Study of the structural characterization of TMEM165

It is widely recognized that proteins are composed of amino acids that assemble to form polypeptide chains, which then fold into 3D structures through various interactions. However, the precise 3D structure of most proteins remains uncertain or unknown. This is the case of TMEM165, a membrane protein located in the Golgi apparatus of human cells. This cation transporter of Ca2+, Mn2+ and potentially H+ has been linked to a rare genetic disorder called Congenital Disorder of Glycosylation Type II when mutated. The purpose of this master thesis was thus to perform a structural characterization of TMEM165 by determining its oligomerization state and the presence or not of intra or intermolecular disulfide bonds. To do so, the project begun with the production and purification of Δ78TMEM165 in insect cells (Sf9) using a 10-histidine tag or a strep-tag. After that, the quality of the purifications was assessed by analyzing Western Blots and SDS-PAGE gels. In these blots a band between 55 and 70 kDa was hypothesized to represent a dimer of TMEM165. In addition to that, contaminants could still be detected, even after a double purification (10His-Strep). To eliminate the remaining impurities, an optimization of the purification process has been performed. Unfortunately, the contaminants could not be further separated from TMEM165. This led to the hypothesis that these contaminants could be part of a complex with TMEM165. Intact infusion mass spectrometry analyses were also carried out to obtain information about the oligomeric state of TMEM165, but the purity of our sample was not sufficient to obtain good spectra. After that, the bands of the resulting SDS-PAGE gels were sent to mass spectrometry to find out the presence of potential insect interactants in the purified samples. Finally, after the purifications, a portion of the eluates was treated with different reducing agents to assess the presence of disulfide bonds. Nevertheless, no proof of disulfide bond could be observed in Western Blots. Following purification improvements and structural analyses would represent a major step towards a better knowledge of TMEM165 and its role in Congenital Disorder of Glycosylation Type II.