ISSN 1672-9145 Acta Biochimica et Biophysica Sinica 2005, 37(2): 113–118 CN 31-1940/Q

©Institute of Biochemistry and Cell Biology, SIBS, CAS

Structural Evidence for ααααα-Synuclein Fibrils Using in SituAtomic Force Microscopy

Feng ZHANG1, Li-Na JI1,2, Lin TANG1, Jun HU1,3, Hong-Yu HU2, Hong-Jie XU1, and Jian-Hua HE1*1Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China;

2Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, Shanghai 200031, China;

3Bio-X Research Center, Shanghai Jiaotong University, Shanghai 200030, China

Abstract Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that arebelieved to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson’sdisease, dementia with Lewy bodies and Lewy body variant of Alzheimer’s disease. In this paper, in situatomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solutionusing two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situcontact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and asimilar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrilswere incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentoustopography for over 1 h in the in situ tapping mode atomic force microscopy experiments. The presentresults provide indirect evidence on how β-sheets assemble into α-synuclein fibrils on a nanometer scale.

Key words α-synuclein; atomic force microscopy; tapping mode; contact mode; guanidine hydrochlo-ride

α-Synuclein is the major component of intracellular in-clusions in several neurodegenerative diseases such asParkinson’s disease, dementia with Lewy bodies and Lewybody variant of Alzheimer’s disease, and the conversionof soluble α-synuclein into filamentous aggregates maycontribute to disease pathogenesis [1–3]. In vitro experi-ments have shown that α-synuclein is a cytoplasmic pro-tein of 140 amino acids with unknown function and that itis thermally stable [4,5], and is abundantly expressed andconcentrated in the presynaptic terminals of neuronal cells[6]. Studies using animal models have shown that α-synuclein aggregates can be detected in transgenic mousemodels as well as in transgenic flies. Overexpressed α-synuclein and the formation of α-synuclein aggregatescause neurodegeneration and motor impairment in thesemodels [7].

α-Synuclein is highly dynamic and, therefore, is classi-fied as a natively unfolded protein [5]. In vitro aggregation

of α-synuclein is a nucleation-dependent process [8]. Thislag phase is dramatically reduced by the addition of seedsor nuclei of pre-aggregated α-synuclein [9], and nativelyunfolded α-synuclein protein also undergoes a transitionto partially folded intermediates before forming maturefibrils [10]. However, after aggregation, a number of notyet well-defined regions of α-synuclein undergo a con-formational change and take up a cross-β structure inwhich individual β-strands run perpendicular to the fiberaxis [11].

Atomic force microscopy is a very important tool forthe observation of high-resolution three-dimensional struc-tures and has the advantage of easy sample preparationscompared with electron microscopy or X-ray fiberdiffraction. In situ atomic force microscopy under liquidoffers the possibility of examining α-synuclein fibrils underphysiological conditions in a time-dependent manner, whichallows monitoring of the changes in conformation andaggregation of α-synuclein fibrils. Atomic force micro-scopy can also be used to gain an insight into the structureof α-synuclein and other substances that are of potentialimportance in Parkinson’s disease, which can either in-

Received: November 29, 2004 Accepted: December 30, 2004*Corresponding author: Tel, 86-21-59554730; Fax, 86-21-

59553021; E-mail, hejh@sinap.ac.cn

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hibit or break α-synuclein fibrils. Results from the presentstudy will be useful in evaluating special drugs designed toinhibit or reverse the process, and will lead to an under-standing of how these interactions affect α-synucleinfibrils. Despite the importance of α-synuclein insyncleinophathies, a clear understanding of the structureand mechanism of α-synuclein fibril formation remainselusive.

In the present paper, in situ atomic force microscopyhas been used to study the structural properties of α-synuclein fibrils on a nanometer scale.

Materials and Methods

Expression and purification of ααααα-synuclein

α-Synuclein was expressed in Escherichia coli BL21(DE3) using a pET3a plasmid containing the human α-synuclein gene. The cell homogenate was processed byprecipitation from a 50% saturation of ammonium sulfate.The α-synuclein-containing fraction was loaded onto aDEAE-Sephadex A25 ion-exchange column (Amersham,Uppsala, Sweden) equilibrated with 50 mM Tris-HCl (pH8.0). α-Synuclein was eluted with a linear gradient of 0–0.5 M NaCl in the same buffer and further purified usingfast-performance liquid chromatography (FPLC) on aSuperose-12 column (Amersham, Uppsala, Sweden) withPBS (phosphate-buffered saline containing 100 mMphosphate, 100 mM NaCl, pH 7.5). The homogeneity ofthe purified α-synuclein protein was identified using so-dium dodecyl sulfate-polyacrylamidegel electrophoresis(SDS-PAGE) and mass spectrometry, and the concentra-tions of the proteins were determined by measuring theabsorbance at 280 nm using extinction coefficients calcu-lated according to the amino acid sequence.

ααααα-Synuclein fibrils and ThT fluorescence

High concentration α-synuclein protein (approximately200 µM) was incubated in PBS at 37 °C for 6 days withcontinuous shaking to form α-synuclein fibrils. A thioflavinT (ThT) fluorescence assay and an absorbance recordingat 330 nm (A330) were carried out to detect the formationof filamentous aggregates (data not shown). Atomic forcemicroscopy was also used to check the formation of α-synuclein protein filaments.

Atomic force microscopy

Samples for atomic force microscopy (AFM) imagingwere prepared by depositing 2–5 µl of the mixture on

freshly cleaved mica. After adsorption for 5 min, the micasurface was gently washed with PBS. Samples were driedusing condensed air and stored in a desiccator overnight.The AFM images in air were obtained using MultimodeAFM (NanoScope IIIa, Veeco/Digital Instruments, SantaBarbara, CA, USA) with a J-scanner (125 µm×125 µm) ina tapping mode. Standard etched silicon cantilevers(Ultrasharp NSC/11, MikroMasch, Portland, Oregon, USA)with a force constant of approximately 48 N/m were usedin the air imaging and the surrounding environment waskept at approximately 25 °C and 35%–40% humidity. Theimages were flattened by the plane-fit profile and heightmeasurements were carried out with the section profileusing the NanoScope IIIa offline analysis program.

In situ AFM experiments

A diluted aliquot (5 µl, 40 µM) of the α-synuclein fibrilspreparation was absorbed onto a freshly cleaved mica sub-strate for 5 min. The substrate was rinsed gently withPBS and immediately transferred into the liquid cell of AFMusing a silicon nitride contact cantilever. Imaging experi-ments were carried out in the same buffer. The humidityfor in situ experiments was 70% and the temperature waskept at (25 ± 1) °C. For the dissociation experiments, 10µl of 6 M guanidinium chloride (GdmCl) was added intothe liquid cell (final concentration of 0.6 M) so that stableimaging could be obtained. Both tapping mode AFM andcontact mode AFM were used in the in situ experiments.In tapping mode AFM, the tip does not contact the sampleat all times, but rather scans the sample surface usingvibrations. In the in situ tapping mode AFM experiments,the setpoint value was increased to the maximum to re-duce damage to the sample. In contact mode AFM, the tipcontacts the sample surface at all times when scanning.In the in situ contact mode AFM experiment, the setpointvalue was reduced to the minimum to reduce damage tothe sample.

Results

AFM tip breaks ααααα-synuclein fibrils in contact mode,but not in tapping mode

α-Synuclein fibrils were obtained after incubation asshown by tapping mode AFM (Fig. 1). In the in situ con-tact mode AFM experiments, several α-synuclein fibrilsbroke into fragments with the continuous scanning of theAFM tip and finally disappeared after less than 20 min. α-Synuclein fibrils on the substrate can be classified into

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two types by their orientation relative to the scanningdirection: fibrils lying parallel or almost parallel along thescanning direction (type I) and fibrils lying perpendicularto the scanning direction (type II) (Fig. 2). The two typesof fibrils behaved differently when the AFM tips kept scan-ning in the in situ contact mode experiments. Type II fibrilsbroke into fragments and dissolved gradually into solution

with repetitive scanning. However, type I fibrils wereharder to break into fragments and did not show obviouscracks along their fibril axis. In addition, all fibrils did notbecome thin in all experiments. In the in situ tapping modeAFM experiments neither type I nor type II fibrils showeddistinct changes in their topological features when the α-synuclein fibrils were continuously scanned by a non-con-tact tip for more than 60 min (Fig. 3). There were somefibril tangles (Fig. 3), the height of which was higher thanthe single fibrils on the substrate. The fibril tangles werenot observed in Fig. 2 because we rinsed the substrate afew more times with PBS in the contact mode experi-ments and carefully selected the scanning region to avoidcontamination on the tip.

GdmCl dissociates ααααα-synuclein fibrils

In the in situ tapping mode AFM experiments, α-synu-clein fibrils broke into fragments when the fibrils wereincubated with 0.6 M GdmCl. As shown in Fig. 4, a pieceof α-synuclein fibril gradually dissolves into solutionwithin approximately 34 min. Increased numbers of spotswere deposited on the substrate as the fibrils broke intofragments. Dissociation of α-synuclein fibrils was nothomogenous along the fibril axis. Results revealed that some

Fig. 1 Tapping mode AFM images for ααααα-synuclein oligomersand fibrils in air(A) Fresh α-synuclein protein imaged on bared mica. The arrows point to theoligomers of α-synuclein protein. (B) α-Synuclein fibrils were generated by incu-bating α-synuclein protein at 37 °C for 6 days with continuous shaking. Heightand size bars are shown for both (A) and (B).

Fig. 2 In situ AFM images of ααααα-synuclein fibrils in contact modeHeight bars and scanning size bars are shown for all pictures. Solid line arrows point to type II fibrils and broken line arrows point to type I fibrils. The number of scansis shown in each picture.

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parts of the fibril were fragile and dissociated easily,whereas other parts were harder to dissociate. However,

the dissociating rate of the α-synuclein fibrils was inde-pendent of the orientation of the fibrils (data not shown).

Fig. 3 In situ AFM images of ααααα-synuclein fibrils in tapping modeScanning is continuous. Height bars and scanning size bars are shown for all pictures. The number of scans for (A), (B), (C), (D), and (E) is 1, 4, 6, 9, and 12, respectively.

Fig. 4 In situ tapping mode AFM images for ααααα-synuclein fibrils dissociated by GdmClScanning is continuous. Scanning size scales and height bars are shown for all pictures.

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Discussion

α-Synuclein fibrils break into fragments rather than intothin fibrils along the fibril axis, which reveals the assem-bly manner of the α-synuclein protein in the fibrils (Figs.2 and 4). The inter-strand hydrogen bond distance and theinter-sheet spacing of α-synuclein fibrils have been ob-tained from X-ray fiber diffraction data [8]. In Sunde andBlake’s cross-β sheet model, β-strands are perpendicularto the fibril axis and β-sheets are parallel to the fibril axis[12]. According to the cross-β sheet model, the α-synuclein fibrils are composed of helically twisted β-sheetswith hydrogen bonds. In our experiments, we have shownthat the structure perpendicular to the fibril axis is vulner-able under the interaction between AFM tips and fibrils.Our results support Sunde and Blake’s cross-β sheet model.A further explanation using a simplified cross-β sheet modelof α-synuclein fibrils is outlined below (Fig. 5).

the direction of the AFM fast scanning axis and the inter-sheet hydrogen bond, parallel to the fibril axis, is easilydissociated by the lateral shearing strength exerted by theAFM tip. The AFM tip is very sharp with a radius of cur-vature less than 10 nm, and when such a sharp tip scansα-synuclein fibrils, which lie perpendicular to the scanningdirection, some β-sheets can be pushed out of the fibrils.For type I fibrils, the lateral shearing strength is exertedmainly on β-strands that are difficult to break. The resultsalso indicate that the interaction between AFM tip and α-synuclein fibrils is stronger than the inter-sheet hydrogenbond, but is weaker than the peptide bond in contact modeAFM. However, in tapping mode AFM, there is basicallyno lateral shearing strength exerted on the fibrils and noobvious changes in α-synuclein fibrils were observed.

The process of GdmCl breaking α-synuclein fibrils ap-pears to be similar to that of the contact tip breaking typeII α-synuclein fibrils. However, they differ in the molecu-lar mechanism: one is a chemical process and the otherresults from physical effects. GdmCl can destroy the hy-drogen bond between peptides by forming a new hydro-gen bond with the carbonyl oxygen of amino acids. In thisway, GdmCl of high concentrations break the α-synucleinfibrils into fragments which dissolve in the buffer and theamidocyanogens in GdmCl provide protons to the carbonyloxygen of the peptide bonds.

Our experiment indicates that it is possible to estimatethe energy of chemical bonds directly using the physicaltechnique of AFM on a nanometer scale. AFM provides auseful technique to gain an insight into the surface struc-ture and chemical bonds of macromolecules, of whichwe foresee an application to molecular surgery.

Acknowledgements

We thank Dr. Hong-Tao LI and Hai-Ning DU for theirtechnical assistance, and Dr. Zhi-Xiang ZHANG for help-ful comments and critical reading of the manuscript. Fund-ing from the Research Project of Shanghai Institute ofApplied Physics (Manipulation of Single Molecules) isgratefully acknowledged.

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In contact mode AFM, a lateral shearing strength isexerted on the α-synuclein fibrils when the tip scans thefibrils. Because the peptide bond energy is much higherthan the hydrogen bond energy, it is much easier to breakthe hydrogen bonds. Type II α-synuclein fibrils are easierto break into fragments than type I fibrils (Fig. 2). This isbecause type II α-synuclein fibrils are perpendicular to

Fig. 5 Sketch maps of ααααα-synuclein fibrils broken intofragments by AFM tips(A) and (B) are sketch maps for contact AFM tips breaking α-synuclein fibrils. (C)and (D) are straightforward sketch maps of α-synuclein fibrils. The black rods areβ-strands, and the dotted lines are the hydrogen bonds between the β-strands.

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Edited byZhi-Hong ZHANG