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 maritima was cloned and the protein expressed at high levels in Escherichia coli. Purification of L7/L12 was achieved under non-denaturing conditions via heat treatment and two chromatographic steps. Circular dichroism melting profiles were monitored at 222 nm, showing the melting temperature of the protein at pH 7.5 around 110 degrees C, compared to approximately 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely followed the path of the thermal melting. Dynamic light scattering, gel filtration chromatography, and crosslinking experiments suggested that under physiological buffer conditions Thermotoga maritima L7/L12 exists as a tetramer. The protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (12(1)3) space group with an estimated two and three to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed which improved the mosaic spreads and the resolution limits. For the structure solution isoleucine was mutated to methionine at two separate positions, the mutant forms expressed as selenomethionine variants and crystallized.
Wahl
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MC
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Martinsried, Germany.
Huber
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R
Marinkoviç
S
S
Weyher-Stingl
E
E
Ehlert
S
S
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D012996:Solutions; D013329:Structure-Activity Relationship; D020124:Thermotoga maritima / Q000737:chemistry*
D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D012996:Solutions; C016700:ribosomal protein L7-L12