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G Cesareni

Publications and source records attributed to G Cesareni.

At least 19 recordsLinked to original sources
PubMed1992

Loop mutations can cause a substantial conformational change in the carboxy terminus of the ferritin protein.

Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.

Amino Acid Sequence↗
PubMed1992

Peptide display on filamentous phage capsids. A new powerful tool to study protein-ligand interaction.

Peptides can be displayed on the surface of filamentous bacteriophages by fusion to phage coat proteins. It was recently shown that vast (10(8)) collections of phages, each exposing a variant of the original peptide, can be constructed and utilized as a general source of peptide ligands. By panning these libraries on a target molecule linked to a solid support it is possible to select, out of the hundreds of millions of clones, those few phages that display a peptide that binds the target molecule. Searching these libraries is a powerful tool to be applied in many areas of fundamental and applied biology.

Bacteriophages↗
PubMed1991

Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector.

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

Amino Acid Sequence↗
PubMed1991

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

Amino Acid Sequence↗
PubMed1991

Control of ColE1 plasmid replication by antisense RNA.

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.

Bacterial Proteins↗
PubMed1990

Proton nuclear magnetic resonance assignments and secondary structure determination of the ColE1 rop (rom) protein.

The complete resonance assignment of the ColE1 rop (rom) protein at pH 2.3 was obtained by two-dimensional (2D) proton nuclear magnetic resonance spectroscopy (1H NMR) at 500 and 600 MHz using through-bond and through-space connectivities. Sequential assignments and elements of regular secondary structure were deduced by analysis of nuclear Overhauser enhancement spectroscopy (NOESY) experiments and 3JHN alpha coupling constants. One 7.2-kDa monomer of the homodimer consists of two antiparallel helices connected by a hairpin loop at residue 31. The C-terminal peptide consisting of amino acids 59-63 shows no stable conformation. The dimer forms a four-helix bundle with opposite polarization of neighboring elements in agreement with the X-ray structure.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of the channel and loop sequences of human ferritin H-chain.

Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli. The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence. The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation. Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins. The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues. Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation. The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size. The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified.

Chromosome Deletion↗
PubMed1989

Identification of the ferroxidase centre in ferritin.

Ferroxidase activity in human H-chain ferritin has been studied with the aid of site-directed mutagenesis. A site discovered by X-ray crystallography has now been identified as the ferroxidase centre. This centre is present only in H-chains and is located within the four-helix bundle of the chain fold.

Animals↗
PubMed1989

Recombinant H-chain ferritins: effects of changes in the 3-fold channels.

Human H-chain ferritins bearing sequence changes in the 3-fold channels have been expressed in E. coli to investigate the role of these channels in iron-storage processes. The proteins assemble into shells resembling those of native ferritins. Iron uptake measurements indicate that residues in the 3-fold channels are involved neither in initial Fe(II)-oxidation nor in iron-core nucleation.

Amino Acid Sequence↗
PubMed1989

Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli.

We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase. In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Amino Acid Sequence↗
PubMed1989

Genetic and structural analysis of the ColE1 Rop (Rom) protein.

Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.

Amino Acid Sequence↗
PubMed1989

A strategy to optimize translation initiation in recombinant mRNA: application to the Rop gene.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.

Bacterial Proteins↗
PubMed1989

The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth.

The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.

Base Sequence↗
PubMed1988

Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants.

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.

Chromosome Deletion↗
PubMed1988

SnR30: a new, essential small nuclear RNA from Saccharomyces cerevisiae.

The gene for a previously unidentified small nuclear RNA has been cloned from Saccharomyces cerevisiae and its nucleotide sequence has been determined. The RNA, snR30, was mapped to a unique coding sequence 605 nucleotides long. SnR30 appears to be one of the most abundant snRNAs of S, cerevisiae in that it can be resolved by ethidium bromide staining on one-dimensional denaturing gels of total yeast RNA. Like other snRNAs, snR30 is enriched in nuclei preparations and possesses a trimethyl guanosine cap structure at its 5' end. After substituting one allele of the wild type gene in a diploid strain for a deleted gene, after sporulation, haploid strains carrying the deletion were unable to grow, indicating that snR30 is required for an essential, but as yet, unknown function. The nucleotide sequence close to the initiation site of the SNR30 gene is similar to that of other yeast SNR genes whose transcripts are associated with pre-rRNA, suggesting that snR30 is related to this group of snRNAs.

Base Sequence↗
PubMed1988

Unexpected divergence and molecular coevolution in yeast plasmids.

Four closely related species of yeast possess multicopy nuclear plasmids whose shared molecular architecture demonstrates a common ancestor, despite their lack of discernible DNA sequence homology. Each plasmid encodes three proteins which have equivalent essential functions in plasmid maintenance. These three groups of proteins show markedly different degrees of conservation, so that although we have successfully aligned sequences for two groups, members of the third group have diverged to such an extent that they cannot be aligned. All the proteins are sufficiently different that they function only in conjunction with their encoding plasmid. These proteins have therefore conserved their functional interactions with the relevant DNA sequences of their particular plasmids, despite lack of amino acid sequence conservation. The maintenance of function in the face of DNA sequence divergence is analogous to the coevolution of ribosomal DNA promoters and RNA polymerase I, and suggests that molecular drive may be an important force in the evolution of these plasmids. This view is reinforced by the inconsistent phylogenetic relationships determined from the two alignment sets, and by the contradiction that the two plasmids known to be the closest related taxonomically and by their host interchangeability are suggested to be the most distant by their sequences.

Amino Acid Sequence↗