Effects of Detergent on α-Synuclein Structure: A Native MS-Ion Mobility Study

The intrinsically disordered protein α-synuclein plays a major role in Parkinson’s disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.


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Similar to α-syn, β-lac has a high binding capacity for the neutral detergent DDM. β-lac is known to 24 have a hydrophobic cavity where ligands can bind, for example DDM [1]. This might be an indication 25 that the two proteins interact with this detergent in a similar way and mainly through hydrophobic 26 interactions. The binding capacity of β-lac for PC-14 is however significantly lower. In general, for β-lac 27 there seems to be no effect of the chain length of the zwitterionic detergents tested and it binds these 28 detergents poorly. As for α-syn, when CTAB is present only binding of Brand not the cationic detergent 29 could be detected to β-lac. For the anionic detergents DSand CDC -, binding capacity seems to be similar 30 for both proteins with up to two molecules bound for α-syn and three for β-lac. β-lac can be embedded 31 2 in e.g. SDS micelles in a denatured conformation, so it might be that binding of detergent molecules is 32 retained from the micelle rather than individual interacting detergent molecules [2]. For GDCthere is 33 however a significant difference in binding capacity with six molecules bound to α-syn and three for β-34 lac, the latter is similar to the maximum binding stoichiometry of the other anionic detergents for β-lac.

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Detergent binding capacity and stoichiometry 37 Most of the detergents that were included in this study bind to α-syn and their bound states can be 38 clearly detected. For Triton X-100, however, the intensity of the bound states is very low. Supplementary 39 Figure 2 shows the intensity of bound and unbound states of the 7+ (A) and 12+ (B) charge state of α-40 syn monomers when 2x CMC Triton X-100 is present (red spectrum). As the mass of Triton X-100 can 41 differ according to the number of ethylene oxide groups in the tail of the molecule, more than one peak 42 can be found for the bound state with one Triton X-100 molecule bound. The difference of 44 Da, one 43 ethylene oxide unit, is indicated by a bow. For the 12+ charge state, the intensity of the bound states is 44 higher compared to the 7+ charge state. For this higher charge state also more peaks that can be 45 attributed to Triton X-100 molecules with different number of ethylene oxide groups are found. This 46 might indicate that the longer the chain of Triton X-100 is, the more it prefers to bind to more extended 3 When CTAB is added to α-syn or β-lac, no binding can be detected of the intact detergent or the cationic 54 group with detergent properties. Only Bris found to bind to both proteins as is shown in 55 Supplementary Figure 3. The top part shows the full MS spectra of α-syn (A) and β-lac (B) without 56 (black line) and with (red line) 0.2x CMC CTAB present. For β-lac the presence of CTAB results in partial 57 denaturation which results in a shift of the charge state distribution, as the charge state range is 58 increased from 6+ to 16+ instead of 6+ to 9+. CTAB is known as a harsh detergent that can disrupt inter-59 and intramolecular interactions, leading to denaturation [3]. This is similar to what was expected for 60 SDS, however, we did not see similar denaturing effects on β-lac when SDS was present. As α-syn is 61 already partially denatured in its native state, we don't see this denaturing effect of CTAB with α-syn.

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The bottom part of the figures zooms in on specific parts of the MS spectra and Brbinding is here

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Conformational selectivity of detergent binding 74 As can be seen in the previous figure for β-lac, the addition of detergents could lead to a shift in charge 75 state distribution. Supplementary Figure 4 indicates if the intensities of the 7+ and 8+ charge state of α-76 syn increase or decrease when a specific detergent is present. Every spectrum was first normalised to 77 the intensity of the most intense peak. All stoichiometries (bound and unbound) were then summed 78 and compared to the unbound 7+ or 8+ charge state of the control, respectively, as the 100% reference.

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In the presence of detergents the range of observed charge states in the mass spectra did not shift, in 80 4 each case charge states 5+ or 6+ up to 18+ were detected as was seen in Figure

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As it is known that Ca 2+ plays an important role in the interaction between biological membranes and

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In the control panel A a gradual compaction when more Ca 2+ ions bind, with a maximum of three ions 164 bound, is observed as described in earlier studies [4,5]. It can be seen that for PC-14, the binding of 165 additional Ca 2+ ions together with detergent molecules leads to the formation of additional very compact 166 conformations as was seen for DDM in Figure 3F in the main text. However, the resulting 167 conformational pattern is different which indicates a specific effect related to a certain detergent. When 168 CDCbinds, there is an intensity shift towards more compact conformations however only 169 conformations which were already present before. This indicates that for neutral and zwitterionic  is similar to what we saw for the 7+ charge state. For PC-14, of which the protein-detergent interaction 208 was broken at too low trap CE voltages to be able to detect any stabilising effects for the 7+ charge state,

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we see a destabilisation of the three most compact conformational families for the 8+ charge state. As it 210 wasn't clear from the CCS plots alone if and which conformational effects could occur when this 9 detergent is present, it is interesting to see that it has a preference for stabilising of the more extended 212 conformation. In contrast to the drastic stabilizing effect that was seen for the 7+ state, DSdoes not seem