The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure.

Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on αS aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of αS in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-αS in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated αS. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in αS, thus predisposing αS toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.

Direct Visualization of Model Membrane Remodeling by α-Synuclein Fibrillization.

The interaction of α-synuclein (αS) with membranes is thought to be critical in the etiology of Parkinson's disease. Besides oligomeric αS aggregates that possibly form membrane pores, the aggregation of αS into amyloid fibrils has been reported to disrupt membranes. The mechanism by which aggregation affects the integrity of membranes is, however, unknown. Here, we show that whereas mature αS fibrils only weakly adhere to POPC/POPG giant unilamellar vesicles (GUVs), fibrillization of αS on the membrane results in large-scale membrane remodeling. Fibrils that grow on the vesicle surface stiffen the membrane and make the initially spherical membrane become polyhedral. Additionally, membrane-attached fibrils extract lipids. The lipid extraction and membrane remodeling of growing fibrils can consume the complete bilayer surface and results in loss of vesicle content. These observations suggest that there are several mechanisms by which growing fibrils can disrupt membrane function.

Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.

α-Synuclein Oligomers Stabilize Pre-Existing Defects in Supported Bilayers and Propagate Membrane Damage in a Fractal-Like Pattern.

Phospholipid vesicles are commonly used to get insights into the mechanism by which oligomers of amyloidogenic proteins damage membranes. Oligomers of the protein α-synuclein (αS) are thought to create pores in phospholipid vesicles containing a high amount of anionic phospholipids but fail to damage vesicle membranes at low surface charge densities. The current understanding of how αS oligomers damage the membranes is thus incomplete. This incomplete understanding may, in part, result from the choice of model membrane systems. The use of free-standing membranes such as vesicles may interfere with the unraveling of some damage mechanisms because the line tension at the edge of a membrane defect or pore ensures defect closure. Here, we have used supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPC/POPS) to study the membrane damage caused by αS oligomers. Although αS oligomers were not able to initiate the disruption of POPC/POPS vesicles or intact SLBs, oligomers did stabilize and enlarge pre-existing SLB defects. The increased exposure of lipid acyl chains at the edges of defects very likely facilitates membrane-oligomer interactions, resulting in the growth of fractal domains devoid of lipids. Concomitant with the appearance of the fractal membrane damage patterns, lipids appear in solution, directly implicating αS oligomers in the observed lipid extraction. The growth of the membrane damage patterns is not limited by the binding of lipids to the oligomer. The analysis of the shape and growth of the lipid-free domains suggests the involvement of an oligomer-dependent diffusion-limited extraction mechanism. The observed αS oligomer-induced propagation of membrane defects offers new insights into the mechanisms by which αS oligomers can contribute to the loss in membrane integrity.

Oligomers of Parkinson's Disease-Related α-Synuclein Mutants Have Similar Structures but Distinctive Membrane Permeabilization Properties.

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Alpha-synuclein amyloid oligomers act as multivalent nanoparticles to cause hemifusion in negatively charged vesicles.

Multivalent membrane binding sites on the α-synuclein oligomer result in clustering of vesicles and hemifusion of negatively charged model membranes. These multivalent, biological nanoparticles are reminiscent of inorganic nanoparticles in their interactions with membranes. Alpha-synuclein oligomers induce lipid exchange efficiently, with fewer than 10 oligomers/vesicle required to complete hemifusion. No full fusion or vesicle content mixing is observed.

Amyloids of alpha-synuclein affect the structure and dynamics of supported lipid bilayers.

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71-82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.

Locally resolved membrane binding affinity of the N-terminus of α-synuclein.

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.

Nanophotonic control of the Förster resonance energy transfer efficiency.

We have studied the influence of the local density of optical states (LDOS) on the rate and efficiency of Förster resonance energy transfer (FRET) from a donor to an acceptor. The donors and acceptors are dye molecules that are separated by a short strand of double-stranded DNA. The LDOS is controlled by carefully positioning the FRET pairs near a mirror. We find that the energy transfer efficiency changes with LDOS, and that, in agreement with theory, the energy transfer rate is independent of the LDOS, which allows one to quantitatively control FRET systems in a new way. Our results imply a change in the characteristic Förster distance, in contrast to common lore that this distance is fixed for a given FRET pair.

Fabrication of cell container arrays with overlaid surface topographies.

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

Porous multilayer-coated AFM tips for dip-pen nanolithography of proteins.

A simple and novel method for fabricating nanoporous-structure-coated silicon nitride tips for dip-pen nanolithography (DPN) by using the layer-by-layer (LbL) technique has been developed. The pore sizes can be adjusted by treating the LbL films coated onto the amino-terminated self-assembled monolayer (NH(2)-SAM)-functionalized AFM tip surface with a base solution for different periods of time. This hydrophilic porous material can absorb biomolecules easily and also provides a larger-volume ink reservoir compared with a bare silicon nitride tip. Proof-of-concept of the porous AFM tip is demonstrated by using fluorescent proteins as ink molecules to fabricate protein patterns at the micrometer and submicrometer length scales.

Temperature-modulated quenching of quantum dots covalently coupled to chain ends of poly(N-isopropyl acrylamide) brushes on gold.

A thermo-responsive polymer/quantum dot platform based on poly(N-isopropyl acrylamide) (PNIPAM) brushes 'grafted from' a gold substrate and quantum dots (QDs) covalently attached to the PNIPAM layer is presented. The PNIPAM brushes are grafted from the gold surface using an iniferter-initiated controlled radical polymerization. The PNIPAM chain ends are functionalized with amine groups for coupling to water-dispersible COOH-functionalized QDs. Upon increasing the temperature above the lower critical solution temperature (LCST) of PNIPAM the QD luminescence is quenched. The luminescence was observed to recover upon decreasing the temperature below the LCTS. The data obtained are consistent with temperature-modulated thickness changes of the PNIPAM layer and quenching of the QDs by the gold surface via nonradiative energy transfer.

Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices.

The putative function of the Parkinson's disease-related protein alpha-Synuclein (alphaS) is thought to involve membrane binding. Therefore, the interaction of alphaS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of alphaS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but alphaS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative-charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of alphaS. Thus, the interaction of alphaS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of alphaS to membranes. These findings contribute to our understanding of the details and structural consequences of alphaS-membrane interactions.

Creating nanopatterns of His-tagged proteins on surfaces by nanoimprint lithography using specific NiNTA-histidine interactions.

Directed assembly of the DsRed FT protein is demonstrated on self-assembled monolayers (SAMs) on silicon substrates patterned by nanoimprint lithography. Initially, the DsRed protein is attached using electrostatic interactions on both topographical (polymer) templates with an amino functionalization and on chemically patterned (flat) substrates. In a second experiment, a patterned NiNTA SAM is used in order to attach the DsRed FT protein via supramolecular interactions, taking advantage of the histidine functionalization of the DsRed FT protein. The NTA SAM is formed on silicon oxide using a multistep covalent process. Patterning of the NTA SAM is performed using nanoimprint lithography. The DsRed FT protein is attached on the patterned NTA layer after treating this with a Ni(II) solution. Moreover, the histidine-NiNTA binding may be reversed by removing the Ni using EDTA or by competition using imidazole. The regeneration and reuse of the substrate by subsequently attaching and removing two different histidine-functionalized proteins from the patterned NTA is shown by fluorescence microscopy.

Micromechanical bending of single collagen fibrils using atomic force microscopy.

A new micromechanical technique was developed to study the mechanical properties of single collagen fibrils. Single collagen fibrils, the basic components of the collagen fiber, have a characteristic highly organized structure. Fibrils were isolated from collagenous materials and their mechanical properties were studied with atomic force microscopy (AFM). In this study, we determined the Young's modulus of single collagen fibrils at ambient conditions from bending tests after depositing the fibrils on a poly(dimethyl siloxane) (PDMS) substrate containing micro-channels. Force-indentation relationships of freely suspended collagen fibrils were determined by loading them with a tip-less cantilever. From the deflection-piezo displacement curve, force-indentation curves could be deduced. With the assumption that the behavior of collagen fibrils can be described by the linear elastic theory of isotropic materials and that the fibrils are freely supported at the rims, a Young's modulus of 5.4 +/- 1.2 GPa was determined. After cross-linking with glutaraldehyde, the Young's modulus of a single fibril increases to 14.7 +/- 2.7 GPa. When it is assumed that the fibril would be fixed at the ends of the channel the Young's moduli of native and cross-linked collagen fibrils are calculated to be 1.4 +/- 0.3 GPa and 3.8 +/- 0.8 GPa, respectively. The minimum and maximum values determined for native and glutaraldehyde cross-linked collagen fibrils represent the boundaries of the Young's modulus.

Parkinson's Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics.

Binding of human α-Synuclein, a protein associated with Parkinson's disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50-100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.

α-Synuclein oligomers distinctively permeabilize complex model membranes.

α-Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic neurons in Parkinson's disease. The toxicity mechanism of α-synuclein oligomers likely involves membrane permeabilization. Even though it is well established that α-synuclein oligomers bind and permeabilize vesicles composed of negatively-charged lipids, little attention has been given to the interaction of oligomers with bilayers of physiologically relevant lipid compositions. We investigated the interaction of α-synuclein with bilayers composed of lipid mixtures that mimic the composition of plasma and mitochondrial membranes. In the present study, we show that monomeric and oligomeric α-synuclein bind to these membranes. The resulting membrane leakage differs from that observed for simple artificial model bilayers. Although the addition of oligomers to negatively-charged lipid vesicles displays fast content release in a bulk permeabilization assay, adding oligomers to vesicles with compositions mimicking mitochondrial membranes shows a much slower loss of content. Oligomers are unable to induce leakage in the artificial plasma membranes, even after long-term incubation. CD experiments indicate that binding to lipid bilayers initially induces conformational changes in both oligomeric and monomeric α-synuclein, which show little change upon long-term incubation of oligomers with membranes. The results of the present study demonstrate that the mitochondrial model membranes are more vulnerable to permeabilization by oligomers than model plasma membranes reconstituted from brain-derived lipids; this preference may imply that increasingly complex membrane components, such as those in the plasma membrane mimic used in the present study, are less vulnerable to damage by oligomers.

Imaging the static dielectric constant in vitro and in living cells by a bioconjugable GFP chromophore analog.

A fluorescent probe structurally similar to the GFP chromophore is demonstrated to report the local static dielectric constant. This probe can be chemically functionalized for selective targeting at the intracellular level.

Kinetic measurements give new insights into lipid membrane permeabilization by α-synuclein oligomers.

Interactions of oligomeric aggregates of the intrinsically disordered protein α-synuclein with lipid membranes appear to play an important role in the development of Parkinson's disease. The permeabilization of cellular membranes by oligomers has been proposed to result in neuronal death. The detailed mechanisms by which α-synuclein oligomers permeabilize lipid bilayers remain unknown. Two different mechanisms are conceivable. Oligomers may either insert into membranes forming pores through which small molecules can cross the membrane or their interaction with the membrane may disorder the lipid packing, giving rise to membrane defects. Here we show, using kinetic leakage measurements, that α-synuclein oligomer induced impairment of membrane integrity is not limited to the formation of permanent membrane spanning pores. Fast membrane permeabilization could be observed in a fraction of the large unilamellar vesicles. We have also observed, for the first time, that α-synuclein oligomers cause an enhanced lipid flip-flop. In neuronal cells, most of the α-synuclein is not expected to be present in an oligomeric form, but as monomers. In our in vitro experiments, we find that membrane bound monomeric α-synuclein can only delay the onset of oligomer-induced membrane permeabilization, implying that α-synuclein monomers cannot counteract oligomer toxicity.