Generation of chromophore Tyr-containing mutants of the ribosomal protein L7/L12 from Escherichia coli by site-directed mutagenesis and characterization.
The ribosomal protein L7/L12 from Escherichia coli has two domains with different structure-a globular C-terminal domain and a non-globular elongated N-terminal domain. The N-terminal domain of the protein has been subjected to site-directed mutagenesis to introduce the chromophore Tyr and to study its structure by spectroscopic methods. The mutant proteins S1Y, M14Y and M26Y were expressed at high levels in Escherichia coli and purified to homogeneity by ion-exchange chromatography and gel-filtration. Growth and purification protocols were optimized to allow reproducible and efficient production of mutant proteins. The effects of the replacements were assessed by UV, far-UV circular dichroism (CD), DSC and 1H-NMR studies. The spectroscopic characteristics (far-UV CD and 1H-NMR) and the thermal unfolding (far-UV CD and DSC) of the mutants have shown that these single mutations in the N-terminal region of the protein have no appreciable effect on its secondary and tertiary structures. 1H-NMR spectroscopy was used to show that the Tyr mutants retain their dimer structure. The initiating Met is the first amino acid in the mutant protein S1Y. Y2(S1) is located in a structurally disordered region of the N-terminal domain of the protein S1Y and does not seem to have close amino acid neighbours. Y14(M14) and Y26(M26) participate in the structurally ordered regions of the molecule. Phe30 is situated in the surroundings of Y14. The unchanged structure resulting from the mutations makes these proteins highly suitable for structural studies by multidimentional NMR to determine the structure of the N-terminal domain of protein L7/L12.
Todorova
R
R
Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D012269:Ribosomal Proteins; D014443:Tyrosine; C016700:ribosomal protein L7-L12