Study of tyrosine-containing mutants of ribosomal protein L7/L12 from Escherichia coli.

Three mutant forms of the ribosomal protein L7/L12 with replacements of Ser1, Met14 and Met26 to Tyr were studied by the methods of fluorescence spectroscopy, circular dichroism and microcalorimetry. The amino-acid residue Tyr14 in the protein L7/L12 Tyr14 is located in a region with a more organized structure than Tyr26 in protein L7/L12 Tyr26. The replacements Ser1-->Tyr1 and Met14-->Tyr14 do not affect the secondary structure of protein L7/L12. The replacement Met26-->Tyr26 stabilizes the secondary structure of protein L7/L12. A pH-induced temperature transition was observed in the pH range 5.0-7.3 in protein L7/L12 Tyr14 by tyrosine fluorescence. Analogous transitions were observed for protein L7/L12 Tyr26 by Tyr fluorescence and for the wild type protein L7/L12 by Phe fluorescence. Three pH-dependent states of protein L7/L12 and its mutant forms L7/L12 Tyr1 and L7/L12 Tyr14 were found on the microcalorimetric melting curves. The characteristics of protein L7/L12 Tyr14 are very close to the wild type protein L7/L12 and it is a suitable object for studying the structure of the N-terminal part of molecule by two-dimentional 1H-NMR.

Structure of the N-terminus of the Escherichia coli ribosomal protein L7/L12.

Three mutant forms of the ribosomal protein L7/L12 (S1Y, M14Y and M26Y) were constructed in order to use the Tyr residues as internal labels at the N-terminal region. The proteins were studied by the methods of 1H-NMR, Fluorescence, Circular dichroism spectroscopy and Differencial scanning calorimetry. The secondary structure of N-terminus (residues 1-36) was predicted by the programme "Globule" [1, 2]. Y1 is located in a structurally disordered region at the free end of the molecule. M14 and M26 are located in structurally ordered regions. The replacement M14 does not change the structure of the protein. The replacement M26 disturbs the helicity in the region of the mutation. The first 1-5 positions from the N-terminus of L7/L12 are disordered, the position 7-14 participate in an alpha-helical/beta-structural region and position 19-30 in a strongly alpha-helical region.

[Preparation, isolation, and study of mutant forms of ribosomal protein L7/L12]. / Poluchenie, vydelenie i issledovanie mutantnykh form ribosomnogo belka L7/L12.

Three mutant forms of the ribosomal protein L7/L12 with Ser1, Met14 and Met26 substituted by the Tyr residue were constructed for studying the protein's N-terminal domain. Three point mutations were introduced into the L7/L12 gene by means of the phage M13mp18 system, the mutant genes were expressed in Escherichia coli cells, and methods of the proteins' purification were developed. The mutant proteins L7/L12 are very close, in structure and properties to the wild type protein and represent suitable objects for the 1H-NMR study of the N-terminal domain.

[Three mutant forms of ribosomal protein L7/L12]. / Tri mutantny formy ribosomnogo belka L7/L12.

Three mutant forms of the ribosomal protein L7/L12 with Ser1, Met14, and Met26 replacements with Tyr were constructed for studying the N-terminal domain of the protein. Three point mutations in the gene L7/L12 on the phage M13mp18 were generated. Recombinant plasmids containing the mutant genes were constructed on the base of expression vector pKK223-3 and plasmid pUC19. The mutant proteins were expressed in Escherichia coli cells, and methods of their purification were developed. It was found that the mutant proteins L7/L12 do not differ from the wild-type protein L7/L12 and represent a suitable object for 1H-NMR study of the L7/L12 protein. The three mutant proteins bind with the E. coli ribosome.