Abstract
Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson’s disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å) layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could potentially be related to its physiological functions as well as to the onset of Parkinson’s diseases.
Highlights
Introduction aSynuclein is a small 140-residue protein that is highly conserved in vertebrates and is preferentially expressed in presynaptic nerve terminals in various regions of the brain [1,2,3]
A-synuclein aggregation is implicated in Parkinson’s disease and in a number of other neurodegenerative disorders [9,10,11]. a-Synuclein belongs to a unique class of proteins known as natively unfolded or Intrinsically Disordered Proteins (IDPs), which has no persistent structure and confronts the conventional sequence-structure-function paradigm [12,13,14,15,16]. aSynuclein consists of three distinct modular domains: (i) positively charged N-terminal region from residues 1–60 has propensity to bind to membranes; (ii) hydrophobic NAC-domain from residues 61–95 is responsible for aggregation [17]; (iii) negatively charged Cterminal region from residues 96–140 is highly flexible and facilitates interaction with Ca2+ ion and other molecules
Studies using a diverse array of biophysical tools involving circular dichroism (CD) [23,24,25], nuclear magnetic resonance (NMR) [26,27,28,29,30], electron paramagnetic resonance (EPR) [31,32,33,34,35,36] and fluorescence [37,38,39,40,41] have shown that a-synuclein undergoes a profound conformational transition from a random coil state to a-helical state upon interaction with membrane mimetic such as sodium dodecyl sulfate and anionic phospholipid membranes
Summary
Synuclein is a small 140-residue protein that is highly conserved in vertebrates and is preferentially expressed in presynaptic nerve terminals in various regions of the brain [1,2,3]. A-synuclein aggregation is implicated in Parkinson’s disease and in a number of other neurodegenerative disorders [9,10,11]. ASynuclein consists of three distinct modular domains: (i) positively charged N-terminal region from residues 1–60 has propensity to bind to membranes; (ii) hydrophobic NAC-domain (non-Ab component of Alzheimer’s disease amyloid) from residues 61–95 is responsible for aggregation [17]; (iii) negatively charged Cterminal region from residues 96–140 is highly flexible and facilitates interaction with Ca2+ ion and other molecules Recent single molecule experiments have provided direct and compelling evidence in favor of the two switchable conformational states [48,49,50,51]
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