conditions on αS aberrant fibrillation have to been considered.

ASSOCIATED CONTENT 17



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Supporting Information Available: Figure S1: 19F NMR spectra of 3F-tyrosine, αS and its variants in 10% (v/v) D2O and 100% D2O. Figure S2: 19F exchange spectra of Y133 and Y136 variants in the absence of NaCl, respectively. Figure S3: Intra-molecular PRE intensity ratios of amide protons in spin-labeled αS. Figure S4: Inter-molecular PRE intensity ratios of amide protons in spin-labeled αS. Figure S5: The Rh of wild-type αS and its variants. Figure S6-7: 1H-15N HSQC and 19F NMR spectra of αS variants (A30P, E46K, A53T) in the presence or absence of 150 mM NaCl, respectively. Figure S8: R2 for backbone 15N nuclei of αS variants. Table S1-2: The measured 19F R1, R2 of S variants in the presence or absence of 150 mM NaCl. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This work is supported by Ministry of Science and Technology of China grant 2013CB910200, National Natural Sciences Foundation of China grants 21173258 and the 1000 Young Talents Program of China.

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REFERENCES

(1)

Goedert, M. Alpha-synuclein and neurodegenerative diseases. Nat. Rev.

Neurosci. 2001, 2, 492-501. (2) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. [alpha]-Synuclein in Lewy bodies. Nature 1997, 388, 839-840. (3) Uversky, V. N. Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation. J. Neurochem. 2007, 103, 17-37. (4) Bertoncini, C. W.; Jung, Y. S.; Fernandez, C. O.; Hoyer, W.; Griesinger, C.; Jovin, T. M.; Zweckstetter, M. Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 1430-1435. (5) Lee, J. C.; Gray, H. B.; Winkler, J. R. Tertiary contact formation in alpha-synuclein probed by electron transfer. J. Am. Chem. Soc. 2005, 127, 16388-16389. (6) Dedmon, M. M.; Lindorff-Larsen, K.; Christodoulou, J.; Vendruscolo, M.; Dobson, C. M. Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 2005, 127, 476-477. (7) Uversky, V. N.; Li, J.; Fink, A. L. Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J. Biol. Chem. 2001, 276, 10737-10744. (8)

Spillantini, M. G.; Crowther, R. A.; Jakes, R.; Hasegawa, M.; Goedert, M.

alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6469-6473. (9)

Munishkina, L. A.; Cooper, E. M.; Uversky, V. N.; Fink, A. L. The effect of 19



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

Page 20 of 30

macromolecular crowding on protein aggregation and amyloid fibril formation. J. Mol. Recognit. 2004, 17, 456-464. (10) Shtilerman, M. D.; Ding, T. T.; Lansbury, P. T., Jr. Molecular crowding accelerates fibrillization of alpha-synuclein: Could an increase in the cytoplasmic protein concentration induce Parkinson's disease? Biochemistry 2002, 41, 3855-3860. (11)

Binolfi, A.; Rasia, R. M.; Bertoncini, C. W.; Ceolin, M.; Zweckstetter, M.;

Griesinger, C.; Jovin, T. M.; Fernandez, C. O. Interaction of alpha-synuclein with divalent metal ions reveals key differences: A link between structure, binding specificity and fibrillation enhancement. J. Am. Chem. Soc. 2006, 128, 9893-9901. (12) Hoyer, W.; Antony, T.; Cherny, D.; Heim, G.; Jovin, T. M.; Subramaniam, V. Dependence of alpha-synuclein aggregate morphology on solution conditions. J. Mol. Biol. 2002, 322, 383-393. (13) Lee, J. H.; Lee, I. H.; Choe, Y. J.; Kang, S.; Kim, H. Y.; Gai, W. P.; Hahn, J. S.; Paik, S. R. Real-time analysis of amyloid fibril formation of alpha-synuclein using a fibrillation-state-specific fluorescent probe of JC-1. Biochem. J. 2009, 418, 311-323. (14) Bousset, L.; Pieri, L.; Ruiz-Arlandis, G.; Gath, J.; Jensen, P. H.; Habenstein, B.; Madiona, K.; Olieric, V.; Bockmann, A.; Meier, B. H.; et al. Structural and functional characterization of two alpha-synuclein strains. Nat. Commun. 2013, 4, 2575. (15) Danielson, M. A.; Falke, J. J. Use of 19F NMR to probe protein structure and conformational changes. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 163-195. (16)

Eccleston, J. F.; Molloy, D. P.; Hinds, M. G.; King, R. W.; Feeney, J.

Conformational differences between complexes of elongation factor Tu studied 20


Page 21 of 30

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


19F-NMR spectroscopy. Eur. J. Biochem. 1993, 218, 1041-1047. (17) Suzuki, Y.; Brender, J. R.; Hartman, K.; Ramamoorthy, A.; Marsh, E. N. G. Alternative pathways of human islet amyloid polypeptide aggregation distinguished by 19F nuclear magnetic resonance-detected kinetics of monomer consumption. Biochemistry 2012, 51, 8154-8162. (18) Suzuki, Y.; Brender, J. R.; Soper, M. T.; Krishnamoorthy, J.; Zhou, Y.; Ruotolo, B. T.; Kotov, N. A.; Ramamoorthy, A.; Marsh, E. N. Resolution of oligomeric species during the aggregation of Abeta1-40 using (19)F NMR. Biochemistry 2013, 52, 1903-1912. (19) Conway, K. A.; Lee, S. J.; Rochet, J. C.; Ding, T. T.; Williamson, R. E.; Lansbury, P. T., Jr. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: Implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 571-576. (20) Hoyer, W.; Antony, T.; Cherny, D.; Heim, G.; Jovin, T. M.; Subramaniam, V. Dependence of α-Synuclein Aggregate Morphology on Solution Conditions. J. Mol. Biol. 2002, 322, 383-393. (21) Li, C.; Lutz, E. A.; Slade, K. M.; Ruf, R. A.; Wang, G. F.; Pielak, G. J. 19F NMR studies of alpha-synuclein conformation and fibrillation. Biochemistry 2009, 48, 8578-8584. (22)

Jones, J.; Wilkins, D.; Smith, L.; Dobson, C. Characterisation of protein

unfolding by NMR diffusion measurements. J. Biomol. NMR 1997, 10, 199-203. (23) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. NMRPipe: A 21



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

Page 22 of 30

multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 1995, 6, 277-293. (24)

Goddard, T. D.; Kneller, D. G. In SPARKY 3, University of California, San

Francisco, 2007. (25)

Fernandez, C. O.; Hoyer, W.; Zweckstetter, M.; Jares-Erijman, E. A.;

Subramaniam, V.; Griesinger, C.; Jovin, T. M. NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation. EMBO J. 2004, 23, 2039-2046. (26) Rasia, R. M.; Bertoncini, C. W.; Marsh, D.; Hoyer, W.; Cherny, D.; Zweckstetter, M.; Griesinger, C.; Jovin, T. M.; Fernandez, C. O. Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4294-4299. (27)

Battiste, J. L.; Wagner, G. Utilization of site-directed spin labeling and

high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry 2000, 39, 5355-5365. (28) Gillespie, J. R.; Shortle, D. Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. paramagnetic relaxation enhancement by nitroxide spin labels1. J. Mol. Biol. 1997, 268, 158-169. (29) Gath, J.; Bousset, L.; Habenstein, B.; Melki, R.; Böckmann, A.; Meier, B. H. Unlike twins: An NMR comparison of two α-synuclein polymorphs featuring different toxicity. PLoS ONE 2014, 9, e90659. 22


Page 23 of 30

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(30) Zarranz, J. J.; Alegre, J.; Gómez-Esteban, J. C.; Lezcano, E.; Ros, R.; Ampuero, I.; Vidal, L.; Hoenicka, J.; Rodriguez, O.; Atarés, B.; et al. The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia. Ann. Neurol. 2004, 55, 164-173. (31) Kruger, R.; Kuhn, W.; Muller, T.; Woitalla, D.; Graeber, M.; Kosel, S.; Przuntek, H.; Epplen, J. T.; Schols, L.; Riess, O. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat. Genet. 1998, 18, 106-108. (32) Polymeropoulos, M. H.; Lavedan, C.; Leroy, E.; Ide, S. E.; Dehejia, A.; Dutra, A.; Pike, B.; Root, H.; Rubenstein, J.; Boyer, R.; et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997, 276, 2045-2047.

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Figure 1. 1H-15N HSQC and

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F NMR spectra of α-synuclein in the presence or

absence of NaCl. (A) 1H-15N HSQC spectra of α-synuclein in the presence (blue) or absence (red) of 150mM NaCl, respectively. Residues with large chemical shift change were labelled in the Figure. (B) The chemical shift perturbation (CSP) analysis of αS in presence or absence of 150mM NaCl. The N-terminal-, NAC- and C-terminal-regions are coloured red, green and blue, respectively. (C) four tyrosines (Y39, Y125, Y133, Y136) of α-synuclein was labelled with 3F-tyrosine, for its variants, only label one tyrosine residue at each time, and the others all changing to phenylalanine. (B)

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F spectra of wild-type α−synuclein and its variants in the

presence of 150 mM NaCl. (D) 19F spectra of wild-type α-synuclein and its variants in the absence of NaCl. All spectra were acquired at 15 ℃.

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Figure 2. R1 (A), R2 (B) and steady state NOE (C) relaxation parameters for backbone

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N nuclei of α-synuclein in the presence (blue) or absence (red) of 150

mM NaCl. All spectra were obtained at 15 ℃.

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Figure 3. Secondary structure propensity of α-synuclein in the presence (blue) or absence (red) of 150 mM NaCl at 15 ℃. Positive values indicate preference for α-helix. Negative values represent the preference for β-sheet. The grey dashed lines indicate the separation of the N-terminal, NAC, and C-terminal regions.

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Figure 4. The distance of amide protons from the spin-labeled site in the presence (A-C) and absence (D-F) of 150 mM NaCl, respectively. Variants A19C, A90C and G132C were labeled with MTSL. The distance was calculated from the intramolecular PRE data. The N-terminal- (residues 1-60), hydrophobic NAC- (residues 61-95) and C-terminal-regions (residues 96-140) are colored red, green and blue, respectively.

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Figure 5. Fibrillation of α-synuclein and its Y133 variant monitored by 19F NMR and ThT fluorescence as a function of time. 19F spectra of α-synuclein in the presence (A) or absence (B) of 150 mM NaCl.

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F spectra of Y133 variant in the presence (C) or

absence (D) of 150 mM NaCl. All the

19

F spectra were acquired at 37 ℃ .

(E) Fibrillation of αS monitored by ThT fluorescence in the absence and presence of 150 mM NaCl, respectively. Fluorescence intensity was normalized to 100 units. (F) The integrated 19F signals as a function of time. The points connected with solid lines integrate the whole 19F signals. The points connected with dashed lines integrate only the major peak (D, asterisk) of Y133 variant in the absence of NaCl. All the curve lines were fitting to equation (1).

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Figure 6. Schematic illustration of α-synuclein aggregation pathway in different solution conditions. αS with structural plasticity adopt different structures depending on its environment. The different initial structures of αS in the presence and absence of 150 mM NaCl are illustrated in the figure. Two different aggregation pathways are described, one initial structure is closer to that of intermediates or nucleus, resulting faster fibrillation and more dynamical aggregates. (A) The aggregation pathway for αS in the presence of 150 mM NaCl. (B) The aggregation pathway for αS in the absence of NaCl.

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TABLE OF CONTENTS (TOC) IMAGE

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Impact of the Î±-Synuclein Initial Ensemble ... - ACS Publications

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