Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia

Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed to specific recognition of the A chain by diphtheria toxin's translocation pathway because the translocation pathway also accommodates folded myoglobin.     

Dynamics of N2O in vicinity of plant residues: a microsensor approach

Plant and Soil - Plant residues decomposing within the soil matrix are known to serve as hotspots of N2O production. However, the lack of technical tools for microscale in-situ N2O measurements...     

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS^ΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.     

Immunity protein is not required for the bactericidal activity of colicin E3

Plasmid pLAX3, carrying the colicin E3 gene, was used to direct the in vitro synthesis of a colicin E3* molecule totally devoid of its immunity protein. We established that this molecule is able to kill sensitive Escherichia coli cells in the total absence of immunity protein. Therefore, all of the information required for colicin E3 action is located on the colicin polypeptide itself. Furthermore, our studies indicated that immunity protein protects the C-terminal enzymatic part of native colicin E3 protein against proteolytic degradation before or during its translocation across the cell envelope. These results are discussed in relation to the mode of entry of colicin E3 into bacterial cells.     

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab¹ shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.     

Estimating crop N uptake from organic residues using a new approach to the 15N isotope dilution technique

Experiments were conducted to test a new approach to the ¹⁵N isotope dilution technique for estimating crop N uptake from organic inputs. Soils were pre-labelled with ¹⁵N fertiliser and a carbon source. These were then incubated until there was stabilisation of the ¹⁵N abundance of the inorganic N pool and resumption of inorganic N concentrations. Residues were then applied to the soils and planted with ryegrass (Lolium perenneL.) to determine the nitrogen derived from the residue (Ndfr) using the isotope dilution equations. This method was compared with the direct method, i.e. where ¹⁵N-labelled residues were added to the soil and Ndfr in the ryegrass calculated directly. Estimates of percentage nitrogen derived from the residue (%Ndfr) alfalfa (Medicago sativaL.) in the ryegrass, were similar, 22 and 23% for the direct and soil pre-labelling methods, respectively, in the Wechsel sandy loam. Also, estimates of the %Ndfr from soybean (Glycine max (L.) Merr) residues in the Krumbach sandy loam were similar 34% (direct) and 36% (soil pre-labelling approach). However, in the Seibersdorf clay loam, the %Ndfr from soybean was 49% using the direct method and 61% using the soil pre-labelling method; yet Ndfr from common bean residue was 46% using the direct approach and 40% using the pre-labelling, not significantly different (P > 0.05). The soil pre-labelling approach appears to give realistic values for Ndfr. It was not possible to obtain an estimate of Ndfr using the soil pre-labelling method from the maize residues (Zea mays L.) in two of the soils, as there was no increase in the total N of the ryegrass over the growing period. This was probably due to microbial immobilisation of inorganic N, as a result of the wide C:N ratio of the residue added. The results suggest that the new soil pre-labelling method is feasible and that it is a potentially useful technique for measuring N release from a wide range or organic residues, but it requires further field-testing. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cationic surfactant mediated fibrillogenesis in bovine liver catalase: a biophysical approach

Protein aggregation into oligomers and mature fibrils are associated with more than 20 diseases in humans. The interactions between cationic surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) with varying alkyl chain lengths and bovine liver catalase (BLC) were examined by various biophysical approaches. The delicate coordination of electrostatic and hydrophobic interactions with protein, play imperative role in aggregation. In this article, we have reconnoitered the relation between charge, hydrophobicity and cationic surfactants DTAB and TTAB on BLC at pH 7.4 and 9.4 which are two and four units above pI, respectively. We have used techniques like turbidity, Rayleigh light scattering, far-UV CD, ThT, ANS, Congo red binding assay, DLS, and transmission electron microscopy. The low concentration ranges of DTAB (0–600 μM) and TTAB (0–250 μM) were observed to increase aggregation at pH 9.4. Nevertheless, at pH 7.4 only TTAB was capable of inducing aggregate. DTAB did not produce any significant change in secondary structure at pH 7.4 suggestive of the role of respective charges on surfactants and protein according to the pI and alkyl chain length. The morphology of aggregates was further determined by TEM, which proved the existence of a fibrillar structure. The surfactants interaction with BLC was primarily electrostatic as examined by ITC. Our work demystifies the critical role of charge as well as hydrophobicity in amyloid formation.     

Comparing the efficiency of sensory systems: A biophysical approach

This article is an exposition of the view that the complexity of biological systems permitsintensive (as opposed toextensive) research on any level. Specifically, (i) new views onstructural information theory used to compare the efficiency of sensory systems in measuring the theoretically derived maximum amount of information in the sensory environment, (ii) the concept of the brain system involving the amygdaloid complex and the hypothalamus as acoupled harmonic oscillator system, and (iii) the concept of the cerebral cortex as ane.m. interferometric (holographic) structure, are outlined.     

Mutagenesis of murine granulocyte/macrophage-colony-stimulating factor reveals critical residues near the N terminus

A number of cDNAs encoding mutant forms of the murine haemopoietic growth factor, granulocyte/macrophage-colony-stimulating factor (GM-CSF), have been derived by in vitro mutagenesis and expressed in simian COS cells. Determination of the biological activity of the mutant factors revealed that residues within the regions 11-15, 24-37, 47-49 and 81-89 are required for generating a functional GM-CSF molecule. In particular, truncation of either of two strongly predicted alpha helices near the N terminus of the molecule severely depresses the activity of the factor.     

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin.

The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7. Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively. Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins. Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin. In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8. This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins.     

Study of the interaction between fisetin and human serum albumin: a biophysical approach

The binding of fisetin with human serum albumin (HSA) has been studied at different pH using UV-Vis, FTIR, CD and fluorescence spectroscopic techniques. The binding constants were found to increase with the rise in pH of the media. The negative ΔH° (kJ mol-1) and positive ΔS° (J mol-1 K-1) indicate that fisetin binds to HSA via electrostatic interactions with an initial hydrophobic association that result in a positive ΔS° . In presence of potassium chloride (KCl) the binding constants were found to be decrease. The α-helical content of HSA increased after binding with fisetin as analyzed from both CD and FTIR methods. The site marker displacement studies using fluorescence anisotropy suggest that fisetin binds to the hydrophobic pocket (Site 1, subdomain IIA) of HSA which is in good accordance with the molecular docking study. The change in accessible surface area (ASA) of residues of HSA was calculated to get a better insight into the binding.     

Structure-function relationship of lapemis toxin: a synthetic approach

The synthetic approach to the structure-function relationship of lapemis toxin has been very useful in clarifying the important binding regions. To identify the neurotoxic binding domain(s) of lapemis toxin, several peptides were synthesized using the 9-fluorenylmethoxycarbonyl protocols. These peptides were based on the sequence of lapemis toxin, a 60-amino-acid, short-chain postsynaptic neurotoxin found in sea snake (Lapemis hardwickii) venom. The peptides were purified using high-performance liquid chromatography and sequenced to verify the correct synthesis, isolation, and purity. The synthetic peptide names and single letter sequences were Peptide A1 (15 mer) CCNQQSSQPKTTTNC Peptide B1 (18 mer) CYKKTWSDHRGTRIERGC Peptide B2 (16 mer) YKKTWSDHRGTRIERG Peptide C1 (12 mer) CPQVKPGIKLEC Peptide NS (20 mer) EACDFGHIKLMNPQRSTVWY. The peptide NS (nonsense peptide) sequence was arbitrarily determined and used as a control peptide. Biological activities of the synthetic peptides were determined by in vivo as well as by in vitro assay methods. For the in vivo assay, lethality was determined by intravenous injection in mice (Swiss Webster). For the in vitro assay, peptide binding to the Torpedo californica nicotinic acetylcholine receptor was determined. The peptides were found to be nontoxic at approximately 114 times the known LD50 of lapemis toxin. Binding studies with 125I-radiolabeled lapemis toxin and tyrosine-containing peptides indicated that lapemis toxin and peptide B1 bound the receptor, while the other peptides had no detectable binding. The central loop domain of lapemis toxin (peptide B1) plays a dominate role in the toxin's binding ability to the receptor. These results and the hydrophilicity analysis predict peptide B1 may serve as an antagonist or antigen to neutralize the neurotoxin effects in vivo.     

Multistep binding of transition metals to the H-N-H endonuclease toxin colicin E9

We report the first stopped-flow fluorescence analysis of transition metal binding (Co(2+), Ni(2+), Cu(2+), and Zn(2+)) to the H-N-H endonuclease motif within colicin E9 (the E9 DNase). The H-N-H consensus forms the active site core of a number of endonuclease groups but is also structurally homologous to the so-called treble-clef motif, a ubiquitous zinc-binding motif found in a wide variety of metalloproteins. We find that all the transition metal ions tested bind via multistep mechanisms. Binding was further dissected for Ni(2+) and Zn(2+) ions through the use of E9 DNase single tryptophan mutants, which demonstrated that most steps reflect conformational rearrangements that occur after the bimolecular collision, many common to the two metals, while one appears specific to zinc. The kinetically derived equilibrium dissociation constants (K(d)) for transition metal binding to the E9 DNase agree with previously determined equilibrium measurements and so confirm the validity of the derived kinetic mechanisms. Zn(2+) binds tightest to the enzyme (K(d) approximately 10(-)(9) M) but does not support endonuclease activity, whereas the other metals (K(d) approximately 10(-)(6) M) are active in endonuclease assays implying that the additional step seen for Zn(2+) traps the enzyme in an inactive but high affinity state. Metal-induced conformational changes are likely to be a conserved feature of H-N-H/treble clef motif proteins since similar Zn(2+)-induced, multistep binding was observed for other colicin DNases. Moreover, they appear to be independent both of the conformational heterogeneity that is naturally present within the E9 DNase at equilibrium, as well as the conformational changes that accompany the binding of its cognate inhibitor protein Im9.     

Identification of specific residues in colicin E1 involved in immunity protein recognition

The basis of specificity between pore-forming colicins and immunity proteins was explored by interchanging residues between colicins E1 (ColE1) and 10 (Col10) and testing for altered recognition by their respective immunity proteins, Imm and Cti. A total of 34 divergent residues in the pore-forming domain of ColE1 between residues 419 and 501, a region previously shown to contain the specificity determinants for Imm, were mutagenized to the corresponding Col10 sequences. The residue changes most effective in converting ColE1 to the Col10 phenotype are residue 448 at the N terminus of helix VI and residues 470, 472, and 474 at the C terminus of helix VII. Mutagenesis of helix VI residues 416 to 419 in Col10 to the corresponding ColE1 sequence resulted in increased recognition by Imm and loss of recognition by Cti.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo , whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37°C. At 0°C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37°C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37°C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.     

Surface aspartate residues are essential for the stability of colicin A P-domain: a mechanism for the formation of an acidic molten-globule

The pore-forming domains of members of a family of bacterial toxins, colicins N and A, share > 50% sequence identity, identical folds and yet display strikingly different behavior in acid conditions. At low pH colicin A forms a molten globule state while colicin N retains a native fold. This is relevant to in vivo activity since colicin A requires acidic phospholipids for its toxic activity but colicin N does not. The pI of colicin A (5.25) is far lower than that of colicin N (10.2) because colicin A contains seven extra aspartate residues. We first introduced separately each of these acidic amino acids into homologous sites in colicin N, but none caused destabilization at low pH. However, in the reverse experiment, the sequential replacement of these acidic side chains of colicin A by alanine revealed six sites where this change destabilized the protein at neutral pH. Some of these residues, which each contribute less than 4% to the total negative charge, appear to stabilize the protein via a network of hydrogen bonds and charge pairs which are sensitive to protonation. Other residues have no clear interactions that explain their importance. The colicin A is thus a protein that relies upon acid sensitive interactions for its stability at neutral pH and its in vivo activity.