Mobility of the terminal regions of flagellin in solution.

The mobility of the disordered terminal regions of flagellin was examined in detail based on 1H NMR chemical shifts and spin-lattice relaxation times in the rotating frame. Proteolytic fragments of flagellin with terminal deletions of different sizes were used to compare the dynamical properties of various N- and C-terminal segments. We found that dynamic properties of different terminal segments were similar to each other and were close to those of the heat-denatured state of flagellin. The main chain of these terminal segments undergoes rapid motions with effective correlation times of 1.3-4.1 x 10(-9) s. The terminal regions contain no large segments with well-defined structure. However, comparison with the random-coiled state of poly-L-lysine suggests significant structural constraints in the terminal regions (as well as in the heat-denatured flagellin) which may reflect the existence of some highly fluctuating secondary structure, as suggested by earlier CD studies.     

Interaction of the disordered terminal regions of flagellin upon flagellar filament formation

Helical filaments of bacterial flagella are built up by a self-assembly process from thousands of flagellin subunits. To clarify how the disordered terminal regions of flagellin interact upon filament formation, polymerization ability of various terminally truncated fragments was investigated. Fragments deprived of 19 N-terminal residues were able to bind to the end of filaments, however, only a single layer was formed. Removal of C-terminal segments or truncation at both ends resulted in the complete loss of binding ability. Our observations are consistent with the coiled-coil model of filament formation, which suggests that the alpha-helical N- and C-terminal regions of axially adjacent subunits form an interlocking pattern of helical bundles upon polymerization.     

Role of the disordered terminal regions of flagellin in filament formation and stability

Terminal regions of flagellin from Salmonella typhimurium, residues 1 to 65 and 451 to 494, have no ordered tertiary structure in solution, which makes them very susceptible to proteolytic degradation. Flagellin was subjected to mild controlled proteolytic treatment with highly specific proteases to remove terminal segments from the disordered regions. It is demonstrated here that various fragments can be readily prepared that differ from each other in 1 x 10(3) to 2 x 10(3) Mr segments in their NH2- or COOH-terminal regions. Terminally deleted fragments of flagellin were used to clarify the role of the disordered regions in the self-assembly of flagellin. The polymerization ability of the fragments was tested by inducing filament formation with ammonium sulfate. We found that fragments of flagellin containing large terminal deletions could form straight filaments, although the stability of these filaments required high salt concentrations. Even a fragment lacking the whole mobile COOH-terminal part of flagellin and 36 residues from the NH2-terminal region could form long filaments. The fragments could be also polymerized onto native flagellar seeds, suggesting that the subunit packing of the filaments of fragments is similar to that of the native ones. The fragments could also copolymerize with native flagellin, resulting in various helical forms. Filaments of fragments were found to be straight at both pH 4.0 and pH 12.5, indicating that they might have lost their polymorphic ability. Our results show that the major part of the disordered terminal regions of flagellin is not essential for polymerization, but it does play an important role in stabilization of the filaments and in influencing their polymorphic conformation.     

Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin

The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.     

Conformational adaptability at GABA-sensitive inhibitory synapses.

Receptors for γ-aminobutyric acid (GABA) and its agonists display a considerable tolerance to the size of the agonist molecule. By considering the potencies of four rigid GABA analogues, it is possible to construct a model for the elasticity of the receptor. Using this model in conjunction with the probability distributions of the charge separations of 12 GABA agonists, based on classical potential energy calculations, interaction probabilities are calculated which enable the molecular structure of the agonists to be correlated with their pharmacological activity.     

Plant adaptability in karst regions

Journal of Plant Research - Karst ecosystems are formed by dissolution of soluble rocks, usually with conspicuous landscape features, such as sharp peaks, steep slopes and deep valleys. The plants...     

Terminal regions of flagellin are disordered in solution

Limited proteolysis of flagellin from Salmonella typhimurium SJW1103 by subtilisin, trypsin and thermolysin results in homologous degradation patterns. The terminal regions of flagellin are very sensitive to proteolysis. These parts are degraded into small oligopeptides at the very early stage of a mild digestion that yields a relatively stable fragment with a molecular weight of 40,000. Further proteolytic degradation results in a stable 27,000 Mr fragment. The 40,000 Mr tryptic fragment has been identified as residues 67 to 446 of the flagellin sequence, while the 27,000 Mr fragment involves the 179 to 418 segment. The NH2-terminal sequence positions for the corresponding fragments produced by subtilisin are 60 and 174 for the 40,000 Mr and 27,000 Mr fragments, respectively. The fragments lost their polymerizing ability. Structural properties of flagellin and its 40,000 Mr tryptic fragment were compared by circular dichroism spectroscopy and differential scanning calorimetry. Analysis of the calorimetric melting profiles suggests that terminal parts of flagellin have no significant internal stability and they are in extensive contact with water. However, these regions contain some secondary structure, probably alpha-helices, as revealed by comparison of the circular dichroic spectra in the far-ultraviolet region. Our results indicate that, although the terminal regions of flagellin may contain some alpha-helical secondary structure of marginal stability, they have no compact ordered tertiary structure in solution. On the contrary, the central region of the molecule involves at least two compact structural units.     

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition. The other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.     

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases.

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases.     

Conformational studies of peptides corresponding to the coeliac-activating regions of wheat alpha-gliadin.

The structures of four peptides corresponding to parts of the coeliac-activating protein A-gliadin were studied by structure prediction and c.d. spectroscopy. Three of the peptides corresponded to parts of the coeliac-activating N-terminal region (residues 3-55, 3-19 and 39-45) and contained two tetrapeptide motifs common to all coeliac-active regions (Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro). The Pro-Ser-Gln-Gln sequence was also present in the fourth peptide, on the basis of the C-terminal part of the molecule (211-217). These studies showed that beta-reverse turns were the predominant structural feature in all peptides and were predominantly of type I/III in two of the N-terminal peptides and type II in the C-terminal peptide. These turns form when the peptide is dissolved in solvents of low dielectric constant (trifluoroethanol) and high dielectric constant (water and iso-osmotic saline), although their presence in the N-terminal peptides may be masked in the latter solvents due to equilibrium with a poly-L-proline II structure favoured at lower temperatures.     

Collagen fibril formation in vitro. The role of the nonhelical terminal regions.

We showed previously that fibril formation in vitro from rat tail tendon collagen requires a temperature-dependent initiation (Step 1) following which linear assembly to form thin filaments (Step 2) proceeds as rapidly at 4 degrees C as at 26 degrees C. Step 3, lateral assembly of filaments to form fibrils, is again temperature-dependent. We now find that Step 1 is complete in 6 min at 26 degrees C and the time is independent of collagen concentration in the range 0.08 to 0.39 mg/ml. Collagen treated with pepsin, which removes the nonhelical ends but leaves the triple helix intact, forms fibrils by a similar mechanism. However, Step 1 is altered or absent and early temperature changes produce a complex response consistent with an alternate, counterproductive pathway. Assembly is also much slower, particularly Step 2, and the fibrils formed are abnormal in that native banding is often absent and short tactoidal forms are common. These results suggest that in the assembly of fibrils from normal collagen the nonhelical ends are involved in an early conformational change and critically regulate later steps.     

Nucleotide sequences of the retroviral long terminal repeats and their adjacent regions.

The nucleotide sequences of the LTRs and their adjacent regions from 19 type C and one type B retrovirus were compared. Salient features are: (a) The R regions in the genomes of most of the type C retroviruses begin with GC and end with CA. (b) The mammalian type C retroviruses have a polyadenylation signal "AATAAA" in the R region, and most have a "CAT" box and a "TATA" box in the U3 region. (c) The avian type C retroviruses have an AATAAA sequence, and some also have "CAT-like" and "TATA-like" boxes, in the U3 region. (d) As with many transposable elements, the IR regions of the proviruses begin with TG and end with CA, and the DR sequences in the host genomes flanking the proviruses are different from one another. Although SNV is an avian retrovirus, the nucleotide sequences in the R, U5, TBS, and PU region are more similar to the mammalian type C than to the avian type C retroviruses.     

Evolution of the terminal regions of the Streptomyces linear chromosome

Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities.     

Resonant and localized breathing modes in terminal regions of the DNA double helix.

A Green's function approach is used in constructing a dynamic model of a semi-infinite length of the DNA homopolymer B poly(d) . poly(d). Considerable attention is focused on the hydrogen bond stretching close to the terminus. A melting (or breathing) coordinate (M) is defined as an average over the three linking hydrogen bond stretches in a unit cell. The thermal mean squared amplitude of (M) is enhanced at the chain end compared with the interior. Spectral branches at 69, 80 and 105 cm-1, as well as a local mode at 75 cm-1, are primary contributors to the enhancement. We suggest that this fact can affect the thermal melting of a DNA double helical homopolymer, enhancing the tendency to start from an end (if one is available). We show how certain infinite chain modes with small (M) amplitude can turn into breathing modes near the terminus, and suggest that the same phenomenon may occur near other specific base-pair sequences. There is also considerable attention paid to the low microwave region from approximately 0 to 1.75 cm-1. The thermally activated modes in this frequency region contribute approximately (0.02 A)2 to [M2(0)] at 40 K, approximately two orders of magnitude greater than for [M2(infinity)]. Most important however, is the existence of narrow resonant modes in this frequency region. Particularly pronounced resonances near 0.03 cm-1 and 0.08 cm-1 (approximately 0.9 and 2.4 GHz) amplify M2(0) at the terminus by about for orders of magnitude over the infinite chain value M2(infinity).     

Symmetric-strand packaging of recombinant parvovirus LuIII genomes that retain only the terminal regions.

LuIII is an autonomous parvovirus which encapsidates either strand of its genome with similar efficiency in NB324K cells. Two parvoviruses closely related to LuIII, minute virus of mice (MVM) and H-1 virus, encapsidate primarily the minus strand of their genome when grown in the same cell type. It has been postulated that an AT-rich region unique to LuIII is responsible for symmetric encapsidation of plus- and minus-strand genomes by LuIII. To address this hypothesis, recombinant LuIII-luciferase genomes containing or lacking the AT-rich sequence (AT) were packaged into LuIII virions. Hybridization of strand-specific probes to DNA from these virions revealed that either strand of the genome was packaged regardless of the presence of AT. In addition, encapsidation of both strands of the AT+ LuIII-luciferase genome into MVM and H-1 virions was observed, suggesting that MVM and H-1 viral proteins are not responsible for the minus-strand packaging bias of these two viruses. Alignment of the published LuIII and MVMp sequences shows that AT exists as an insertion into an element that, in MVM, binds cellular proteins. We suggest that in LuIII, AT disrupts binding of these cellular proteins, allowing encapsidation of either strand.     

Conformational regions of Boc-Ala-Aib-Ala-OMe. Sampling with molecular dynamics simulations using time averaging of distance restraints.

The method of time averaging of distance restraints in molecular dynamics simulations is applied to Boc-Ala-Aib-Ala-OMe in order to demonstrate the improved sampling properties of this method compared to conventional distance restraining. Two conformational regions, beta-turn type II and gamma-turn, are seen during MD runs at a simulation temperature of 500 K, while in simulations with conventional distance restraining, no conformational transitions could be observed for temperatures up to 1000 K.