Using Site-Specific Vibrational Spectroscopy to Determine Structure of Membrane-Bound N-Terminally Acetylated Alpha-Synuclein

Abstract

Despite a demonstrated connection to the etiology of Parkinson's Disease (PD), the structure and function of the neuronal brain protein alpha-synuclein (aS) remain elusive and confuse attempts to elucidate the link between the protein and PD. Structure determination is difficult because aS is an intrinsically-disordered protein in solution and because the recently ubiquity of N-terminal acetylation in vivo questions the relevance of many previous experiments that used unacetylated aS. Most previous research on aS membrane-bound structure, primarily using NMR, also lacks the ability to directly assess conformational dynamics on very short timescales. Site-specific vibrational spectroscopy presents a new technique that could reveal new details of the membrane-bound conformational distribution of aS. Here, we first attempted to obtain N-terminally acetylated aS (Nt-Ac aS) via the coexpression of human aS and yeast N-acetyltransferase B (NatB) in E. coli cells but failed to complete the acetylation. An adapted expression protocol using a sequence of increasing-volume cultures (according to the lab of David Eliezer) and E. coli cells pretransformed to express NatB before transformation of aS (according to the lab of Elizabeth Rhoades) successfully yielded several single-cysteine mutants of N-terminally acetylated aS, and we converted the thiol to the SCN vibrational probe group. We used infrared spectroscopy to measure solvent exposure of the SCN group at several sites along membrane-bound unacetylated and acetylated aS and compared the results. SCN-labeled variants of aS should be useful to explore aS's membrane-bound conformations in contact with a wide variety of lipid systems.