To read the full-text of this research, you can request a copy directly from the authors.
... In this review, we analyze a recently proposed model developed for bacterial systems  which is becoming popular  that aims to integrate cotranslational enzymes into the overall mechanism of protein synthesis. Prior to the new model, the physical association of the NME machinery with the ribosome had been considered transient or nonexistent . Focusing on the specific case of PDF and MAP, we address several important questions raised by this new model, which we name the 'concerted model', such as how general the proposed mechanism could be (i.e. whether the positioning of RPBs in relation to the ribosome is conserved either in bacteria or in other living systems). ...
... One of the key findings reported  is the demonstration that PDF molecules physically interact with non-translating E. coli ribosomes in vitro. Furthermore, PDF activity has been detected early on in the process for translating ribosomes of Bacilllus subtilis and, to a lesser extent, E. coli . Nevertheless, immunoblotting experiments with specific antibodies showed that PDF was associated with the postribosomal fraction rather than the ribosomes themselves in E. coli . ...
... This would also suggest that type II PDF would interact differently with other proteins of the ribosome. Note that B. subtilis represents the only organism where PDF binding to the ribosome has been shown . As type II PDF seems to be four to six times more abundant in B. subtilis than type IB , it could be argued that higher concentrations of a PDF that does not naturally bind to ribosomes might compensate for the absence of a ribosome binding site. ...
Recent major advances have been made in understanding how cotranslational events are achieved in the course of protein biosynthesis. Specifically, several studies have shed light into the dynamic process of how nascent chains emerging from the ribosome are supported by protein biogenesis factors to ensure both processing and folding mechanisms. To take into account the awareness that coordination is needed, a new 'concerted model' recently proposed simultaneous action of both processes on the ribosome. In the model, any emerging nascent chain is first encountered by the chaperone trigger factor (TF), which forms an open cradle underneath the ribosomal exit tunnel. This cradle serves as a passive router that channels the nascent chains to the first cotranslational event, the proteolysis event performed by the N-terminal methionine excision machinery. Although fascinating, this model clearly raises more questions than it answers. Does the data used to develop this model stand up to scrutiny and, if not, what are the alternative mechanisms that the data suggest?
... Extracts from certain microorganisms [E. coli, B. stearothermophilus, and B. subtilis (1,115,378)] were found to contain enzymes cleaving fMet-peptides into formate and Met-peptides. A protein fraction from E. coil which contained such a peptide deformylase was found to liberate formate from proteins synthesized in vitro. ...
... Extracts from E. coil and B. subtilis contain an aminopeptidase capable of removing a Met residue from Met-puromycin (but not from fMetpuromycin), and from the N-terminal hexapeptide of f2 coat protein (but only after the formyl residue has been cleaved off by treatment with the deformylase). These observations indicate that if a fMet residue is removed from a nascent protein, this takes place in two steps: first, the formyl residue is cleaved off, and subsequently, the Met residue (378). ...
... At least some of the proteins specified by bacteriophage T4-infected E. coli are initiated with N-formylmethionine (183,184,251). Some of the procaryotic cells, other than E. coli, in which the occurrence of fMet-tRNA is established are the following: B. subtilis (20,162,378), B. stearothermophilus (1,295), Micrococcus lysodeicticus, Pseudomonas aeruginosa, Anacystis nidulans (a blue-green alga) (20), Mycoplasma laidlawii B, M. gallisepticum A 5969, and Mycoplasma species (caprine strain; 149). The dependence of the derepression pattern on the formylating capacity in Salmonella typhimurium (27) and the fact that Met-tRNA from E. coil B can be formylated by extracts of Lactobacillus leichmanii, Pseudomonas species, Streptomyces antibioticus, and Clostridium tetanomorphum (84) suggest, but do not prove, the involvement of fMet-tRNA in protein synthesis in these organisms. ...
...  Peptide deformylase (PDF), a metalloprotease that removes the N-formyl group present in all newly synthesized bacterial polypeptides, is required for proper protein maturation in prokaryotes but not mammalian cells. 4 However, PDF is an unexploited clinical target as a novel antimicrobial agent. 5 GSK1322322 is a potent inhibitor of PDF, discovered by a combination of structure-based drug design and iterative medicinal chemistry ( Figure 1). ...
GSK1322322 is the first in a new class of antibiotics that inhibit peptide deformylase, necessary for bacterial protein maturation. Previously, low absolute bioavailability was observed for the 1500-mg oral tablet formulation, resulting in a less than dose-proportional increase from the 1000-mg dose. Furthermore, high variability of pharmacokinetic (PK) parameters within cohorts was suggested to be associated with differences in body weight. This open-label, randomized, 4-period, crossover, single-dose phase I study in healthy individuals compared the PK, safety, and tolerability of free base oral tablets under fasted or fed conditions with intravenous and oral mesylate salt solution of GSK1322322 under fasted conditions. Absolute bioavailability of GSK1322322 1500-mg free base tablets under fasted conditions, fed conditions, and oral mesylate salt solution was 57%, 77%, and 92%, respectively. Moderate–fat/calorie food intake increased area under the concentration-time curve (AUC0-∞) by 36%, maintained maximum observed concentration (Cmax), and delayed time to Cmax. It appeared that AUC0-∞ decreased with body weight, whereas clearance increased. GSK1322322 administration resulted in only mild-to-moderate adverse events. These results support future clinical investigations of the free base oral tablet formulation of GSK1322322 1500 mg after intake of a moderate–fat/calorie meal, including further investigation of a potential weight-based dosage change.
... It is therefore considered to be an early cotranslational process (Pine, 1969;Ball and Kaesberg, 1973). Takeda and Webster (1968) attempted to account for the high efficiency of PDF by suggesting that it might be bound to ribosomes. However, no such association was observed in E. coli (C. ...
Peptide deformylase was discovered 30 years ago, but as a result of its unusually unstable activity it was not fully characterized until very recently. The aim of this paper is to review the many recent data concerning this enzyme and to try to assess its potential as a target for future antimicrobial drugs.
... (2) Our purified recombinant SNase (pro − ) might not be completely homogeneous, which gives rise to apparently parallel folding pathways. For example, the N-terminal sequence of a recombinant protein expressed in E. coli is widely recognized to start with formyl-methionine , which is in most cases subsequently processed by deformylase enzyme [18,19] and removed by methionine aminopeptidase to finally produce the N-terminal methionine-free recombinant protein. Removal of the N-terminal extra methionine, however, does not always occur. ...
The folding mechanism of proline‒free staphylococcal nuclease (SNase (pro−)) (P11A, P31A, P42A, P47T, P56A, P117G) was investigated using the double‒jump stopped‒flow method (interrupted refolding). This method has enabled us to specifically monitor the amount of the native molecules during the refolding. The results indicate that the middle and slow phases observed in the refolding kinetics represent the formation of the native state (IM→N, IS→N) and that the folding mechanism of SNase (pro−) is not represented by a single sequential pathway, but at least two parallel pathways are required for interpreting the results.
... Le caractère co-traductionnel de la voie de la NME a été décrit chez les bactéries il y a près de 50 ans . Il avait alors été montré que la déformylation de la méthionine initiatrice, suivie de son excision, ont lieu très tôt dans la synthèse des protéines, dès que le polypeptide en cours de synthèse émerge du tunnel de sortie du ribosome, lorsqu'il dépasse une taille d'environ 40 ± 5 acides aminés, pouvant aller jusqu'à 60 résidus  . Cependant, il a longtemps été admis que la PDF n'interagissait pas avec le ribosome, car cette interaction n'était pas détectable, mais aussi parce que l'enzyme est capable de déformyler un peptide en absence de ribosomes 90  . ...
Les protéines en cours de synthèse subissent des modifications très précoces de leur extrémité N-terminale, dès lors que celle-ci émerge du tunnel de sortie du ribosome. La première modification est l’excision de la méthionine initiatrice, assurée par une méthionine aminopeptidase (MetAP), précédée de sa déformylation par une enzyme peptide déformylase (PDF) chez les bactéries et dans les mitochondries et chloroplastes. Ce processus est ubiquitaire et essentiel, et a été décrit dans tout le règne du vivant. Chez les bactéries, les PDFs de type 1B se fixeraient au ribosome à proximité de l’extrémité du tunnel de sortie du peptide naissant, via son hélice α C-terminale. Or des analyses métagénomiques récentes ont révélé la présence insoupçonnée de gènes codant des PDFs putatives chez des virus marins. De manière inattendue, toutes les PDF virales présentent des séquences C-terminales très courtes et dépourvues de l’hélice α3. L’identification de ces PDFs atypiques soulève alors de nouvelles questions quant à leur possible interaction au ribosome et à leur fonction biologique. L’objectif de ma thèse a donc été de réaliser la caractérisation complète et intégrée de la peptide déformylase du bactériophage Vp16T, dont la séquence est l’une des plus courtes connues à ce jour. J’ai montré que le phage Vp16T code une protéine active, in vivo et in vitro, et qu’elle peut se lier au ribosome malgré l’absence d’hélice α C-terminale. La caractérisation structure-fonction de Vp16PDF a révélé des caractéristiques uniques qui pourraient alors expliquer sa fonction au cours de la réplication du phage. Ainsi j’ai montré que l’expression de Vp16PDF chez E. coli modifie la structure de l’enveloppe, induit l’accumulation d’agrégats et finalement inhibe la croissance bactérienne. De plus, l’étude de souches bactériennes mutantes a montré que Vp16PDF interfère spécifiquement avec le repliement et l’adressage de protéines membranaires. Cette dernière fonction pourrait permettre de déstabiliser la membrane de l’hôte et ainsi favoriser la libération des particules virales.
... The N-terminus of a recombinant protein newly synthesized in Escherichia coli is initiated with N-formylmethionine , which is subsequently processed by peptide deformylase, and removed by methionine aminopeptidase (MetAP) to finally produce the N-terminal methionine-free mature protein [18,19]. However, incomplete cleavage of the N-terminal methionine often takes place because the specificity and efficiency of MetAP is influenced by the size of the side chain of the second or penultimate amino acid residue and there are numerous cases where E. coli-expressed proteins contain the extra N-terminal methionine residue . ...
The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core-shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.
... b Two overlapping sequences amounting to Ͼ95% of the total repetitive yield were obtained. This is likely due to contaminating activity of methionine-aminopeptidase, as previously reported (21,32). ...
The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the
lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology
of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its
fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into
the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107
has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized
membrane. Moreover, an allele, S105ΔTMD1, with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type
allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather
than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion
protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105
but restored when both S105ΔTMD1 and holin carried the substitution.
... This is a common modification caused by the initiation of translation and limited to the N-terminal methionine . Usually, the formyl group is removed by a deformylase  followed by cleavage of the whole methionine residue, provided there is an amino acid with a small side chain (such as alanine) in the second position . According to the crystal structure of PSII [3,5], the N-termini of all PSII subunits with an N-terminal formyl-methionine seem to orientate themselves towards the lumenal space [5,56]. ...
The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27-Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (<10kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using (15)N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50μmol photons m(-2) s(-1)). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
... In this regard, peptide deformylase (PDF) may prove an intelligent choice because of the prokaryotic protein synthesis which universally initiates with an N-formylmethionine residue . It is an essential metalloenzyme in most pathogens, which removes the formyl group from methionine at the N terminus of nascent polypeptide chains followed by protein maturation . This protein is expressed in all pathogenic bacteria, along with Mycoplasma and Chlamydia species. ...
Objective: Increasing prevalence of antibiotic resistant pathogenic bacteria has reached the alarming level that poses a need to identify new drugs and drug targets. Peptide Deformylase may prove to be a wise choice since it is crucial for native protein functioning in most pathogenic bacteria.
Methods: In present study, we demonstrated preexisting drug Thiazides, primarily used for hypertension and edema, as an antibacterial agent. We screened the binding affinity of Thiazides and its derivatives against seven peptide deformylases (PDF’s) models retrieved from different pathogenic bacteria using Autodock 4.2 (version1.5.6). Further their interaction pattern was analyzed using Lig Plot+.
Results: S-Bendroflumethiazide (CID_6604206) and Quinethazone (DB01325) showed a considerable binding affinity against all selected protein models. Analysis of their interaction pattern revealed that DB01325 is interacting with protein models 1BSK and 1SZZ through more number of H-bonds with their active site residues. An almost same pattern was observed for CID_6604206 with 1N5N and 1SZZ protein models. The binding affinity of these ligands was significantly better than that of Actinonin (CID_443600) which is a well known natural PDF inhibitor.
Conclusion: The binding pattern of Thiazide derivatives with peptide deformylase protein models may provide a clue for designing a new potent drug to control the diseases caused by newly evolved antibiotic resistant pathogenic bacterial strains.
... The following different types of functions have been found for proteinases; (i) maturation of proteins by removal of the N-terminal methionine residue from newly synthesized proteins (33); (ii) specific modifications of individual proteins resulting in activation or inactivation of a particular enzyme activity (6) or modification of enzyme activity or specificity (30); (iii) selective elimination of defective protein molecules (9); (iv) protein turnover for supplying amino acids and energy in the case of starvation and cell differentiation (sporulation) at the expense of unneeded cellular protein (10,24); and (v) utilization of peptides as sources of amino acids and nitrogen (in the case of peptidases [29 ]). ...
A mutant of yeast lacking proteinase C (carboxypeptidase Y) activity has been found by using a histochemical stain to screen mutagenized colonies. This defect segregates 2:2 in meiotic tetrads. Cell extracts lacked the esterolytic, amidase, and proteolytic activities associated with proteinase C. The absence of proteinase C does not affect mitotic growth and has no obvious effect on the formation of viable ascospores or meiotic segregation. The mutant grows on peptides known to be cleaved by proteinase C in vitro. This finding is consistent with the idea that other enzymes exist in vivo with overlapping substrate specificities.
Suspensions of single cells were prepared from normal adult rat liver by perfusion of liver with and shaking in buffered solutions of 0.1% hyaluronidase and 0.05% collagenase. l-[1-¹⁴C]Leucine and l-[1-¹⁴C]methionine incorporated into protein could be measured by precipitating the cells on filter paper disks without prior homogenization. No incorporation of amino acids into protein was observed if the cells were incubated under nitrogen. Optimal incorporation was obtained in 3 mm to 12 mm phosphate buffer at pH 7.17. The incorporation was inhibited at higher phosphate concentrations, and it was also less efficient in Tris-HCl buffer, Tris-maleate buffer, and Tris-phosphoric acid buffer.
The incorporation of l-[1-¹⁴C]leucine into protein was directly proportional to the concentration of the cells in the suspension. High doses of l-leucine or l-methionine (∼2 mm) did not inhibit the incorporation of amino acids into protein. The cells were freely permeable for l-leucine and l-methionine. The system incorporating amino acids into protein was half-saturated with 7.2 x 10⁻⁵ml-leucine and 5.1 x 10⁻⁵ml-methionine. An inhibition of 50% of the incorporation of l-leucine or l-methionine into protein was observed at a puromycin concentration of 1.4 µg per ml, or 0.8 µg per ml, respectively. Changing the incubation temperature from 22° to 32° caused a 4-fold increase of the initial rate of incorporation of l-leucine or l-methionine.
This review analyzes the concept according to which the pathway of synthesized peptide from the ribosome peptidyl transferase center to the exit domain goes along the tunnel of the large subparticle. Experimental data on the accessibility of the nascent polypeptide chain to molecules of modifying agents and fluorescence quenchers are considered. Results of localization of the exit site for the nascent peptide on the ribosome surface, possible conformational states of the peptide, and its mobility and folding on the ribosome are analyzed. The analysis is based on the ribosomal tunnel parameters obtained using X-ray crystallography of whole ribosomes and large ribosomal subparticles. Special attention is given to data that do not fit in the concept of the "tunnel for peptide exit" and to results already obtained before the reliable tunnel visualization using X-ray crystallography was achieved.
1.1. N-Acylamino acid amidohydrolase (EC 184.108.40.206), or aminoacylase, was isolated from the seeds of Palo Verde (Parkinsonia aculeata L.). The enzyme was localized within the cotyledons and embryo of the seed. An acetone powder preparation from the combined cotyledons and embryo furnished an active extract which was purified greater than 75-fold. Fractionation consisted of treatment with (NH4)2SO4, cold acetone, freeze-thaw (which removed a cold-labile, inhibitory protein), and chromatography on DEAE-Sephadex A-25. The specific activity was 2650 μmoles/h per mg protein nitrogen.2.2. Co2+ was shown to enhance activity and also to provide stability to the enzyme during the (NH4)2SO4 fractionation.3.3. was the best substrate and exhibited a vmax/Km = 2.9·105 at pH 7.2. The acetyl derivatives of l-methionine, l-valine, and l-leucine inhibited the enzyme at concentrations above 10 mM. The hydrolyses of and exhibited non-Michaelis-Menten kinetics with an indication of two possible binding sites.4.4. The molecular weight was estimated by gel filtration to be 79 500.5.5. The enzyme was stable between pH 6.5 and 10.0, and up to a temperature of 50° for 10 min. The temperature optimum was 52°.6.6. The enzyme catalyzed the hydrolysis of P-nitrophenyl acetate and was inhibited by some classical sulfhydryl reagents. Mercaptoethanol enhanced enzyme activity.
The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat α-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant α-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.
Escherichia coli harboring the gene encoding human interleukin-2 (IL-2) produces a mixture of two recombinant IL-2 species: one with the amino-terminal alanine (rIL-2) and the other having an additional methionine residue at the amino terminus (Met-rIL-2). Ways to increase the amount of rIL-2 and its proportion to the total IL-2 were tried. Among E. coli K-12 derivatives, N4830 was an effective producer of recombinant IL-2. The production of the mixture was greatly increased by optimizing the medium ingredients or culture conditions. However, the percentage of rIL-2 in the product decreased almost linearly with an increase of the total production of recombinant IL-2 and was less than 10% under optimal culture conditions. By adding 4.1 × 10−5 M maganese and 7.4 × 10−5 M ferric ions to the medium, we succeeded in raising the percentage of rIL-2 to 50% without any decrease of the total production.
When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme).
The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as
the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of
hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase,
as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding
yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed
closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus
of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy
and its activity were almost identical with those of native hen egg-white lysozyme.
Two homologous 29 amino acid-long highly hydrophobic membrane miniproteins were identified in the Bligh-Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e., comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.
Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology™ toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNAi, resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture.
Ausgehend von einer neuen His-Tag PS2-Variante konnten durch den Einsatz sehr leistungsfähiger Chromatographietechniken verschiedene PS2-Subkomplexe in großen Mengen isoliert und in Kombination mit unterschiedlichen massenspektrometrischen Verfahren analysiert werden. Es konnte gezeigt werden, dass ein potenzielles PS2-Assemblierungsintermediat die bisher nicht näher charakterisierte Psb27 Untereinheit enthielt, bei welcher erstmals eine kovalente Lipidmodifikation nachgewiesen werden konnte. Ausgehend von Psb27 als Modellprotein konnten über einen bioinformatischen Ansatz weitere potenzielle Lipoproteine aus dem Bereich der Photosynthese in Cyanobakterien identifiziert werden. Darüber hinaus wurden mit der isolierten PsbO-Untereinheit ESR- und NMR-spektroskopische Untersuchungen durchgeführt, die neue Einblicke in die strukturelle Dynamik des Proteins ermöglichten. Zusätzlich konnten Informationen zur Interaktion der PsbO-Untereinheit mit potentiellen Liganden erhalten werden.
Peptide deformylase (EC 220.127.116.11) is a mononuclear iron enzyme that cleaves the formyl group of the N-terminal formyl-methionine residue of nascent polypeptide chains in eubacteria. It is strictly stereospecific for a l-amino acid as the first residue of formyl-peptide substrates and strongly prefers substrates with at least two or more residues. The native enzyme from Escherichia coli is a monomeric protein of 168 amino acid residues that contains one tightly bound Fe²⁺ ion. The metal ion is tetrahedrally coordinated by a water molecule and the side chains of residues Cys90, His132, and His136. Similar to thermolysin, the two histidines are part of the HEXXH motif and the glutamate residue is required for enzymatic activity. The native iron form of peptide deformylase is extremely sensitive to oxidative destruction. On substitution of the iron ion by Ni²⁺ or Co²⁺ the enzyme is insensitive to oxidation and maintains its activity, whereas the Zn²⁺ form is nearly inactive.
This chapter describes initiation as the act preceding the start of polypeptide chain synthesis, and which consists in a series of events ensuring the “recognition” of particular regions of the messenger RNA chain, called the “initiation signals,” that specify the place where decoding of the template RNA begins. During this step, the tRNA providing the N-terminal aminoacyl residue of the nascent chain is positioned opposite the initiation triplet, present within the initiation signal. It is the formation of such an initiation complex that is critical in phasing the “readout” of the messenger RNA and that is probably the rate-limiting step in protein synthesis. It also reviews that initiation also represents an obvious and important control point in translation. Although results obtained from several systems hint that such phenomena may play a role in biological regulations, the evidence presently available is far from conclusive. The chapter also emphasizes on the molecular involvement of the initiation components, as currently conceived. It concludes that some ribosomal proteins are now available in reasonable amounts and in a high degree of purity, which results in the ribosome becoming a choice material for physical chemists. In the not-too-distant future, the general principles governing all these interactions should be established.
Initiation of protein synthesis has been studied in the presence of the tetrahydrofolic acid analogues trimethoprim or aminopterin in Bacillus subtilis. This bacterium can grow in the presence of the inhibitors, when the medium is supplemented with the low molecular weight products of tetrahydrofolate-dependent pathways. In an attempt to show whether formylation of initiator tRNA is a prerequisite for the iniation of protein synthesis in procaryotic cells, the amount of N-formylmethionine in tRNA and in protein has been determined. The level of formylation of methionyl-tRNA was found to be 70% in control cells and approximately 2% in inhibitor-treated cells. The content of formyl groups in protein has also been found to be drastically reduced. Trimethoprim or aminopterin did not alter the amount of tRNAMet nor the degree of aminoacylation of tRNAMet in vivo. These results indicate that in B. subtilis inititation of protein synthesis is possible without prior formylation of initiator tRNA.
Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.
This chapter focuses on the current assessment of peptide utilization by microorganisms from the standpoints of cleavage and transport. It also lays emphasis on the effects of peptides on other aspects of microbial physiology. It is clear that prior to their nutritional utilization peptides must be hydrolyzed to yield free amino acids, and that this cleavage may occur before or after absorption. Variations in these two essential features of cleavage and transport produce circumstances in which particular peptides may give a growth response that is greater than, equal to, or less than an equivalent mixture. Simple peptides offer a vast reservoir of biologically active compounds and techniques exist for the synthesis of any desired sequence. It is hoped that studies on the structural requirements of microbial peptide permeases and peptidases, and of the effects of peptides on microbial growth, may be useful not only in their immediate context, but they may also act as a model system to provide information relevant to peptide interactions in more complex biological systems of free amino acids.
A comparison is made of the N- and C-terminal amino acids from 96 published protein sequences, 26 from prokaryotes, 70 from eukaryotes. The observed frequencies of the N-terminal amino acids methionine, alanine and serine in prokaryotes, and alanine and serine in eukaryotes are significantly higher than expected for a random arrangement of amino acids. At the C-terminal end, the observed frequencies of lysine, asparagine and glutamine in prokaryotes and phenylalanine, asparagine and glutamine in eukaryotes exceed random expectation. These results could be explained by specific proteolytic cleavage during protein synthesis.
Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.
An N-acylamino acid acylase was partially purified from tobacco (Nicotiana tabacum) leaves and some of its properties are described. It hydrolyses N-acetylarginine, N-acetylmethionine, N-acetylcysteine and to a lesser extent N-formylmethionine. It does not appreciably hydrolyse N-formyl peptides and is therefore unlikely to be involved in protein synthesis.
A peptidase from Escherichia coli B has been prepared in a highly pure form and characterized with respect to its substrate specificity, requirements for activity, size and subunit structure. This enzyme preferentially catalyzes the hydrolysis of certain methionyl dipeptides and for this reason is referred to as dipeptidase m. Of the substrates tested with the homogeneous enzyme methionylalanine and methionylserine were the most rapidily hydrolyzed. Other substrates were cleaved more slowly, if at all, by this peptidase. The substrate specificity of this enzyme suggests that it may be involved in the removal of NH2-terminal methionine from newly initiated E. coli proteins.
In the cell newly synthesized polypeptides are subjected to enzymatic processing, chaperone-assisted folding, and targeting to translocation pores at membranes concurrently with their synthesis by the ribosome (Figure 1). The major players in these events are, (i) ribosome-associated chaperones, (ii) nascent-chain-processing enzymes, (iii) the signal recognition particle — a complex that recognizes ribosomes that are translating membrane and some secretory proteins and targets them to the membrane — and (iv) the membrane-protein-insertion machinery — a large multi-subunit trans-membrane complex responsible for protein insertion into or translocation across membranes. The ribosome plays a major role in governing the interplay between the various factors involved. Using electron microscopy, crystallography and biochemical approaches, we investigated the structural and mechanistic aspects of the interaction between these factors and the ribosome.
In the preceding chapters the reader has already encountered the concept that the mRNA (messenger RNA) is an intermediary in the expression of that portion of the genetic information in the DNA that encodes proteins. The present chapter presents a detailed consideration of the process of translation of the mRNA. Although this will be prefaced with a summary of the main features of translation, the reader is directed to suitable textbooks of biochemistry (e.g. ) for a more elementary account of this topic.
Aminopeptidases (AP) catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. These enzymes generally have broad specificity, occur in several forms, and are widely distributed throughout the plant and animals kingdoms. Over 100 APs have been purified and/or studied, and over 50 genes have been cloned and characterized. Several forms of these enzymes have been found in many tissues or cells, on cell surfaces, and in soluble cytoplasmic or secreted forms in plants and animals1–7 (see chapters 2–8). In some cells they constitute a substantial proportion of enzyme protein.4,8,9
Peptide deformylase, which catalyzes the removal of N-formyl groups from the initiating N-formyl-methionine of nascent polypeptides, has recently been characterized from several plants, including rice, tomato and Arabidopsis thaliana. The two Arabidopsis thaliana DEF genes, AtDEF1 and AtDEF2, encode enzymes which are functionally active both in vitro and in vivo and are catalytically inactivated by the naturally-occurring peptide deformylase inhibitor actinonin, a product of a soil-borne actinomycete. Actinonin has profound herbicidal effects when applied to many plant species both pre- and postemergence. Transgenic tobacco plants were engineered to over-express each of the AtDEF proteins. These plants were completely resistant to the herbicidal effects of actinonin. This data provides the first unequivocal evidence that the lethality of actinonin to plants in vivo is strictly a consequence of the inhibition of peptide deformylase activity. This work also confirms that peptide deformylase is a valid target for both the development of novel broad-spectrum herbicides, and the engineering of herbicide selectivity in plants without the use of foreign genes.
The ribosome is the cell's protein-making factory, a huge protein-RNA complex, that is essential to life. Determining the high-resolution structures of the stable "core" of this factory was among the major breakthroughs of the past decades, and was awarded the Nobel Prize in 2009. Now that the mysteries of the ribosome appear to be more traceable, detailed understanding of the mechanisms that regulate protein synthesis includes not only the well-known steps of initiation, elongation, and termination but also the less comprehended features of the co-translational events associated with the maturation of the nascent chains. The ribosome is a platform for co-translational events affecting the nascent polypeptide, including protein modifications, folding, targeting to various cellular compartments for integration into membrane or translocation, and proteolysis. These events are orchestrated by ribosome-associated protein biogenesis factors (RPBs), a group of a dozen or more factors that act as the "welcoming committee" for the nascent chain as it emerges from the ribosome. In plants these factors have evolved to fit the specificity of different cellular compartments: cytoplasm, mitochondria and chloroplast. This review focuses on the current state of knowledge of these factors and their interaction around the exit tunnel of dedicated ribosomes. Particular attention has been accorded to the plant system, highlighting the similarities and differences with other organisms.
Our laboratory has a long interest in methionine (Met) biosynthesis, metabolism and its role in oxidative damage. Although the primary goal of this review was to summarize more recent studies on the role of Met oxidation in proteins on cellular function, we have used this opportunity to describe some of the early studies that elucidated the unique and important functions that Met has in one carbon metabolism, and as an initiator of protein synthesis. Oxidative damage, our main focus, is believed to be a major factor in age related diseases and the aging process. The oxidation of Met residues in proteins to methionine sulfoxide has turned out to be an important biomarker of oxidative damage since there are specific enzyme systems, methionine sulfoxide reductases, that can repair this damage and which play an important role in protecting cells against oxidative damage.
Staining for peptidase activities on starch gel after the electrophoresis of lysates of rabbit red blood cells or reticulocytes and the identification of the reaction products revealed the existence of the following three enzymes: (a) An enzyme that hydrolyzes NH2-terminal methionine from methionyl peptides longer than tetrapeptides and from shorter methionyl peptides with blocked carboxy ends. This enzyme does not hydrolyze several peptides tested without methionine at the NH2 termini. (b) An enzyme that cleaves formylmethionine from formylmethionyl peptides. This enzyme can also react with acetylmethionyl peptides but does not hydrolyze several peptides tested without an acylmethionine at the NH2 termini. (c) An enzyme that deformylates formylmethionine. This enzyme can hydrolyze various formyl- and acetylamino acids and may be identical with the nonspecific dipeptidase in red cells. These three enzymes exist also in other tissues and are not bound to ribosomes.
NH2-terminal methionine and formylmethionine in the short nascent peptides obtained from reticulocytes were hydrolyzed by hemolysates.
These findings suggest that biological function of the methionine aminopeptidase and the formylmethionine-releasing enzyme is to remove the terminal methionine and formylmethionine from nascent peptides.
Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH2-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH2-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.
N-terminal protein modifications correspond to the first modifications which in principle any protein may undergo, before translation is completed by the ribosome. This class of essential modifications can have different nature or function and be catalyzed by a variety of dedicated enzymes. Here, we review the current state of the major N-terminal co-translational modifications, with a particular emphasis to their catalysts, which belong to metalloprotease and acyltransferase clans. The earliest of these modifications corresponds to the N-terminal methionine excision, an ubiquitous and essential process leading to the removal of the first methionine. N-alpha acetylation occurs also in all Kingdoms although its extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial pathway existing only in eukaryotes. Recent studies dealing on how some of these co-translational modifiers might work in close vicinity of the ribosome is starting to provide new information on when these modifications exactly take place on the elongating nascent chain and the interplay with other ribosome biogenesis factors taking in charge the nascent chains. Here a comprehensive overview of the recent advances in the field of N-terminal protein modifications is given.
The peptide deformylase from Leptospira interrogans (LiPDF) shows many unusual characteristics. The substrate pocket of formate-bound complex adopts an open conformation. However, in the actinonin-bound LiPDF complex, a slightly open substrate pocket is observed. The opening is not large enough for the inhibitor, because the CD-loop restricts the access to the active site. To explore the conformational changes of the substrate pocket, we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free LiPDF and actinonin-bound LiPDF. During the molecular dynamics simulations, extensive conformational changes have taken place. The comparison of the two MD results shows that the CD-loop, hydrophilic inhibitor, and hydrophobic cluster are necessary for the reopening of the substrate pocket. In addition, Tyr71 plays an important role in mediating the movements of CD-loop, and the transition of the substrate pocket from open to semi-open only occurs in the presence of an inhibitor, which are consistent with the experiment very well.
Initiation of protein synthesis appears to be a process of great complexity in both prokaryotic and eukaryotic systems. A possible reason for such complexity, particularly in comparison with the process of elongation of nascent proteins, may reside in gene regulation operating through the selection of mRNA or cistrons by ribosomes and protein factors. It is known that in prokaryotes the main control occurs at the transcriptional level. However, for polycistronic mRNA’s there are a few facts consistent with a regulation at the translational level, and this is particularly shown in the case of RNA phage infection of E. coli (MS2, Qß, R17) where the three cistrons of the phage RNA are translated with different frequencies in vivo  although they are present in equal concentrations. Therefore, it is conceivable that a control exists at the translational level which might have a specific function in fine regulation of bacterial protein synthesis.
A series of divalent zinc and cadmium complexes ([(beppa)Zn](ClO4)2 (1), [(bmppa)Zn](ClO4)2 (2), and [(bmppa)Cd(ClO4)](ClO4)·MeOH (3)) of amide-appended N2S2 ligands (beppa = N-bis-2-(ethylthio)ethyl-N-(6-pivaloylamido-2-pyridylmethyl)amine; bmppa = N-bis-2-(methylthio)ethyl-N-(6-pivaloylamido-2-pyridylmethyl)amine) have been synthesized and characterized. Treatment of these complexes with 1 equiv of Me4NOH·5H2O in MeOH results in quantitative amide alcoholysis.
Sodium borohydride-reduced, carboxymethylated holotryptophanase was digested with trypsin, and the resulting peptides were purified by column chromatography on basic and acidic ion exchange resins, and by paper chromatography and electrophoresis. The isolation and sequence determinations of 13 peptides, including the NH2- and COOH-terminal peptides, those containing carboxymethylcysteine or tryptophan, and that containing the pyridoxyllysine residue (Lys(Pxy)) are reported here, together with a summary of the sequences of a total of 54 tryptic peptides present in the digest. The NH2-terminal peptide, Met-Glu-Asn-Phe-Lys, and the COOH-terminal peptide, Leu-Lys-Glu-Val, were isolated, and an extended sequence at the COOH-terminal end of tryptophanase was proposed to be His-Thr-Phe-Ala-Lys-Leu-Lys-Glu-Val on the basis of carboxypeptidase A experiments and the sequence of an isolated pentapeptide. The amino acid sequence of an extended portion of the peptide chain around the reduced pyridoxal 5-phosphate residue was determined to be Tyr-Ala-Asp-Met-Leu-Ala-Met-SerAla-Lys-Lys(Pxy)-Asp-Ala-Met-Val-Pro-Met-Gly-Gly-Leu-Leu-Cys(Cm)-Met-Lys. Tryptophanase contains 2 tryptophan and 6 cystine residues per subunit of 55,000 molecular weight, and two distinct tryptophan peptides and five distinct carboxymethylcysteine peptides were isolated and sequenced. The studies thus support the view that this subunit contains a single peptide chain. Of a total of about 474 amino acid residues per subunit, 411 were recovered in the 54 tryptic peptides so far studied.
Methionine aminopeptidases with a universal specificity have been revealed from the sequences of the amino-terminal region of mutant forms of yeast iso-1-cytochrome c and from a systematic examination of the literature for amino-terminal sequences formed at initiation sites. The aminopeptidase removes amino-terminal residues of methionine when they precede certain amino acids, with a specificity that appears to be determined mainly by the residue adjacent to the methionine residue at the amino terminus. The result with the mutationally altered iso-1-cytochromes c and the results from published sequences of other proteins from a wide range of prokaryotes and eukaryotes suggest that the aminopeptidase usually cleaves amino-terminal methionine when it precedes residues of alanine, cysteine, glycine, proline, serine, threonine and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine or methionine. We suggest that the specificity is almost always determined simply by the size of the side chain of the penultimate residue; methionine is usually cleaved from residues with a side chain having a radius of gyration of 1·29 Å or less, but is not cleaved from residues with larger side chains.
The NH2-terminal residues of Bacillus subtilis soluble and ribosomal proteins were determined by the dinitrophenyl method. A nonrandom distribution of amino acids was observed. Alanine occurred most frequently, followed by methionine, serine, threonine, glutamic acid, and aspartic acid; smaller quantities of leucine, isoleucine, valine, and glycine were also found at the terminal position. A marked difference between these results and those obtained for Escherichia coli was noted. A slight difference in the frequency of distribution of the amino acids was also detected between log and stationary phase soluble proteins. An analysis of radioactive proteins newly synthesized by B. subtilis cells also indicated a preferential incorporation of alanine into the NH2-terminal residues.
Aided by grants AM-01845, AM-08953, and FR-05399 from the National Institutes of Health,
U.S. Public Health Service, the Jane Coffin Childs Fund for Medical Research, and E. I. dii Pont
de Nemours and Company, Inc.
Two amber mutations in the coat gene of the phage f2 have been shown to occur at the sites specifying amino acid residue 6 (sus-3) and residue 70 (sus-11) in the intact coat protein. RNA containing these amber codons directs the synthesis in vitro of suppressible peptide fragments, while RNA from wild-type phage directs mainly the synthesis of whole coat protein. These fragments have been identified as the NH2-terminal portions of the coat protein, which have been terminated at the residue coded for by the amber codon and, therefore, must have been produced by the translation of the amber codon as a signal to terminate protein synthesis. With the amber mutation at site 6 (sus-3), the translation of the amber codon as a chain termination signal leads to the release of the coat protein fragment (a hexapeptide) with its carboxy-terminus free of any attached tRNA. The amber codon can be translated in vitro as a chain terminating signal.
In cells of the zona fasciculata externa of the adrenal cortex of mice, the maximal value of nuclear volume is observed in evening and night time, while the mitotic peak occurs in the early part of the day. Ten day subcutaneous injection of 1.5 units of ACTH twice in 24 hr produced nuclear hypertrophy and stimulation of mitotic activity of cells of the zona fasciculata externa. The circadian periodicity of nuclear volume in mice injected with ACTH is disturbed, while the circadian rhythm of mitotic activity is retained.
The purified complement, the G factor of Nishizuka and Lipmann (1), and salt-washed ribosomes were, by themselves, virtually free of guanosine triphosphatase (GTPase) activity. By saturation with one component, GTP hydrolysis was linearly dependent upon the amount added of the other. The Km for GTP for the maximally active system was 2 × 10−6m, and the Ki for guanosine diphosphate was 7.6 × 10−6m. Addition of poly U and other polynucleotides and of sRNA stimulated GTPase.The GTPase was inhibited by sulfhydryl-blocking agents. The amino acid polymerization and GTPase functions of the ribosome were dissociated by brief heating to 55 °, which “uncoupled” by abolishing the synthetic but enhancing the hydrolytic effect.The function of GTP in amino acid polymerization is discussed.
Extracts of Escherichia coli B contain highly specific N-formyl-l-methionine amidohydrolase activity. With the exception of N-acetyl-l-methionine, all other formylated and acetylated amino acids tested were hydrolyzed at rates which were small compared with the rate of hydrolysis of N-formyl-l-methionine.
Extracts of an ornithine-requiring mutant of E. coli in which N-formyl-l-methionine amidohydrolase activity is only 1% of that observed with E. coli B have been used to demonstrate the existence of an amidohydrolase which removes the formyl group from dipeptides without prior hydrolysis of the peptide bond. This enzyme exhibits specificity for methionine as the formylated N-terminal amino acid.
The amidohydrolase activity observed in the ornithine-requiring mutant is also present in E. coli B. Thus extracts of E. coli B can readily hydrolyze formyl groups from N-formyl-l-methionine as well as from N-formyl-l-methionyl-l-alanine. It is concluded that these two activities are probably due to separate enzymes.
Extracts from Escherichia coli and Bacillus stearothermophilus contain enzymes which liberate amino groups from formylmethionyl peptides. Both extracts cleave formyl-Met-Ala-Ser to yield formate and Met-Ala-Ser, but not formylmethionine, as products. A number of formylmethionyl peptides are hydrolyzed much faster than other formyl peptides, acetyl-Met-Ala or formylmethionine. Furthermore, a protein fraction from E. coli which contains the peptide deformylase removes formate from protein made in vitro. These findings argue that the function of the peptide deformylases is to remove the formyl group from nascent protein. Another enzyme in the E. coli extract hydrolyzes formylmethionine, but the presence of this enzyme probably is not significant, because it has been identified as acetylornithine deacetylase. The peptide deformylase of E. coli is quite labile under a variety of experimental conditions and is inhibited strongly by thiols; so far the instability has prevented any significant purification.
Coliphage MS2 RNA directs the synthesis in Escherichia coli extracts of a protein product the tryptic peptides of which correspond to the peptides of the phage coat protein. Fingerprint analysis of the synthetic protein labeled with various [14C]amino aeids indicated that in each case only those coat protein peptides which contain a particular amino acid were labeled. The formation of whole coat protein molecules as the predominant product was shown by co-chromatography on Sephadex with authentic phage coat subunit. Sequential synthesis of coat protein ending at the carboxyl end was observed by pulse-labeling with radioactive amino acid and by analysis of the peptides formed in the presence of puromycin. In addition to phage coat protein, MS2 RNA directed the synthesis of other proteins. These could be preferentially labeled with histidine, an amino acid lacking in the coat protein, and partially separated on Sephadex.
Atherosclerosis is a complex disease with both genetic and environmental determinants. Apolipoprotein (Apo) E-deficient mice have been created that are highly susceptible to atherosclerosis. In order to assess the role of human apolipoprotein (hApo) A-I and high density lipoprotein (HDL) in atherosclerosis susceptibility, transgenic mice overexpressing the hApo A-I gene were crossed with Apo E-deficient mice. Apo E-/-, hApo A-I mice with two-fold elevation in HDL cholesterol have markedly diminished atherosclerosis with less fibroproliferative lesions by 8 months of age. A strong reciprocal relationship between HDL cholesterol levels and atherosclerosis was found with HDL levels accounting for 78% of the observed variance in mean lesion area. The effect of HDL on atherosclerosis resistance was independent of non-HDL cholesterol.
Bacterial sRNA has been fractionated into two methionine-accepting species, only one of which could be formylated. The binding of each charged species to ribosomes under the direction of a variety of trinucleoside diphosphates has been studied. The codeword AUG could be assigned to both species, whereas the related trinucleoside diphosphates GUG and UUG caused binding only of the species which could be formylated. The presence of the formyl group on this methionyl-sRNA did not alter its binding characteristics. The results of the binding studies were supported by investigations using a cell-free system directed by poly UAG or poly UG. Methionine was incorporated from the charged species, which could be formylated into the N-terminal position of polypeptides by both polymers whether this species was formylated or not. Poly UAG, but not poly UG, stimulated the incorporation of methionine into polypeptide from the methionyl-sRNA species, which could not be formylated. The analysis of this polypeptide product identified methionine in internal positions.
Synthesis and characterization are described of a new GTP analogue, 5′-guanylyl methylenediphosphonate. A methylene bridge is substituted for oxygen between the β and γ phosphorus atoms, thus preventing enzymic cleavage at this position.The effects of this analogue on protein biosynthesis have been studied in an in vitro system from Escherichia coli. It inhibits the homopolynucleotide-directed syntheses of polyphenylalanine, polylysine and polyproline. The inhibition takes place in a resolved system, starting with phenylalanyl sRNA, and is reversed by the addition of excess GTP. The degree of inhibition depends upon the concentration ratio of GTP and the analogue. The analogue does not affect the activity of amino acyl sRNA synthetases or pyruvate kinase. It is concluded that this analogue acts as a specific, competitive inhibitor of the GTP reaction in protein synthesis.
Binding of formyl methionyl-tRNA to ribosomes, directed by RNA extracted from the f2 bacteriophage, is dependent upon initiation factors, GTP and the presence of the formyl group. Binding of f2 RNA to ribosomes is, however, independent of initiating factors, amino acyl-tRNA and GTP.
A rapid, sensitive method is described for measuring C14-aminoacyl-sRNA interactions with ribosomes which are specifically induced by the appropriate RNA codewords prior to peptide-bond
formation. Properties of the codeword recognition process and the minimum oligonucleotide chain length required to induce
such interactions are presented. The trinucleotides, pUpUpU, pApApA, and pCpCpC, but not dinucleotides, specifically direct
the binding to ribosomes of phenylalanine-, lysine-, and proline-sRNA, respectively.
Since 5'-terminal, 3'-terminal, and internal codewords differ in chemical structure, three corresponding classes of codewords
are proposed. The recognition of each class in this system is described. The template efficiency of trinucleotide codewords
is modified greatly by terminal phosphate. Triplets with 5'-terminal phosphate are more active as templates than triplets
without terminal phosphate. Triplets with 3'- or 3' (2')-terminal phosphate are markedly less active as templates. These findings
are discussed in relation to the probable functions of terminal codewords. The modification of RNA and DNA codewords, converting
sense into missense or nonsense codewords, is suggested as a possible regulatory mechanism in protein synthesis.
The reaction between methionine and S-RNA of Escherichia coli has been investigated. It has been demonstrated that, after the initial attachment to its specific S-RNA, the free α-amino group of the attached methionine may become formylated.
Anderson, J. S., J. E. Dahlberg, M. S. Bretscher, M. Revel, and B. F. C. Clark, Nature,
216, 1072 (1967).
18 Ohta, T., S. Sarkar, and R. E. Thach, these PROCEEDINGS, 58, 1638 (1967).
I Marcker, K. A., and F. Sanger, J. Mol. Biol., 8, 835 (1964).
J M Adams
Adams, J. M., J. Mol. Biol., 33, 571 (1968).
7Horikoshi, K., and R. H. Doi, Arch. Biochem. Biophys., 122, 685 (1967).
9 Leder, P., and H. Bursztyn, Biochem. Biophys. Res. Commun., 24, 233 (1966).
10 Takeda, M., and F. Lipmann, these PROCEEDINGS, 56, 1875 (1966).
M Personal Communication
11 Ochoa, S., personal communication.
12 Nirenberg, M., and P. Leder, Science, 145, 1399 (1964).
J E Allende
H Weissbach Hershey
R E Monro
M R Lamborg
Allende, J. E., and H. Weissbach, Biochem. Biophys. Res. Commun., 28, 82 (1967).
20 Hershey, J. W. B., and R. E. Monro, J. Mol. Biol., 18, 68 (1966).
21 Fry, K. T., and M. R. Lamborg, J. Mol. Biol., 28, 423 (1967).