Alteration of the Conformational Dynamics of a DNA Hairpin by α‐Synuclein in the Presence of Aqueous Two‐Phase Systems

Abstract The effect of an amyloidogenic intrinsically disordered protein, α‐synuclein, which is associated with Parkinson's disease (PD), on the conformational dynamics of a DNA hairpin (DNA‐HP) was studied by employing the single‐molecule Förster resonance energy transfer method. The open‐to‐closed conformational equilibrium of the DNA‐HP is drastically affected by binding of monomeric α‐synuclein to the loop region of the DNA‐HP. Formation of a protein‐bound intermediate conformation is fostered in the presence of an aqueous two‐phase system mimicking intracellular liquid‐liquid phase separation. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved. Hence, in addition to toxic amyloid formation, α‐synuclein may alter expression profiles of disease‐modifying genes in PD. Furthermore, these findings might also have significant bearings on the understanding of the physiology of organisms thriving at high pressures in the deep sea.

DNA-protein interactions and the conformational stability of DNA are crucial for normal cell function. [1] It has been reported that neuro-proteins, such as a-synuclein( a-Syn), which is directly linked with Parkinson's disease, and other amyloidogenic proteins may be involved in the conformational stability of DNA. [2][3][4][5] However,t ow hat extenta nd how the local conformational dynamics of DNA is altered upon binding with such neuro-proteins is still largely unknown. [5,6] Altered gene expression in most of the human diseases like Parkinson's disease (PD) and Alzheimer's disease (AD) is observed due to confor-mationalc hanges of the DNA. [7,8] It has been found that the conformation of the DNA in PD-affected human postmortem brain cells is alteredf rom the normalB -form to an alteredBconformation, and in AD, ac onformational transition occurs from the B-form to the Z-form. [7,8] The factors responsible for these conformational fluctuations are still not clear.A sa-Syn acts as at ranscription modulator, [9] it has asignificant influence on gene expression profiles, which cause also neuronal cell dysfunction. [9][10][11] DNA has also been reportedt os timulate amyloid formation (fibrillization) in vitro. [10] Remarkably,only alimited number of studies has been carriedo ut on the interaction between monomeric a-synucleina nd nucleic acids. In this respect, the conformational landscape of non-canonicalD NA structures, such as DNA hairpins (DNA-HPs), is of particular interest because they regulate gene expression alongw ith playing as ignificant role in DNA recombination and transposition. [12][13][14] Hence, these structures are very attractive from abiophysical point of view for investigating the conformationald ynamics of a-Syn-DNA interactions, [15,16] which is the purpose of this study.
In recenty ears, it has become increasingly clear that the physicochemical properties of biomolecules maya lso get significantly alteredi nt he crowdedi nv ivo situation when compared to those in dilute solution. [17][18][19][20][21] Furthermore, regulation of biochemical processes is frequently achieved through the compartmentalization of the cellularm ilieu. In this respect, non-membrane bound compartments consistingo fp hase-separated liquid-liked roplets of proteins and protein-RNA mixtures have been shown to be of particular importance,w hich are supposed to significantly alter cellular reactions as well. [22][23][24] Due to the lack of ap hysical barrier,s uch as al ipid membrane, these liquid condensates are able to exchange their components rapidlyw ith the surroundingm edium. The effect of liquid-liquid phase separation (LLPS) as observed in artificial aqueoust wo-phase systems (ATPS) on the conformational dynamics of biomolecules, including DNA,i sh ardly explored, however, [25] and has therefore been included in this study.
To yield am olecular level understanding of the conformational dynamics of a-Syn interacting with DNA hairpins (DNA-HPs), single-molecule Fçrsterr esonance energy transfer (smFRET) experiments have been carried out. SmFRET has emerged as one of the most powerful techniques for elucidating dynamical properties of biomolecules as it provides mechanistic information on the underlying molecular level interactions, which are otherwise averaged out in ensemble-based experiments.
[a] Dr.S.K.Mukherjee, + J.-M. Knop As ac onsequence of intra-strand hybridization between complementary sequences, oligonucleotides are able to form hairpin structures, where the duplex region generated upon hybridization forms the stem of the hairpin and the nucleotides in between form the hairpin loop. In solution, equilibrium is established between oligonucleotides in hairpin conformation and oligonucleotides that are not self-hybridized, establishing an open structure. The chemical environments urrounding the DNA-HPi se xpected to influencei ts hybridization abilities extensively.H ere, we focus on applicationso fs mFRET to study the conformational dynamics of aD NA-HPw hich contains 32 adenine residues in the loop ( Figure 1A)u pon interacting with the disordered monomeric protein a-Syn in neat buffer andu nder liquid-liquidp hase separation conditions.
To reveala dditional mechanisticd etails of the interaction process,p ressure modulationh as been used as well. Next to its biological relevance for understandingt he physiology of deep-sea organismsl iving at high hydrostatic pressure (HHP) conditions of severalh undreds of bar, pressure-axis experiments have been shown to enablem odulationo fi ntra-and inter-molecular interactions and reveald etails of the freeenergy and conformational landscape of biomolecules. [26][27][28][29][30] According to Le Châtelier's principle, an increaseo fp ressure shifts an equilibrium towards the state that occupiest he smallest possible overall volume. The pressuree ffect on ag iven reversible reactionf ollows the relation( dlnK/dp) T = ÀDV/(RT), where K is the pressure-dependente quilibrium constant and DV is the associated volumec hange of the transition, which depends sensitively on the packing and hydration properties of the biomolecule. [26][27][28][29][30][31] Figure 1B shows smFRET measurements of the DNA-HP under freely diffusing conditions in the presence of different concentrations of monomeric a-Syn (for details of sample preparation and technical aspects, please refer to the Supporting Information). The FRET histograms of the DNA-HP display two FRET distribution peaks. They are relatedt oc onformations with differents eparations, R,o ft he two attached dyes and thus different FRET efficiencies, E,a sE = R 6 0 /(R 6 0 + R 6 ): The Fçrster radius, that is, the distance at which 50 %o ft he excited donor molecules will be deactivated, is R 0 = 6.5 nm for the fluorophores used (Atto 550 and Atto 647 N). [25,32] The two peaks are located at E % 0.3 and E % 0.9, respectively.T he peak at the lower FRET efficiency represents the open state, where the donora nd acceptor distance is maximum, and the higherF RET efficiency represents the closed conformation of the DNA-HP, where donor and acceptor are at ap roximal distance. At ambient temperature and pressure conditions, the ratio of the open to closed state is approximately 0.81. [33] As shown in Figure 1B Figure 1C).
Recently,i th as been shown that HHP application on the DNA-HP in neat buffer solution gradually populates the low-FRET distribution species( E % 0.3), which is due to the unfolding of the DNA-HP. [25,32,33] Figures 1D and Figure 2d isplay the pressuree ffecto nt he conformational states of the DNA-HP in the presence of different concentrations of a-Syn (the smFRET histograms are shown in Figure S1). From these and literature data for the DNA-HPs ystem in neat buffer solution (Figures 3  and 4i nR ef. [25]), it is evident that the open conformation (unfolded state) becomes more populated with increasing pressureinthe absence [25] and presence of a-Syn (Figure 2).The intermediate conformationo ft he DNA-HPr emains essentially unaffected by pressure, pointing to formation of ac ompact, void-free and pressure-stable DNA-HP-a-Syn complex. Ta king into account the open and closed conformationo nly,w ithin the accuracyo ft he data, av olumec hange in the ordero ft hat  [25] can be calculated for unfolding of the DNA-HP.
LLPS is known to significantly modulate an array of physiological processes, including protein dynamics, folding, aggregation,a nd activity,t on ame af ew. [23,25,36,37] Here, we used an artificial ATPS composed of ad extran (11wt%)a nd PEG (11wt%)m ixture to mimic intracellular LLPS conditions. The advantage of this system is the absence of ap ressure effect on the stability of the ATPS itself. [25] In ap revious study,w ec ould show that the DNA-HPp artitions insidet he dextran-rich droplets of the ATPS (Figure 1i nR ef. [25]) and that the ATPS significantly modulates the pressure-dependence of the conformational dynamics of the DNA-HP. [25] Partitioning of the DNA-HP in the ATPS markedlyc ounteracts the effect of pressure-induced destabilization of the closed conformation. [25] Conversely,h ere, in the presence of a-Syn at all concentrations measured (from 50 to 200 mm), the FRET efficiency distribution data of the DNA-HP at 50 mm a-Syn concentration exhibits ap ronouncedm aximum at E % 0.60, indicating stabilization of the intermediate conformation ( Figure3and Figure S1), which reaches even % 90 %o ft he overall distribution, independentoft he concentrationo fa-Syn. Further, this population remains essentially unchanged upon pressure application. Upon addition of a-Syn, partitioning of the DNA-HP is changing from the dextran-rich droplet phase to the PEG-rich phase, which contains about 14 %P EG. [36] As revealed by complementarym easurements in 30 wt % dextran andi n1 1wt% PEG, serving as simple crowding agents only,a tc oncentrations near to those encountered in the different phases of the ATPS (Figures S2 and S3), the ratio of folded and intermediate conformations is shifted to the folded species when compared with the corresponding ATPS data (Figure 3). The change from approximately 50 %f olded and 30 %i ntermediate states in the neat crowders to about 0% folded and 90 %i ntermediate conformers in the PEG-rich phase of the ATPS is quite dramatic. These data suggest that the molecular properties of the PEG-rich phase in the ATPS are differentf rom those of the crowding agents alone.S uch differences of DNA-protein interactions in the presence of droplet condensates of the ATPS compared to simple crowding agents may be of importance for understanding biochemical processes in cellulo.
Intrinsically disordered proteins that bind to nucleic acids with high affinity represent ag enetically controllable strategy for modulating the conformation and dynamics of nucleic acids in ac ellular environment. Collectively,o ur data reveal that a-Syn is able to alter the structuralp attern and formation of DNA-HPs, thereby changing the conformational dynamics and stabilizing altered conformations, which may have significant biological effects on the gene expression patterns.
The amino acid sequence of a-Syn can be partitioned into three regions, namely,1 )the N-terminal region (residues 1-60) which contains four regions of 11 imperfect repeatsw ith the KTKGEVc onsensus sequence and is responsible for interaction with negatively charged biomolecules such as lipids and nucleic acid backbones,2 )the central NAC region (residues 61-95), showinghigh sequence hydrophobicity that has been implicated in the aggregation and amyloid formation of the protein, and 3) the C-terminal region (residues 96-140), whichi sv ery acidic, containing ten glutamate and fivea spartate residues and predominantly hydrophilic, its role still not being clearly understood. [37][38][39] It has been shown that a-Syn is able to bind to DNA double strandsa nd that upon protein binding the DNA persistence length increases, but base-pairingd oes not seem to be disturbed. [10] However,i ts interaction with oligonucleotides and noncanonical DNA structures is still largely unknown. We show that also unaggregated monomeric a-Syn has as evere effect on the dynamical eventso fD NA hairpins, largely affecting their conformational transition between the closed and the open, non-selfhybridized conformation, and inducinga lternate conformations upon protein binding.T his is in line with results of Naraynan et al., [40] showing that substates of nucleic acids are separated by low free energyb arriersi narather flat and broad energys urface, that is, support the notion that the folding free-energy landscape of DNA-HPs is ar ugged one rather than aw ell-defined two-state system. As the concentration of a-Syn is increased, non-native conformationso ft he DNA-HP becomep rogressively populated. These additional conformational states are formed by strong interactions with a-Syn, most likely through the N-terminus of the protein and the phosphate backbonea long with aw eakening of base stacking by the NAC region through hydrophobic interactions ( Figure 4). Furthermore, the pressure-dependentm easurements reveal stronga nd compact (void-free) binding of a-Syn  In the presence of the ATPS, stabilization of the altered protein-bound DNA-HPc onformation is observed, reaching population levels of approximately 90 %o ft he overall distribution ( Figure 3). This might be due to the fact that the conformational subspace of the DNA-HPb ecomes strongly restricted in the phase-separated dropletp hase. Owing to the strong excluded volume effect and possibly also additional enthalpic interactions with the constituents of the condensate,c onformational fluctuations of the DNA-HP become restricted, favoring compacts tates and hence ad ecrease of the population of the open state. Upon pressurization, the distribution of conformations remains unchanged,r eflecting the notion that compact conformations with high packing efficiency and hence small partial volumes are favored.
In conclusion, smFRET data as shown here are able to provide au nique spectrals ignature for capturing local conformational changes, thereby enabling one to decipher non-specific interactions between nucleic acids and proteins. Furthermore, we think that such approach using smFRET spectroscopy in concert with pressure modulation in studies of DNA-protein interactions, in the absence and presence of liquid condensates inducedb yL LPS, provide av aluable tool to infer ab asic comprehension of hidden mechanismso fc ell science. They can help to explore the conformational andf ree energy landscape of biomolecular systemsi ncludingt he existence of conformationals ubstates inducedb yc hanging the solution conditions, that is, the cellular milieu, which are not easily accessible by otherm eans. Notably, learninga bout pressure effects on such biomolecular assemblies can also help us to gain ab etter appreciation of pressure effects on DNA-protein interactions and gene regulation in general, as, for example, relevant for a better understanding of the physiology of organisms thriving at multi-hundred bar pressures in the deep sea.