Probing Conformational Change of Intrinsically Disordered α

Jan 2, 2014 - Probing Conformational Change of Intrinsically Disordered α-Synuclein to Helical Structures by Distinctive Regional Interactions with L...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Probing Conformational Change of Intrinsically Disordered α‑Synuclein to Helical Structures by Distinctive Regional Interactions with Lipid Membranes Shin Jung C. Lee,† Jong Wha Lee,† Tae Su Choi,† Kyeong Sik Jin,‡ Seonghwan Lee,† Changill Ban,† and Hugh I. Kim*,†,§ †

Department of Chemistry, ‡Pohang Accelerator Laboratory, §Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang, North Gyeongsang 790-784, South Korea S Supporting Information *

ABSTRACT: α-Synuclein (α-Syn) is an intrinsically disordered protein, whose fibrillar aggregates are associated with the pathogenesis of Parkinson’s disease. α-Syn associates with lipid membranes and forms helical structures upon membrane binding. In this study, we explored the helix formation of αSyn in solution containing trifluoroethanol using small-angle X-ray scattering and electrospray ionization ion mobility mass spectrometry. We then investigated the structural transitions of α-Syn to helical structures via association with large unilamellar vesicles as model lipid membrane systems. Hydrogen−deuterium exchange combined with electrospray ionization mass spectrometry was further utilized to understand the details of the regional interaction mechanisms of α-Syn with lipid vesicles based on the polarity of the lipid head groups. The characteristics of the helical structures were observed with α-Syn by adsorption onto the anionic phospholipid vesicles via electrostatic interactions between the N-terminal region of the protein and the anionic head groups of the lipids. α-Syn also associates with zwitterionic lipid vesicles and forms helical structures via hydrophobic interactions. These experimental observations provide an improved understanding of the distinct structural change mechanisms of α-Syn that originate from different regional interactions of the protein with lipid membranes and subsequently provide implications regarding diverse protein−membrane interactions related to their fibrillation kinetics. α-Synuclein (α-Syn, Scheme S1, Supporting Information) is a small amyloidogenic protein and one of the intrinsically disordered proteins (IDPs) that can be aggregated into amyloid fibrils. Amyloid fibrillation of α-Syn is generally considered to be associated with the pathogenesis of Parkinson’s disease.1,2 αSyn is particularly abundant in presynaptic terminals3 and is believed to regulate the synaptic vesicles involved in the transfer of neurotransmitters.4,5 Its structure has been characterized as disordered when it is a freely solvated monomer in an aqueous solution. However, α-Syn can be structurally confined by intermolecular interactions. Recent investigations have reported that α-Syn forms an α-helical structure through the formation of a tetrameric complex6,7 although some studies challenge a tetrameric conformation under native in vivo conditions.8,9 A study employing the use of solution nuclear magnetic resonance (NMR) has revealed that the broken-helix structure, helix-link-helix, is formed by an interaction with detergent micelle.10 Additionally, a number of studies has provided evidence for an increase in the helical propensity of the protein via binding to anionic phospholipids.11−13 In addition to the fact that α-Syn has a physiological function with membrane interactions, the lipid-induced structures of α-Syn have been receiving attention for their pathologic significance. Helically folded α-Syn has resistance against amyloidogenic fibrillation, © 2014 American Chemical Society

while partially folded helices accelerate the formation of fibers.14−16 Interactions between α-Syn and anionic phospholipids particularly enhance the fibrillation process of the protein via structural rearrangement in the N-terminal and nonamyloid component (NAC, residues 61−95) regions.11,13,17 Moreover, α-Syn displays disruptive effects on membranes by forming annular oligomers.18 To clarify the structural transitions of α-Syn via interactions with lipid membranes, numerous experimental techniques have been utilized. Due to its structural heterogeneity and fluctuation, it is challenging to investigate the conformations of this IDP using conventional tools for structural biology, such as X-ray crystallography or NMR, especially in multiphase. Although NMR has been widely used to investigate interactions and IDP structures in lipid membranes, it requires labeling uniformly for the whole proteins or specifically over the regions of interest.11,19 Other spectroscopic methods such as surface plasmon resonance,20 double electron−electron resonance,21 single molecule fluorescence,12,22,23 and spin-labeling electron Received: December 14, 2013 Accepted: January 2, 2014 Published: January 2, 2014 1909

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

paramagnetic resonance24 have also been utilized to investigate α-Syn structures and interactions in lipid membranes. Recently, Han and co-workers reported that the central region of membrane-bound α-Syn can be penetrated below lipid head groups using 1H Overhauser dynamic nuclear polarizationenhanced NMR.25 However, most studies have approached αSyn in membrane systems by probing transitions of ensembleaveraged structures or specific regional changes based on the interactions between labels in the specific amino acid residues. To understand and estimate reactions at the virtual membrane interface, studying the overall picture of α-Syn, along with its site-specific interactions in lipid membranes, is highly required. Structures of desolvated proteins in the gas phase have been extensively studied in relation to their solution phase structures. A number of studies have revealed the structural relevance of protein ions in the gas phase to their solution phase structures using electrospray ionization mass spectrometry (ESI-MS).26,27 Once the protein is transferred from the solution phase to the gas phase by ESI, isolated freeze-out structures moderately reflect the number of existing protein states in the solution phase.26−28 The charged residues of the folded protein are accommodated by self-solvation, such as intramolecular hydrogen bonding and salt-bridge formation in the gas phase, and the folded structures are represented as low charge states in the ESI-MS spectrum. Conversely, high charge states represent denatured or extended structures of proteins as there is no media to attenuate Coulomb repulsion in vacuum.29,30 Electrospray ionization ion mobility mass spectrometry (ESIIM-MS) enables the identification of individual conformers in heterogeneous conformational distributions of IDPs based on variances in the collision cross section (ΩD) of their gas-phase conformations.31−33 A number of applications for characterizing the conformational conversions and assembly mechanisms of amyloidogenic IDPs has also been demonstrated.34,35 However, the structures of IDPs in heterogeneous systems, such as lipid membranes, have yet to be thoroughly investigated. In the present study, solution-phase structures of α-Syn are probed using circular dichroism (CD) spectroscopy and smallangle X-ray scattering (SAXS). Also, structural characteristics reflected in ESI-IM-MS are discussed. Then, interactions and secondary structure changes of intrinsically disordered α-Syn in large unilamellar vesicles (LUVs) as model lipid membrane systems are investigated using ESI-IM-MS and hydrogen− deuterium exchange mass spectrometry (HDX-MS).

SAXS-Based Modeling. To generate realistic solution structures based on experimental SAXS profiles, conformational pools with approximately 150 000 structures of α-Syn were constructed through simulated annealing. From the constructed structural pool, final candidates were determined as criteria of Rg and discrepancy (χ2) between experimental and theoretical SAXS profiles. Further details of sample preparation, SAXS experiments, simulations, ESI-IM-MS parameters, HDX-MS experiments, and CD spectroscopy are included in the Supporting Information.

■

RESULTS AND DISCUSSION Solution-Phase Structures of α-Syn. As one of the IDPs, α-Syn is known to be highly dynamic in solution.9,36,37 The structural diversity of α-Syn in solution is reduced using trifluoroethanol (TFE), a cosolvent to stabilize helical structures by strengthening intramolecular hydrogen bonds and simultaneously minimizing interactions between proteins and solvent molecules.38 The TFE-induced structure is widely used as a model structure for membrane-associated proteins.14,15 The structural properties in solution were investigated using CD spectroscopy and SAXS. To obtain information regarding secondary structural components, CD spectroscopy was utilized. In the absence of TFE, a CD spectrum characteristic of highly populated random structures was observed. However, the helical propensity of α-Syn gradually increased as the TFE content was increased (Figure S1a, Supporting Information). More detailed information on the overall shape and molecular dimensions was obtained from SAXS (Figure S2a, Supporting Information). The Kratky plot39 indicates that αSyn was present as random dynamic structures in water. Additionally, no evidence of the rigid structure of α-Syn in TFE 40% was observed from the Kratky plot, implying that randomness is present despite its enhanced helicity. The radius of gyration (Rg) values derived from Guinier fit (Figure S2c, Supporting Information) of α-Syn in water and in 40% TFE solution are 38.4 and 30.3 Å, respectively, implying that the protein forms a more compact structure in 40% TFE solution than in water. On the basis of the pair distance distribution function (P(r)), ab initio envelopes of α-Syn in water (Figure S2e, Supporting Information) and 40% TFE solution (Figure S2f, Supporting Information) were constructed. Further details of SAXS analysis are described in the Supporting Information. To investigate the molecular fluctuation and conformational properties in detail, MD simulations were performed on the basis of the experimental SAXS profiles. Figure S4, Supporting Information, shows the candidate structures of α-Syn in water and TFE 40% which have the minimum discrepancy from SAXS profiles. SAXS data indicate that α-Syn in water is largely fluctuating and thus cannot be refined in a specific shape. The MD-simulated structures whose theoretical scattering curves have good agreement with the experimental curve (χ2 < 20) also show highly diverse random structural characteristics. Despite the highly dynamic nature of the protein, the theoretical Rg values of α-Syn in water are close to 40 Å (Figure S5b, Supporting Information), which is smaller than fully elongated conformation of the protein (Rg ∼ 54 Å).40 This is attributed to random, but significant, intramolecular interactions of α-Syn. As shown by the CD spectrum, α-Syn in water is suggested to be freely solvated without defined secondary structure but not to be fully extended.

■

EXPERIMENTAL SECTION SAXS and ESI-IM-MS. Solution-phase SAXS experiments were performed using the 4C SAXS II beamline at Pohang Accelerator Laboratory (PAL). A Waters Synapt G2 HDMS traveling wave ion mobility orthogonal acceleration time-offlight mass spectrometer (Waters, Manchester, UK) was utilized for ESI-IM-MS in the positive ion modes. HDX-MS. Freshly prepared α-Syn and LUVs were exposed to D2O for deuteration and directly quenched with formic acid. After the addition of the quenching agent, the sample was immediately injected into the mass spectrometer. HDX-MS of synthetic 12-residue peptides was conducted in the same way. HDX-MS of peptic-digested α-Syn was performed with HDX and quenching followed by pepsin digest in an ice bath. Backward exchanged hydrogens were corrected using fully deuterated α-Syn and LUVs, hydrated with D2O. 1910

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

Figure 1. (a) Positive ion mode ESI-MS spectra of α-Syn with different portions of TFE and charge-state distribution for each spectrum. (b) ESI-IMMS spectra for +16 charged α-Syn with different portions of TFE, plotted on ΩD, and (c) simulated structures of α-Syn and theoretical ΩD corresponding to each structure.

MD-simulated structures for α-Syn in 40% TFE, which were also chosen on the basis of agreement between the experimental and theoretical scattering curves (χ2 < 10), show a horseshoe-like shape with high helicity (>30%) (H2 and H3 in Figure S4b and Table S1, Supporting Information). As the structural fluctuation is present in the helix region, it is difficult to accurately locate the exact helicity in amino acid residues; however, MD-simulated structures in solution commonly show helical structures in the N-terminal and NAC regions. These structural characteristics are similar to those of micelle-bound α-Syn, which comprises two long helices linked by a kink region10 (H1 in Figure S4b, Supporting Information). ESI-MS Reflects the Solution-Phase Structures of αSyn. The structural diversity of α-Syn in various solution conditions has been previously resolved using the charge-state distributions in ESI-MS.41,42 These studies reported that the heterogeneity of α-Syn structures is resolved by multiple distributions of charge states. To verify the use of ESI-MS for investigating the structural changes of α-Syn in solution, we investigated α-Syn in four different TFE/water solutions (0, 5, 20, and 40% TFE). In the positive ion mode, ESI-MS of α-Syn in water (0% TFE), bimodal charge-state distributions were observed, with one envelope centered on the +8/+9 charge states and another centered on the +15/+16 charge states (Figure 1a). With the addition of TFE to the sample solution, a gradual shift to high charge states was observed. Compared with the 0% TFE, α-Syn in the 40% TFE has a distinctly small distribution in the low charge states. We also verified similarities between the positive ion mode and the negative ion mode regarding the charge-state distributions of α-Syn and the changes observed in both due to the addition of TFE (Figure S6, Supporting Information). Assuming that the conformational distribution in ESI-MS represents the population of individual conformers of the protein, the observed changes in the charge-state distributions (Table S2, Supporting Information) indicate conformational transitions induced by the addition of TFE.27,28

In consideration of helicity enhancement in CD spectrum, the observed change in the charge-state distributions of α-Syn in the MS spectra can be correlated with the structural change of the protein in solution. The observed bimodal distributions of α-Syn in water indicate that α-Syn is present as a mixture of extended and compact structures, which result in highly charged and low charged states in the MS spectrum, respectively.30 Subsequently, as the propensity to form an αhelical structure is increased by TFE, the abundance of the low charge-state distribution, which represents the compact structure of the protein, is suppressed. It is thought that the observed high charge-state distribution of α-Syn in the absence of TFE may represent random extended structures based on the CD measurements. However, the high charge-state distribution of the protein in the presence of TFE may represent its helical structure. Resolving Coexisting Structures of α-Syn in the Gas Phase. To understand the difference between the highly charged α-Syn in solution with and without TFE, the gas-phase structures of the protein were investigated using ion mobility mass spectrometry (IM-MS) combined with molecular dynamics (MD) simulations. Figure 1b shows the IM-MS distributions plotted as ΩD with deconvolved curves. As seen in Figure 1b, multiple peaks (ΩD = 3048, 3125, 3176, and 3253 Å2) are determined from the IM-MS distributions for +16 charged α-Syn, which is the most prominent in all ESI-MS spectra. The relative abundances of these peaks vary with TFE content. In the absence of TFE, ions with the largest cross section (ΩD = 3253 Å2) are dominant. However, their relative abundance decreases, while the relative abundances of ions with smaller cross sections increase as the TFE content increases. This result supports the observation that high charge distributions from different TFE concentrations (Figure 1a) originate from different structures of α-Syn. To understand the origin of the observed structural diversity of α-Syn ions with the same charge state, we generated several gas-phase structures through in vacuo simulations. To describe disordered and helically induced α-Syn, several model 1911

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

structures, adopted on the basis of the fit against SAXS profiles, were set as initial geometries (Figure S4a, H2 and H3 in Figure S4b, Supporting Information). The broken-helix (H1 in Figure S4b, Supporting Information) was also set as the initial geometry to investigate the helical structures of the protein. Additionally, a similar approach with the study of Bowers and co-workers31 was taken into consideration with some extreme models (Figure S7c, Supporting Information). Although it is hard to expect that our models are exact solution-phase structures due to the flexible nature of α-Syn, repeated simulations and comparisons to the simulation results from the known NMR structures10 proved to be sufficiently reliable to describe the observed diversity of α-Syn structures in the gas phase. Figure 1c shows the MD simulated structures of α-Syn along with their theoretical ΩD. The unstructured α-Syn (ΩD > 3200 Å2, indicated as U) has larger ΩD than the structured ones; the helical propensity in the initially helical αSyn is partially conserved during the simulations, resulting in smaller ΩD (ΩD < 3200 Å2, designated as H1, H2, and H3), which originate from helically folded α-Syn in solution. This theoretical analysis indicates that the solution structures of αSyn significantly affect its gas phase structures, verifying the ability of the IM-MS method to isolate structural isomers of αSyn and to monitor its structural changes in solution. Specifically, initial geometries with high helical propensity (>30%) are transited to gas-phase structures (>23%, Table S1, Supporting Information). To identify structural isomers in the low charge-state distribution, the IM-MS spectrum of +10-charged α-Syn was also analyzed (Figure S8, Supporting Information). As +10charged ions are relatively not abundant in 40% TFE, it is difficult to discern the protein signal from the background interference. However, resolved peaks appear in the range from 2600 to 2900 Å2 in both conditions. Two unstructured (U1′ and U2′) and two helically folded (H1′ and H2′) conformations with a low charge state (+10) were investigated using MD simulations (Figure S8b, Supporting Information) from the compact initial structures (Figure S7e,f, Supporting Information) based on SAXS profiles (Figure S9, Supporting Information). Although the overall helicity is significantly different among the unstructured and helix-folded models in solution (Table S1, Supporting Information), no significant difference in ΩD is found in the gas phase. Because of its intrinsic fluctuation, some α-Syn molecules may be trapped in the electrostatically folded state during the ESI process and, thus, present low charge states in the gas phase, whereas other α-Syn molecules may maintain a disorderedly extended state with associated high charge.27,31 However, it is inferred that these compact structures still do not lose the structural origin in the solution phase (Table S1, Supporting Information). Monitoring Structural Changes of Membrane-Embedded α-Syn Using ESI-IM-MS. α-Syn is a peripheral membrane binding protein, which is abundant in presynaptic terminals.3,4 The membrane association triggers the helical folding of α-Syn, and this conformational transition is considered to be involved in the neurotransmitter release43 and fibrillation processes16,44,45 of α-Syn. To understand the structural changes and conformational properties of α-Syn in relation to its interactions with lipid membrane systems, we introduced α-Syn to large unilamellar vesicles (LUVs) as model membrane systems. LUVs comprise different ratios of zwitterionic 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) and the anionic 1-palmitoyl-2-oleoyl-sn-phosphatidyl-

serine (POPS) and L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) (Scheme S2, Supporting Information), which are widely used to mimic mammalian neural vesicles.46 With the exception of the PIP2 micelle, dynamic light scattering measurements show overall uniformity among the diameter of LUVs regardless of the phospholipid composition (Table S3, Supporting Information). The ESI-MS spectra of α-Syn with LUVs also show bimodal charge-state distributions, indicating the presence of mixed structures in solution (Figure 2a). Except for the LUVs

Figure 2. (a) ESI-MS spectra with charge-state distributions and (b) ESI-IM-MS spectra for α-Syn with PIP2 micelle and LUVs. IM-MS spectra are isolated for +16 charged α-Syn and plotted on the basis of ΩD.

comprising only POPC, the gradual suppression of low chargestate distributions is observed as the anionic strength of the vesicle is increased. Compared with the ESI-MS spectra of αSyn in TFE, the relative abundance of low charge-state distributions is similar to that of the 5−20% solutions of TFE (Table S4, Supporting Information). This result implies that conformational changes of α-Syn occur via interactions with LUVs. Additionally, an increase in the anionic strength of the LUVs that comprise zwitterionic and anionic phospholipids induces an enhanced helical propensity in the protein. A dramatic suppression of the low charge-state distribution of αSyn in the PIP2 micelle, in which α-Syn is most likely present as a broken helical structure (H1 in Figure S4b, Supporting Information), strongly supports this analysis. This result is in good agreement with previous studies that demonstrated that α-Syn was adsorbed onto the lipid membrane via electrostatic interactions between the anionic head groups of phospholipids and the basic amino acid residues of α-Syn.11 The IM-MS distributions support the observed structural transitions of α-Syn from a random structure to a helix due to the lipid vesicles. Multiple peaks with similar ΩD (≅3048, 3099, 3125, 3176, and 3253 Å2) observed from the TFE cosolvents (Figure 1b) are also observed from the IM-MS distributions for +16 charged α-Syn with LUVs (Figure 2b). A slightly higher abundance of the protein peak with large ΩD (>3200 Å2) is 1912

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

Figure 3. Normalized deuteration level for (a) full sequence α-Syn without and with LUVs, (b) peptic digested α-Syn without and with LUVs, and (c) 12-residue synthetic peptides without and with LUVs. Sequences for synthetic peptides are collected from N-terminal (1−12), amyloid core (71−82), and C-terminal (129−140) α-Syn. Deuteration time for all sample sets are adjusted as 10 s.

observed compared with the IM-MS spectra of α-Syn in the absence of TFE. In IM-MS of α-Syn with LUVs, it is thought that this relatively abundant structure with large ΩD is originated from α-Syn unbound to LUVs. However, other structures with smaller ΩD indicate the presence of helical structures that are due to the interactions of α-Syn with the lipid vesicles in solution. Relatively more abundant structures with smaller ΩD (≅3176 and 3099 Å2) induced from the αhelical structure of PIP2 micelle-bound α-Syn further support this analysis. It is notable that the low charge-state distribution of α-Syn was also significantly suppressed by the LUVs composed of only zwitterionic POPC in the ESI-MS spectrum. The IM-MS distributions also show the major presence of α-Syn as helical structures in the solution. It is also inferred that there are strong interactions between α-Syn and the LUVs composed of POPC. CD measurements were carried out to examine the structural changes of α-Syn that were probed by ESI-IM-MS (Figure S10b, Supporting Information). The helical characteristics of αSyn are apparent only with the strongly anionic LUVs (POPS and POPS/PIP2 (99:1 by mol)) and the PIP2 micelles. No characteristic CD ellipticity was observed for the α-helix with the LUVs composed of only POPC and a mixture of POPC and POPS (1:1 by mol), in contrast with the results from the ESI-IM-MS study. ESI-IM-MS has been proven to isolate individual structural isomers of proteins by charge states and cross sections, as discussed earlier.28,47 However, CD spectroscopy measures the averaged ensemble states of the protein structures. It is inferred that the observed structural changes of α-Syn due to neutral or low negatively charged LUVs occurred in a small specific region of the protein. In the CD spectrum of α-Syn with much higher concentration of POPC vesicles (300fold higher than the concentration of α-Syn), the helically folded species appears dominantly (Figure S11, Supporting Information). This supports that helix conformation is definitely induced by interactions with zwitterionic lipids even if it is not overwhelmingly abundant. Thus, ESI-IM-MS is sufficiently sensitive to probe structural transitions in the minor population which cannot be captured with ensemble measurements using CD spectroscopy. Previous studies also reported similar discrepancies between CD measurements and observation with other techniques.48,49 HDX-MS Reveals Regional Interactions of α-Syn with Membrane Lipids. Although α-Syn is generally believed to have high binding affinity toward anionic lipids, an association

with neutral lipids is also possible.22,49,50 However, the binding events between α-Syn and neutral lipid vesicles are not clearly understood. We performed HDX experiments to investigate the association of α-Syn with anionic POPS and zwitterionic POPC vesicles to understand the observed structural changes that were due to both lipid vesicles. HDX-MS can provide the relative magnitude of interactions between proteins and lipids in the membrane systems.51,52 Combined with the peptic digestion process, it is also possible to identify interactions in the specific regions of the protein.53 Figure 3a shows the relative deuteration level of α-Syn incubated for 10 s in water with and without LUVs (see Figure S12, Supporting Information, for MS spectra). Free α-Syn demonstrated a high HDX level indicative of high solvent exposure, while a suppressed HDX was observed from α-Syn with POPS vesicles, indicating strong interactions with POPS vesicles. It is notable that α-Syn with POPC vesicles also showed a greater level of retarded deuteration than free α-Syn. This result indicates that α-Syn has significant interactions with both anionic and zwitterionic LUVs, although the protein has slightly weaker or smaller regional interactions with neutral LUVs than with anionic LUVs. Additional experiments with different exposure times to the deuterated solvent further indicate that α-Syn undergoes more retarded exchange in both anionic and zwitterionic LUVs than α-Syn in water (Figure S13, Supporting Information). Figure 3b depicts the relative deuteration level of the Nterminal (residues 4−38) and hydrophobic central (residues 55−80) residues of α-Syn with and without LUVs, obtained from the ESI-MS spectrum (Figure S14, Supporting Information) of the peptic digests of α-Syn. Overall, lower exchange levels in the N-terminal and hydrophobic central residues of α-Syn are observed with both LUVs compared to αSyn without LUVs. Specifically, similar HDX levels are observed from the N-terminal region with both POPS and POPC vesicles. However, a lower HDX level is observed from the central residues of the protein with the neutral lipid vesicles than with the anionic lipid vesicles. To verify the observed regional interactions in peptic digested α-Syn, we additionally performed HDX with and without LUVs using 12-residue peptides from the N-terminal (1−12), central (71−82), and C-terminal (129−140) regions of α-Syn (Figure 3c, MS spectra in Figure S15, Supporting Information). A study using small peptides with regional sequences and lipid vesicles can provide a clear picture of their 1913

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

broken-helix (residues 3−92) with residues 38−44 as the linker.10 However, membrane-bound α-Syn is suggested to adopt an extended-helix from the N-terminus to the NAC region rather than a broken-helix.11,12,21,23 Our HDX experiments indicate that the major driving force for the association of α-Syn with anionic lipid vesicles is electrostatic interactions. Additional hydrophobic interactions between the central residues and the POPS vesicles were observed. The observed transition of the structure from random to α-helix using ESI-IM-MS is thought to occur through the electrostatic stabilization of the N-terminal region of the protein and the extension of the helix dipole up to the central region (Scheme S3a, Supporting Information).11 In contrast, the hydrophobic interaction is the driving force for the association of α-Syn with neutral lipid vesicles. The absence of an electrostatic interaction with lipid head groups enables a deeper penetration of the central region of the protein into the lipid membrane. A recently published study indicates that some hydrophobic residues located in NAC can pull down the NAC region below the lipid heads and assist the penetration into hydrophobic acyl chains.25 In addition, the conformational change of the protein to the helical form occurs without electrostatic stabilization by interacting with the lipid membrane (Scheme S3b, Supporting Information). It is suggested that the helical structure of α-Syn can be driven only by hydrophobic interactions in membranes without an expansion of the helix dipole from the N-terminal region. Upon binding to zwitterionic vesicles, the backbone amide groups of the hydrophobic central residues lose their interaction with the solvent water molecules and are preferentially stabilized by the formation of intramolecular hydrogen bonds, leading to the formation of a helical structure. Although it is not large enough to induce a clearly defined helical structure, our ESI-IM-MS and HDX-MS experiments support the appearance of the helical characteristics of α-Syn via adsorption onto the zwitterionic lipid vesicles.

interactions by isolating one region from the other. A considerably lower HDX was observed from α-Syn(1−12) with POPS vesicles, indicating a strong interaction between the peptide and anionic lipids. However, no significant effect on the HDX of the peptide was observed with POPC vesicles. Significantly retarded HDX levels were observed from the peptide corresponding to α-Syn(71−82) with both POPC and POPS vesicles. α-Syn(129−140) shows slightly lower HDX with both LUVs. On the basis of the HDX-MS experiments using α-Syn and small peptides with regional sequences, we can deduce two distinct interaction mechanisms of α-Syn with membranes. For anionic lipid membranes, α-Syn is adsorbed onto the surface of the vesicles due to electrostatic interactions between the Nterminal region of the protein and the lipid head groups along with the hydrophobic interaction between the central region of the protein and the lipid acyl chains. Because the N-terminal region of the protein is adsorbed onto the surface of the lipid vesicles, a lower penetration of the hydrophobic central region into the hydrophobic acyl-chain region occurs. For zwitterionic lipid membranes, the hydrophobic interaction between the central region of α-Syn and the lipid acyl chains is the driving force for embedding the protein inside the membrane. Deep penetration of the central region of α-Syn into the lipid bilayer may induce a weak association of the N-terminal region with the neutral lipids. Electrostatic interactions of the N-terminal region with the anionic lipid head groups are widely accepted as a driving force for the membrane association of α-Syn.11−13,24,54 It is suggested that the helical structure of α-Syn is induced by the alignment of lysine residues with the membrane surface to enhance electrostatic interactions with the anionic head groups. Additional hydrophobic interactions then occur in the central region of the protein with the acyl chains of lipids,11,45,50,55−57 most likely in a concomitant manner. Both interactions are understood to stabilize adsorbed α-Syn on the anionic lipid membrane.11 However, our HDX-MS data indicate that α-Syn can be adsorbed onto the lipid membrane only via hydrophobic interactions. We could not detect peptic digests from the Cterminal region in the present study. However, we suggest that a weak interaction may occur in the C-terminal region based on HDX experiments of α-Syn(129−140). Distinct Structural Change Mechanisms of α-Syn Induced by Different Interactions in the Membrane. Both ESI-IM-MS and HDX-MS suggest that α-Syn undergoes a conformational transition from random to helical structures via interactions with both anionic and zwitterionic lipid vesicles. However, there is a discrepancy in the interaction types and regions of the protein based on the polarity of the lipid head groups. The helix formation of α-Syn on the lipids has been widely investigated experimentally11,13,49,58,59 and theoretically.60,61 It has been generally suggested that electrostatic interactions between α-Syn and anionic lipids dominate the stability of the complex and that hydrophobic interactions further stabilize this complex system.11,44,45,50,55−57 Upon binding to the lipid membrane, the N-terminal region of αSyn adopts a 11/3α-helix conformation (3.67 residues per turn), enabling the positively charged face of the helix to interact electrostatically with the anionic head groups (Figure S16, Supporting Information).11,58 The NAC region interacts with the lipid interior, adjusting its conformation to an α-helix through an enhanced helix dipole from the N-terminal region62,63 The micelle-bound α-Syn is known to adopt a

■

CONCLUSION In the present study, we demonstrated that the secondary structural characteristics of α-Syn in solution can be preserved in the gas phase using CD, solution SAXS, and ESI-IM-MS providing the potential utility of MS for the investigation of the structural dynamics of IDPs. Then, we defined the conformational properties and plausible interacting regions of α-Syn in model membrane systems using ESI-IM-MS and HDX-MS. Our study reveals distinct mechanisms of the membranemediated helix folding of α-Syn based on the polarity of the membrane surface. In particular, α-Syn can adopt a helical structure through deep penetration of the central region in the neutral lipid membrane via hydrophobic interactions. Neutral lipids, such as phosphatidylcholine, are highly abundant in the outer layer of human plasma membranes, whereas anionic lipids are abundant in the inner layer.64 The secondary structures of α-Syn, particularly the structure of the NAC region, are considered important factors in fibrillation kinetics.65,66 As a result, the hydrophobic interactions of the central residues in the membrane are expected to be of importance for the fibrillation processes of the protein. Several previous studies reported that α-Syn fibril formation is accelerated by anionic lipid membranes45,49,67−70 and anionic micelles,71 while it is hindered by interactions with zwitterionic lipid membranes.45 On the basis of our study, the deeply inserted NAC region of αSyn in the hydrophobic environment may prevent the protein 1914

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

(13) Comellas, G.; Lemkau, L. R.; Zhou, D. H.; George, J. M.; Rienstra, C. M. J. Am. Chem. Soc. 2012, 134, 5090. (14) Munishkina, L. A.; Phelan, C.; Uversky, V. N.; Fink, A. L. Biochemistry 2003, 42, 2720. (15) Anderson, V. L.; Ramlall, T. F.; Rospigliosi, C. C.; Webb, W. W.; Eliezer, D. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 18850. (16) Zhu, M.; Fink, A. L. J. Biol. Chem. 2003, 278, 16873. (17) Der-Sarkissian, A.; Jao, C. C.; Chen, J.; Langen, R. J. Biol. Chem. 2003, 278, 37530. (18) Reynolds, N. P.; Soragni, A.; Rabe, M.; Verdes, D.; Liverani, E.; Handschin, S.; Riek, R.; Seeger, S. J. Am. Chem. Soc. 2011, 133, 19366. (19) Madine, J.; Hughes, E.; Doig, A. J.; Middleton, D. A. Mol. Membr. Biol. 2008, 25, 518. (20) Smith, D. P.; Tew, D. J.; Hill, A. F.; Bottomley, S. P.; Masters, C. L.; Barnham, K. J.; Cappai, R. Biochemistry 2008, 47, 1425. (21) Georgieva, E. R.; Ramlall, T. F.; Borbat, P. P.; Freed, J. H.; Eliezer, D. J. Am. Chem. Soc. 2008, 130, 12856. (22) Rhoades, E.; Ramlall, T. F.; Webb, W. W.; Eliezer, D. Biophys. J. 2006, 90, 4692. (23) Trexler, A. J.; Rhoades, E. Biochemistry 2009, 48, 2304. (24) Jao, C. C.; Der-Sarkissian, A.; Chen, J.; Langen, R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8331. (25) Cheng, C.-Y.; Varkey, J.; Ambroso, M. R.; Langen, R.; Han, S. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 16838. (26) Breuker, K.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145. (27) Wyttenbach, T.; Bowers, M. T. J. Phys. Chem. B 2011, 115, 12266. (28) Pierson, N. A.; Chen, L.; Valentine, S. J.; Russell, D. H.; Clemmer, D. E. J. Am. Chem. Soc. 2011, 133, 13810. (29) Jarrold, M. F. Annu. Rev. Phys. Chem. 2000, 51, 179. (30) Bohrer, B. C.; Merenbloom, S. I.; Koeniger, S. L.; Hilderbrand, A. E.; Clemmer, D. E. Ann. Rev. Anal. Chem. 2008, 1, 293. (31) Bernstein, S. L.; Liu, D.; Wyttenbach, T.; Bowers, M. T.; Lee, J. C.; Gray, H. B.; Winkler, J. R. J. Am. Soc. Mass Spectrom. 2004, 15, 1435. (32) Canon, F.; Ballivian, R.; Chirot, F.; Antoine, R.; SarniManchado, P.; Lemoine, J.; Dugourd, P. J. Am. Chem. Soc. 2011, 133, 7847. (33) Harvey, S. R.; Porrini, M.; Stachl, C.; MacMillan, D.; Zinzalla, G.; Barran, P. E. J. Am. Chem. Soc. 2012, 134, 19384. (34) Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J.-E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Nat. Chem. 2009, 1, 326. (35) Dupuis, N. F.; Wu, C.; Shea, J.-E.; Bowers, M. T. J. Am. Chem. Soc. 2009, 131, 18283. (36) Dedmon, M. M.; Lindorff-Larsen, K.; Christodoulou, J.; Vendruscolo, M.; Dobson, C. M. J. Am. Chem. Soc. 2004, 127, 476. (37) Waudby, C. A.; Camilloni, C.; Fitzpatrick, A. W. P.; Cabrita, L. D.; Dobson, C. M.; Vendruscolo, M.; Christodoulou, J. PLoS One 2013, 8, No. e72286. (38) Cammers-Goodwin, A.; Allen, T. J.; Oslick, S. L.; McClure, K. F.; Lee, J. H.; Kemp, D. S. J. Am. Chem. Soc. 1996, 118, 3082. (39) Receveur-Brechot, V.; Durand, D. Curr. Protein Pept. Sci. 2012, 13, 55. (40) Uversky, V. N.; Li, J.; Fink, A. L. J. Biol. Chem. 2001, 276, 10737. (41) Frimpong, A. K.; Abzalimov, R. R.; Uversky, V. N.; Kaltashov, I. A. Proteins 2010, 78, 714. (42) Natalello, A.; Benetti, F.; Doglia, S. M.; Legname, G.; Grandori, R. Proteins 2011, 79, 611. (43) Di Paolo, G.; Kim, T. W. Nat. Rev. Neurosci. 2011, 12, 284. (44) Lee, H.-J.; Choi, C.; Lee, S.-J. J. Biol. Chem. 2002, 277, 671. (45) Zhu, M.; Li, J.; Fink, A. L. J. Biol. Chem. 2003, 278, 40186. (46) Takamori, S.; Holt, M.; Stenius, K.; Lemke, E. A.; Grønborg, M.; Riedel, D.; Urlaub, H.; Schenck, S.; Brügger, B.; Ringler, P.; Müller, S. A.; Rammner, B.; Gräter, F.; Hub, J. S.; De Groot, B. L.; Mieskes, G.; Moriyama, Y.; Klingauf, J.; Grubmüller, H.; Heuser, J.; Wieland, F.; Jahn, R. Cell 2006, 127, 831.

from experiencing hydrophobic protein−protein interactions. We expect that our methods and findings will stimulate further studies and provide an improved understanding, respectively, of the structural and regional characterization of membraneassociated IDPs beyond the limit generated by the intrinsic heterogeneity of the proteins and complex systems.

■

ASSOCIATED CONTENT

S Supporting Information *

Descriptions about materials and experimental details. SAXS data analysis, CD spectra, charge-state distribution analysis, experimental and theoretical SAXS profiles, additional simulated structures, size measurement of LUVs, MS spectra for HDX, additional HDX results, 11/3α-helical wheel projection, pH measurement of α-Syn, ΩD calibration plot, and residual sum of squares analysis of IM-MS deconvolution. 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.

■

ACKNOWLEDGMENTS This work was supported by Basic Science Research (HIK; Grant No. 2013R1A1A2008974) through the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT and Future Planning (MSIP). The synchrotron X-ray scattering measurements at the PAL were supported by the Ministry of Education and Science Technology (MEST). S. J. C. Lee was supported by NRF Grant funded by the Korean Government (NRF-2011-Global Ph.D. Fellowship program). T. S. Choi acknowledges the support from TJ Park Fellowship.

■

REFERENCES

(1) Polymeropoulos, M. H.; Lavedan, C.; Leroy, E.; Ide, S. E.; Dehejia, A.; Dutra, A.; Pike, B.; Root, H.; Rubenstein, J.; Boyer, R.; Stenroos, E. S.; Chandrasekharappa, S.; Athanassiadou, A.; Papapetropoulos, T.; Johnson, W. G.; Lazzarini, A. M.; Duvoisin, R. C.; Di Iorio, G.; Golbe, L. I.; Nussbaum, R. L. Science 1997, 276, 2045. (2) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Nature 1997, 388, 839. (3) Maroteaux, L.; Campanelli, J.; Scheller, R. J. Neurosci. 1988, 8, 2804. (4) Clayton, D. F.; George, J. M. J. Neurosci. Res. 1999, 58, 120. (5) Lotharius, J.; Brundin, P. Nat. Rev. Neurosci. 2002, 3, 932. (6) Bartels, T.; Choi, J. G.; Selkoe, D. J. Nature 2011, 477, 107. (7) Wang, W.; Perovic, I.; Chittuluru, J.; Kaganovich, A.; Nguyen, L. T. T.; Liao, J.; Auclair, J. R.; Johnson, D.; Landeru, A.; Simorellis, A. K.; Ju, S.; Cookson, M. R.; Asturias, F. J.; Agar, J. N.; Webb, B. N.; Kang, C.; Ringe, D.; Petsko, G. A.; Pochapsky, T. C.; Hoang, Q. Q. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17797. (8) Fauvet, B.; Mbefo, M. K.; Fares, M.-B.; Desobry, C.; Michael, S.; Ardah, M. T.; Tsika, E.; Coune, P.; Prudent, M.; Lion, N.; Eliezer, D.; Moore, D. J.; Schneider, B.; Aebischer, P.; El-Agnaf, O. M.; Masliah, E.; Lashuel, H. A. J. Biol. Chem. 2012, 287, 15345. (9) Binolfi, A.; Theillet, F. X.; Selenko, P. Biochem. Soc. Trans. 2012, 40, 950. (10) Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595. (11) Bodner, C. R.; Maltsev, A. S.; Dobson, C. M.; Bax, A. Biochemistry 2010, 49, 862. (12) Ferreon, A. C. M.; Gambin, Y.; Lemke, E. A.; Deniz, A. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5645. 1915

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916

Analytical Chemistry

Article

(47) Shi, H.; Pierson, N. A.; Valentine, S. J.; Clemmer, D. E. J. Phys. Chem. B 2012, 116, 3344. (48) Maiti, N. C.; Apetri, M. M.; Zagorski, M. G.; Carey, P. R.; Anderson, V. E. J. Am. Chem. Soc. 2004, 126, 2399. (49) Jo, E.; McLaurin, J.; Yip, C. M.; St. George-Hyslop, P.; Fraser, P. E. J. Biol. Chem. 2000, 275, 34328. (50) Narayanan, V.; Scarlata, S. Biochemistry 2001, 40, 9927. (51) Demmers, J. A. A.; Haverkamp, J.; Heck, A. J. R.; Koeppe, R. E.; Killian, J. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3189. (52) Hansen, R. K.; Broadhurst, R. W.; Skelton, P. C.; Arkin, I. T. J. Am. Soc. Mass Spectrom. 2002, 13, 1376. (53) Del Mar, C.; Greenbaum, E. A.; Mayne, L.; Englander, S. W.; Woods, V. L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15477. (54) Bartels, T.; Ahlstrom, L. S.; Leftin, A.; Kamp, F.; Haass, C.; Brown, M. F.; Beyer, K. Biophys. J. 2010, 99, 2116. (55) Bussell, R., Jr.; Ramlall, T. F.; Eliezer, D. Protein Sci. 2005, 14, 862. (56) Shvadchak, V. V.; Yushchenko, D. A.; Pievo, R.; Jovin, T. M. FEBS Lett. 2011, 585, 3513. (57) Perrin, R. J.; Woods, W. S.; Clayton, D. F.; George, J. M. J. Biol. Chem. 2000, 275, 34393. (58) Bussell, R., Jr.; Eliezer, D. J. Mol. Biol. 2003, 329, 763. (59) Davidson, W. S.; Jonas, A.; Clayton, D. F.; George, J. M. J. Biol. Chem. 1998, 273, 9443. (60) Perlmutter, J. D.; Braun, A. R.; Sachs, J. N. J. Biol. Chem. 2009, 284, 7177. (61) Ullman, O.; Fisher, C. K.; Stultz, C. M. J. Am. Chem. Soc. 2011, 133, 19536. (62) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X. X.; Oneil, K. T.; Degrado, W. F. Science 1995, 270, 935. (63) Blaber, M.; Zhang, X.; Matthews, B. Science 1993, 260, 1637. (64) Op den Kamp, J. A. F. Annu. Rev. Biochem. 1979, 48, 47. (65) Giasson, B. I.; Murray, I. V. J.; Trojanowski, J. Q.; Lee, V. M.-Y. J. Biol. Chem. 2001, 276, 2380. (66) Bisaglia, M.; Trolio, A.; Bellanda, M.; Bergantino, E.; Bubacco, L.; Mammi, S. Protein Sci. 2006, 15, 1408. (67) Cole, N. B.; Murphy, D. D.; Grider, T.; Rueter, S.; Brasaemle, D.; Nussbaum, R. L. J. Biol. Chem. 2002, 277, 6344. (68) Volles, M. J.; Lee, S.-J.; Rochet, J.-C.; Shtilerman, M. D.; Ding, T. T.; Kessler, J. C.; Lansbury, P. T. Biochemistry 2001, 40, 7812. (69) Zhao, H.; Tuominen, E. K. J.; Kinnunen, P. K. J. Biochemistry 2004, 43, 10302. (70) Haque, F.; Pandey, A. P.; Cambrea, L. R.; Rochet, J.-C.; Hovis, J. S. J. Phys. Chem. B 2010, 114, 4070. (71) Giehm, L.; Oliveira, C. L. P.; Christiansen, G.; Pedersen, J. S.; Otzen, D. E. J. Mol. Biol. 2010, 401, 115.

1916

dx.doi.org/10.1021/ac404132g | Anal. Chem. 2014, 86, 1909−1916