Structural investigations of the highly flexible recombinant ribosomal protein L12 from Thermotoga maritima

Ribosomal protein L7/L12, the only multicopy component of the ribosome, is involved in translation factor binding and in the ribosomal GTPase center. The gene for L7/L12 from Thermotoga maritime was cloned and the protein exp ressed at high levels in Escherichia coli. Purification of L7/L12 was achie ved under non-denaturing conditions via heat treatment and two chromatograp hic steps, Circular dichroism melting profiles were monitored at 222 nm, sh owing the melting temperature of the protein at pH 7.5 around 110 degrees C , compared to similar to 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely follow ed the path of the thermal melting. Dynamic light scattering, gel filtratio n chromatography, and crosslinking experiments suggested that under physiol ogical buffer conditions Thermotoga maritima L7/L12 exists as a tetramer, T he protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (I2((1))3) space group with an estimated two and thre e to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed whi ch improved the mosaic spreads and the resolution limits, For the structure solution isoleucine was mutated to methionine at two separate positions, t he mutant forms expressed as selenomethionine variants and crystallized.