Isolation, functional characterization and crystallization of Aq_1259, an outer membrane protein with porin features, from Aquifex aeolicus

The “hypothetical protein” Aq_1259 was identified by mass spectrometry and purified from native membranes of Aquifex aeolicus. It is a 49.4 kDa protein, highly homologous (> 52% identity) to several conserved hypothetical proteins from other bacteria. However, none of these proteins has been characterized using biochemical or electrophysiological techniques. Based on the sequence and circular dichroism spectroscopy, the structure of Aq_1259 is predicted to be a β-barrel with 16 β-strands. The strands with loops and turns are distributed evenly through the entire sequence. The function of Aq_1259 was analyzed after incorporation into a lipid bilayer. Electrophysiological measurements revealed a pore that has a basic stationary conductance of 0.48 ± 0.038 nS in a buffer with 0.5 M NaH2PO4 at pH 6.5 and 0.2 ± 0.015 nS in a buffer with 0.5 M NaCl at pH 6.5. Superimposed on this is a fluctuating conductance of similar amplitude. Aq_1259 could be crystallized. The crystals diffract to a resolution of 3.4 Å and belong to space group I222 with cell dimensions of a = 138.3 Å, b = 144.6 Å, c = 151.8 Å.

► “Hypothetical protein” Aq_1259 was identified and purify to homogenous. ► Bioinformatics analysis revealed that it was a β-barrel with 16 β-strands. ► The β-barrel structure was confirmed by circular dichroism spectroscopy. ► Its function was analyzed in a lipid bilayer which showed porin-like features.