Isolation, functional characterization and crystallization of Aq-1259, an outer membrane protein with porin features, from Aquifex aeolicus

摘要

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 NaH 2PO 4 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 ? and belong to space group I222 with cell dimensions of a = 138.3 ?, b = 144.6 ?, c = 151.8 ?.