Molecular Details of α-Synuclein Membrane Association Revealed by Neutrons and Photons

Abstract

α-Synuclein (α-syn) is an abundant neuronal protein associated with Parkinson’s disease that is disordered in solution, but exists in equilibrium between a bent- and an elongated-helix on negatively charged membranes. Here, neutron reflectometry (NR) and fluorescence spectroscopy were employed to uncover molecular details of the interaction between α-syn and two anionic lipids, phosphatidic acid (PA) and phosphatidylserine (PS). Both NR and site-specific Trp measurements indicate that penetration depth of α-syn is similar for either PA- or PS-containing membranes (~9–11 Å from bilayer center) even though there is a preference for α-syn binding to PA. However, closer examination of the individual Trp quenching profiles by brominated lipids reveal differences into local membrane interactions especially at position 39 where conformational heterogeneity was observed. The data also indicate that while W94 penetrates the bilayer as deeply as W4, W94 resides in a more polar surrounding. Taken together, we suggest the N- and C-terminal regions near positions 4 and 94 are anchored to the membrane, while the putative linker spanning residue 39 samples multiple conformations, which are sensitive to the chemical nature of the membrane surface. This flexibility may enable α-syn to bind diverse biomembranes in vivo.

Membrane remodeling and mechanics: Experiments and simulations of α-Synuclein

“…Probe-based experiments are typically used to measure the protein's partition depth. These methods include continuous-wave electron paramagnetic resonance (EPR) [36,37,103], and more recently, neutron reflectometry (NR) [104–106], site-specific Trp measurements [105,106], NMR paramagnetic relaxation (PREs) [107], and 1 H Overhauser dynamic nuclear polarization (ODNP), an NMR relaxometry method [38]. Fig.…”

Segmental Deuteration of α-Synuclein for Neutron Reflectometry on Tethered Bilayers

“…41 For this work, we chose an lipid composition of equimolar 1-palmitoyl-2-oleoyl- sn -3-glycero-phosphocholine and 1-palmitoyl-2-oleoyl- sn -3-glycero-phosphate (noted as PC/PA) because PC is the most abundant mammalian phospholipid, PA is the most strongly bound anionic lipid by α-syn, 30 and PC/PA has been well studied in our lab. 6-7, 35 Circular dichroism (CD) spectroscopy, which is highly sensitive to the presence of α-helical structure, 30, 35 was used to confirm membrane binding by the segmentally deuterated α-syn. CD titrations were performed with PC/PA lipid vesicles made by extrusion (average diameter ~ 90 nm).…”

Atomic-level description of protein–lipid interactions using an accelerated membrane model

“…ESR and DEER experiments on α S bound to bilayers instead were consistent with an extended helix model without a kink [144–147]. Interconversion between these two states had been hypothesized [36, 148–150], with special emphasis on the role of specific lipid interactions in regulating the equilibrium between the two populations [36, 151, 152]. Coarse-grained simulations sampled this conformational diversity in part [153, 154], but due to their resolution limitations could not identify specific interactions that anionic membranes might disrupt to allow this transition to take place.…”

“…Furthermore, due to the transient nature of the interaction between peripheral proteins and membrane lipids, crystallizing the membrane-bound complexes of peripheral proteins for X-ray analyses is exceedingly difficult. Other techniques, such as SAXS [16–18], EPR [19–21], NMR [22–24] including high–resolution field–cycling NMR [25–28], FRET [29, 30], fluorescence correlation spectroscopy [31–33], x–ray reflectivity [34, 35], neutron reflectometry [36, 37], and mutagenesis studies can bridge the gap, and provide low-resolution information on protein-lipid interactions such as determination of the binding face of the protein as it interacts with a membrane interface. However, without the guidance of a structural model of the protein-membrane complex, even when structures of constituent proteins are resolved, hypothesis-driven experimental investigations into the biological mechanisms are limited by the uncertainties inherent in missing the contribution of the membrane.…”