Theodora Choli

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.

Blotting of proteins onto immobilon membranes: in situ characterization and comparison with high-performance liquid chromatography

The electrophoretic transfer from polyacrylamide gels to Immobilon [poly(vinylidene difluoride)] membranes of various proteins differing in molecular masses from 14,000 to 200,000 was performed, using both a semi-dry blotting apparatus and a standard blotting chamber. The blotted proteins were analyzed and sequenced with and without staining, and the initial yields of the degradation were examined. Furthermore, protein purification by blotting after one- and two-dimensional gel electrophoresis was compared with conventional HPLC methods. Optimum blotting conditions for in situ enzymatic or chemical cleavages of the proteins on the blots are described, and for the in situ hydrolysis followed by amino acid analysis and cysteine determination.

Extended N‐terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria

We have determined the N‐termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro‐sequencing. The N‐terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r‐) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N‐termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix‐targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.

Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.

Mitochondrial ribosomes of yeast: isolation of individual proteins and N-terminal sequencing

Proteins of the small and large subunits of mitochondrial ribosomes from the yeast Saccharomyces cerevisiae were isolated and characterized by two‐dimensional gel electrophoresis. Ribosomal proteins of the large subunit were separated by reverse‐phase HPLC and up to 37 amino acid residues of the N‐terminal sequences of L3, L4, L9 and L31 were determined. No significant homology to ribosomal protein sequences so far determined from other organisms was found.

The antirepressor of phage P1 Isolation and interaction with the C1 repressor of P1 and P7

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in‐frame start codon. Another open reading frame, icd, 5′ upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene‐carrying plasmids we have overproduced and purified Ant½ in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N‐terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N‐terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co‐precipitate with the Ant½ complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1‐inactivating function by a direct protein—protein interaction.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by GTP

Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, M r = 82 043) was subjected to trypsinolysis alone or in the presence of GTP. Following electroblotting and automated amino acid sequencing of the resulting peptides, the location and the sequential order of the main cleavage sites were identified. Trypsinolysis of IF2 ultimately generates two compact domains: a 24.5 kDa C‐terminal fragment and a 40 kDa G‐fragment which is obtained only in the presence of GTP which strongly protects a cleavage site within the GTP binding domain.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet-tRNA

Translation initiation factor IF2 from Bacillus steaiothermophilus (741 amino acids, M r 82,043) was subjected to trypsinolysis alone or in the presence of fMet‐tRNA. The initiator tRNA was found to protect very efficiently the Arg308‐Ala309 bond within the GTP binding site of IF2 and, more weakly, three bonds (Lys146‐Gln147, Lys154‐Glu155 and Arg519‐Ser520). The first two are located at the border between the non‐conserved, dispensable (for translation) N‐terminal portion and the conserved G‐domain of the protein, the third is located at the border between the G‐ and C‐domains. Since IF2 is known to interact with fMet‐tRNA through its protease‐resistant C‐ (carboxyl terminus) domain, the observed protection suggests that, upon binding or fMet‐tRNA, long‐distance tertiary interactions between the IF2 domains may take place.

The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy‐terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an M r of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.