Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. (1/848)

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.  (+info)

Simplified methods for pKa and acid pH-dependent stability estimation in proteins: removing dielectric and counterion boundaries. (2/848)

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.  (+info)

Structure in the channel forming domain of colicin E1 bound to membranes: the 402-424 sequence. (3/848)

To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402-424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406-416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402-420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408-R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.  (+info)

A cytotoxic ribonuclease targeting specific transfer RNA anticodons. (4/848)

The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.  (+info)

Characterization of nonenterotoxigenic Escherichia coli strains producing F17 fimbriae isolated from diarrheic lambs and goat kids. (5/848)

Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA. Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of D-mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains.  (+info)

A theoretical and empirical investigation of the invasion dynamics of colicinogeny. (6/848)

A mathematical model describing the dynamics of a colicinogenic and a colicin-sensitive population propagated under serial transfer culture conditions was formulated. In addition, a series of in vitro invasion experiments using six representatives of the E colicin group was undertaken, together with the estimation of the growth rates and colicinogenic characteristics of the strains. Growth rates among the strains varied by up to 44%. There were 14-fold differences among strains in their lysis rates and there were up to 10-fold differences in the amount of colicin produced per lysed cell. The in vitro serial transfer invasion experiments revealed that regardless of initial frequency all colicinogenic strains succeeded in displacing the sensitive cell populations. The amount of time required for the colicin-sensitive cell population to be displaced declined as the initial frequency of the colicinogenic population increased and strains producing higher titres of colicin tended to displace the sensitive strain more rapidly. Overall, the observed dynamics of the invasion of colicinogenic strains was adequately described by the theoretical model. However, despite there being substantial differences among the strains in their growth rates and colicinogenic characteristics there were relatively few differences, observed or predicted, in the invasion dynamics of the six colicinogenic strains. These results suggest that the characteristics of different colicinogenic strains cannot be used to explain the extensive variation in the relative abundance of different colicins in natural populations of bacteria.  (+info)

Characterization of colicin S4 and its receptor, OmpW, a minor protein of the Escherichia coli outer membrane. (7/848)

Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.  (+info)

Colicin E1 forms a dimer after urea-induced unfolding. (8/848)

Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.  (+info)

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