Studies on 30S ribosomal protein S1 from E. coli. I. Purification and physicochemical properties.
1. The distribution of ribosomal protein S1 in subcellular fractions of E. coli was determined by radioimmunoassay. It was found that about 70%, 20% and 10% of protein S1 were present in the high salt (1.0 M NH4Cl)-washed ribosomes, the ribosomal wash and the S100 fraction, respectively. 2. Protein S1 was purified from unwashed ribosomes by an improved procedure which included: (i) extraction of protein S1 from unwashed ribosomes with 1.2 M LiCl and 1.0 M NH4Cl, (ii) ammonium sulfate fractionation, (iii) two successive column chromatographies on DEAE-Sephadex, and (iv) hydroxylapatite column chromatography. Purified protein S1 was homogeneous in polyacrylamide gel electrophoresis under native and denatured conditions. 3. The molecular weights determined by sedimentation equilibrium and by SDS-polyacrylamide gel electrophoresis were 83,000 and 70,000 respectively. The sedimentation coefficient was estimated as 3.0S by glycerol gradient centrifugation. The stokes radius determined by Sephadex G-200 gel filtration was 45 A. From these data, the frictional ratio of protein S1 was calculated to be 1.65, assuming the molecular weight and partial specific volume to be 70,000 and 0.736, respectively. Protein S1 had an elongated shape with an axial ratio of approximately 8.5. 4. Protein S1 contained 2 residues of half-cystine and about 10 residues of tryptophan. From CD measurements, the contents of alpha-helix and beta-structure were estimated to be 32 and 27%, respectively. 5. As reported by Kolb et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 2379-2383), and Draper et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 4786-4790), the intrinsic fluorescence of protein S1 was markedly quenched on interaction with poly(U). The maximal quenching was observed when 30 mol of poly(U) (as UMP residues) was added to one mol of the protein.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011072:Poly U; D011485:Protein Binding; D011487:Protein Conformation; D011863:Radioimmunoassay; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000032:analysis; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D012269:Ribosomal Proteins; D011072:Poly U