Palindromic units from E. coli as binding sites for a chromoid-associated protein

Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.     

Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli

We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.     

Phosphate binding sites identification in protein structures

Nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. Many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. Here we describe Pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of known structure. Pfinder uses the Query3D local structural comparison algorithm to scan a protein structure for the presence of a number of structural motifs identified for their ability to bind the phosphate chemical group. Pfinder has been tested on a data set of 52 proteins for which both the apo and holo forms were available. We obtained at least one correct prediction in 63% of the holo structures and in 62% of the apo. The ability of Pfinder to recognize a phosphate-binding site in unbound protein structures makes it an ideal tool for functional annotation and for complementing docking and drug design methods. The Pfinder program is available at http://pdbfun.uniroma2.it/pfinder.     

Studies on the RNA and protein binding sites of the E. coli ribosomal protein L10.

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.     

Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli

A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25% of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport.     

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.     

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin

Summary The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3OH-5P in the direction of the E. coli genetic map is recognized, at the second site the 5P-3OH strand.     

19F NMR evidence for interactions between the c-AMP binding sites on the c-AMP receptor protein from E. coli.

The 19F NMR spectra of 3-fluorotyrosine containing c-AMP receptor protein (CRP) from E. coli have been recorded in the presence of increasing amounts of c-AMP. One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c-AMP and shows both slow and fast exchange behaviour during the titration. This is evidence for interactions between the two c-AMP binding sites on the CRP dimer leading to different dissociation rate constants (less than or equal to 75 s-1; greater than or equal to 350 s-1) for complexes containing one and two c-AMP molecules.     

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).     

大肠杆菌周质蛋白提取工艺的研究

研究了精氨酸缓冲液在不同浓度、pH值及提取时间下的周质蛋白提取率,并以溶菌酶法、渗透压休克法作为对比。结果表明浓度0.4 mol/L,pH值8.0,提取时间为45 min时,周质目的蛋白达到0.89 mg/g湿菌,相比其他方法,周质蛋白提取率分别提高93%、187%。实验得到一种高效、方便的大肠杆菌周质蛋白提取工艺,为周质表达的重组蛋白大规模生产奠定了基础。     

Lipoic acid provokes inhibition and aggregation of the maltose binding protein of Escherichia coli

Lipoic acid provokes aggregation of the monomeric maltose binding protein of Escherichia Coli into dimers and tetramers, and inhibits maltose binding. The sigmoidal shape of the curves showing the dependence of maltose binding versus lipoic acid concentration, and versus maltose concentration (in the presence of lipoic acid) suggests that the inhibition of the maltose binding protein by lipoic acid is a consequence of its aggregation. These results are discussed in relation to recent studies describing dimers of the maltose binding protein purified under certain conditions, and in relation to results suggesting an implication of lipoic acid in the binding protein-dependent transports.     

Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli.

The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.     

Quantitative analysis of ribosome binding sites in E.coli.

185 clones with randomized ribosome binding sites, from position -11 to 0 preceding the coding region of beta-galactosidase, were selected and sequenced. The translational yield of each clone was determined; they varied by more than 3000-fold. Multiple linear regression analysis was used to determine the contribution to translation initiation activity of each base at each position. Features known to be important for translation initiation, such as the initiation codon, the Shine/Dalgarno sequence, the identity of the base at position -3 and the occurrence of alternative ATGs, are all found to be important quantitatively for activity. No other features are found to be of general significance, although the effects of secondary structure can be seen as outliers. A comparison to a large number of natural E.coli translation initiation sites shows the information profile to be qualitatively similar although differing quantitatively. This is probably due to the selection for good translation initiation sites in the natural set compared to the low average activity of the randomized set.     

Interactions between TonB from Escherichia coli and the periplasmic protein FhuD

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.     

Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli

Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.     

The MalK protein of the ATP-binding cassette transporter for maltose of Escherichia coli is accessible to protease digestion from the periplasmic side of the membrane.

The ATP-hydrolyzing subunit MalK of the ATP-binding cassette transporter for maltose of Escherichia coli is demonstrated to be accessible to digestion by proteinase K in right-side-out membrane vesicles. This finding suggests a partial transmembrane orientation of the protein.