Quantitatively Evaluate the Orientation of the Axis of α-Helical α-Synuclein(61-95) in Monolayer with Residue Level Resolution by pMAIRS

Significant scientific attention has been spent on the elucidation of protein’s structure to explain its function. Lots of proteins stay around cell membrane or vesicles to facilitate cellular metabolism. These proteins usually form monolayer structure around cell membrane/vesicles and therefore cause challenges for the measurements of X-ray crystallography and NMR, which cannot provide high resolution results for proteins in monolayer. To address this issue, surface FT-IR techniques were developed to quickly determine the tilt angle of various vibrations. For α-helical peptides/proteins interacting with membrane, the tilt angle of the axis is the key piece of information to address whether the α-helical protein forms transmembrane structure. Among surface FT-IR techniques, p-polarized Multiple Angle Incidence Resolution Spectroscopy (pMAIRS) was recently developed with the capability to quantitatively determine the tilt angle of a vibration, especially the tilt angle of the axis of α-helical protein by the amide I band. It is worth noting that the traditional FT-IR can only provide “low-resolution” results. In this paper, the nonamyloid component (NAC), which spans residues 61–95 of α-synuclein, was used as model peptide in this paper, because α-synuclein interacts with the amphiphilic membrane structure and it directly relates with both Parkinson’s disease and Alzheimer’s disease. A ¹³C isotopic label was introduced into the backbone carbonyl at the position of 93G in the NAC sequence, which formed α-helix in the monolayer at the amphiphilic interface. The ¹³C isotopic labeled carbonyl generated a new ¹³C amide I band, which was then analyzed by pMAIRS to reveal the tilt angle of the axis at 93G to be ~ 0 º. As compared with the overall average tilt angle of the axis of the whole peptide around 30.1 º, 0 º revealed that the axis at 93 G is very parallel to the interface and those at other positions may be more perpendicular. To the best of our knowledge, this is the first report that quantitatively determines the axis of α-helix of a protein in residue level even in monolayer structure. Therefore, pMAIRS can supplement X-ray crystallography and NMR to address a protein’s structure even in monolayer with residue level resolution.