Investigating the structure of alpha-synuclein using mass spectrometry

Abstract

The pathological hallmark of Parkinson’s disease (PD) are Lewy bodies (LBs), insoluble inclusions observed in dopaminergic neurons in the brains of PD patients.

The main protein component of LBs is alpha-synuclein (αSyn), a 140-residue intrinsically disordered protein. Around 10% of PD cases are associated with genetic mutations, including single-point variants of αSyn; and under physiological conditions, the protein carries a constitutive N-terminal acetylation modification.

Thus, the structural and functional properties associated with αSyn are vitally important to investigate in order to further understanding of how this protein contributes to disease.

Here, we report the first full biophysical characterisation and cross-comparison of wild-type (WT) αSyn and a panel of PD-associated variants, using circular dichroism spectroscopy, fluorescence aggregation assays, native mass spectrometry, and ion mobility-mass spectrometry (IM-MS). We uncover that the different variants occupy different conformational spaces in the gas phase, and that the monomeric proteins do not exhibit a completely unfolded structure, as expected for a disordered protein.

The N-terminal acetylated variants of αSyn are a more physiologically relevant model, with the constitutive modification being important for the formation of a transient N-terminal α-helix which mediates the binding of αSyn to various cellular lipid membranes. Here, we studied the effect of this modification on the structure and function of the panel of αSyn variants.

The native state of αSyn is highly disputed, with several reports proposing the existence of naturally occurring multimers of the protein that may be involved in the physiological role of αSyn. However, these species have not been studied extensively and their role is not fully understood. Here, IM-MS and native top-down fragmentation using electron capture dissociation were used to elucidate the structural properties associated with a stable dimeric species of αSyn. In addition, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal to noise ratio and/or reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. Using this new workflow, we were able to infer structural information about this previously unreported αSyn dimer interface.

Overall, this body of work aims to highlight native mass spectrometry as an important tool for investigating challenging structural biology problems, such as intrinsically disordered and aggregating proteins. This work also represents a comprehensive structural study of physiologically relevant WT αSyn and PDassociated variants in the gas phase. We showed that the N-terminal acetylation of αSyn and various PD-associated variants alters all aspects of structure and function of the protein, highlighting the need for physiologically relevant modifications to be used in in vitro studies. We also provide conclusive evidence for a C-C terminal interaction between the monomer units forming the stable dimeric species of αSyn, presenting important structural data on endogenous αSyn multimers.