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1.
We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.  相似文献   

2.
Bae SH  Liu D  Lim HM  Lee Y  Choi BS 《Biochemistry》2008,47(7):1993-2001
Cnu is a nucleoid protein that has a high degree of sequence homology with Hha/YmoA family proteins, which bind to chromatin and regulate the expression of Escherichia coli virulence genes in response to changes in temperature or ionic strength. Here, we determined its solution structure and dynamic properties and mapped H-NS binding sites. Cnu consists of three alpha helices that are comparable with those of Hha, but it has significant flexibility in the C-terminal region and lacks a short alpha helix present in Hha. Upon increasing ionic strength, the helical structure of Cnu is destabilized, especially at the ends of the helices. The dominant H-NS binding sites, located at helix 3 as in Hha, reveal a common structural platform for H-NS binding. Our results may provide structural and dynamic bases for the similarity and dissimilarity between Cnu and Hha functions.  相似文献   

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Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli.  相似文献   

6.
Mukherjee A  Bagchi B 《Biochemistry》2006,45(16):5129-5139
Hydropathy scale is widely used to obtain a measure of the effective interaction between any two amino acid residues in proteins and is based on the assumption that attraction between two hydrophobic groups and repulsion between hydrophilic groups (in water) can be translated straightforwardly to protein environment. Here we employ a recently developed statistical mechanical approach combined with the Protein Data Bank to obtain both distance- and orientation-dependent potential of mean force (ODPMF). This allowed us to explore effective pair potential among many amino acid residues and to examine the validity of the hydropathy scale in modeling the interaction among amino acid residues. We find that in some cases, like Phe-Phe and Lys-Lys, the hydropathy scale approach is largely obeyed. However, we also observe many unexpected pair interactions which defy the trend given by published hydropathy scales. An example of the former is the arginine-arginine (Arg-Arg) pair interaction which is found to be strongly and surprisingly attractive at short separation, even though it is the most hydrophilic residue. Here the head-to-head (see text) interaction is also stabilized. Tryptophan residues also exhibit strong attractive interaction. Equally important, we find strong influence of metal in determining effective interaction among the amino acid residues. It is the behavior of the histidine (His) which is found to be the most unusual. It exhibits a strong attractive interaction with itself which gets significantly enhanced in metalloproteins. These results highlight the important (sometime hidden) role of metals in protein structure and folding.  相似文献   

7.
We previously showed (V. W. Raymond and J. T. Parsons, Virology 160:400-410, 1987) that variants of the Prague A strain of Rous sarcoma virus containing large deletions impinging on a region of the src gene encoding amino acid residues 143 to 169 were defective for transformation of chicken cells in culture. Here we report that introduction of small (tri-and tetrapeptide) deletions into a region of pp60v-src containing amino acid residues 155 to 175 was found to inactivate transformation. In addition, insertion of four, but not one, amino acid residues at position 161 also inhibited transformation. Biochemical analysis of the src proteins encoded by individual transformation-defective variants revealed that the structural alterations introduced into this domain had only marginal effects upon src tyrosine-specific protein kinase activity. However, the src proteins encoded by defective variants exhibited a significantly shorter half-life within the cell, although these proteins efficiently and rapidly associated with cellular membranes. Our results suggest that the structural domain encompassing residues 155 to 177 may influence the stability of pp60src in the cellular membrane, possibly via the interaction of src with a cellular membrane component(s) or substrate(s).  相似文献   

8.
R M Williams  S Rimsky    H Buc 《Journal of bacteriology》1996,178(15):4335-4343
Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.  相似文献   

9.
The primary sequence of H-NS (136 amino acid residues, Mr = 15,402), an abundant Escherichia coli DNA-binding protein, has been elucidated and its quaternary structure has been investigated by protein-protein cross-linking reactions. It was found that H-NS exists predominantly as a dimer, even at very low concentrations, but may form tetramers at higher concentrations and that the protein-protein interaction responsible for the dimerization is chiefly hydrophobic.  相似文献   

10.
Summary Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.  相似文献   

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Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .  相似文献   

13.
Summary A class of trans-acting mutations, which alter the osmoregulated expression of the Escherichia coli proU operon, maps at 27 min on the chromosome in a locus we have called osmZ. Mutations in osmZ are allelic to bglY, pilG and virR, affect gene expression, increase the frequency of the site-specific DNA inversion mediating fimbrial phase variation, stimulate the formation of deletions, and influence in vivo supercoiling of reporter plasmids. We have cloned the osmZ + gene, mapped it at 1307 kb of the E. coli restriction map, identified its gene product as a 16 kDa protein, and determined the nucleotide sequence of the osmZ + gene. The deduced amino acid sequence for OsmZ predicts a protein of 137 amino acid residues with a calculated molecular weight of 15 530. The primary sequence of OsmZ is identical to that of H-NS (H1a), a DNA-binding protein that affects DNA topology and is known to be associated with the bacterial nucleoid. Thus, osmZ is the structural gene for the H-NS (H1a) protein. The nucleotide sequence of osmZ is almost identical to that of hns; however, hns was incorrectly located at 6.1 min on the E. coli linkage map. Increased osmZ gene dosage leads to cell filament formation, altered gene expression, and reduced frequency of fimbrial phase variation. Our results suggest that the nucleoid-associated DNA-binding protein H-NS (H1a) plays a critical role in gene expression and in determining the structure of the genetic material.  相似文献   

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Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor. Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor. Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia. To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken. This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3. Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced. The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE). In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152. Both variants were defective, exhibiting 13 and 27% of normal binding, respectively. In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity. These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.  相似文献   

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Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.  相似文献   

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The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae . Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans -activator protein in Rhodobacter capsulatus , has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli . Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides , XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae . These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.  相似文献   

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