Visualization of the nature of protein folding by a study of a distance constraint approach in two-dimensional models

A distance constraint approach is applied to two-dimensional models of proteins in order to visualize the nature of protein folding and to examine the relative roles of different ranges of interaction. Three different native structures (I, II, and III) are considered; they have two different kinds of residues, viz., hydrophobic and hydrophilic, and different sequences of these residues. We examine how the distance constraint approach functions in the prediction of protein folding when we know the sequence of the residues, the (fixed) bond lengths, the mean distances between residues i and i + 2, and i and i + 3, and the mean distances for hydrophobic–hydrophobic, hydrophobic–hydrophilic, and hydrophilic–hydrophilic contacts between residues i and i + j, where j ≥ 4. This approach involves optimization of an object function with respect to 98 variables and is not free of the multiple-minimum problem. The optimization is always terminated if the chain is entangled and/or the segments (residues) are packed too compactly to move. In order to escape from such situations and to take the excluded-volume effect into account, a Monte Carlo method is used after the optimization is trapped in local minima. Success in the prediction of folding is found to depend on the starting conformations and on the native conformations. Fair success is obtained in predicting the helix-like structure in protein I and the overall structure of protein III, but not the β-like structures of proteins I and II. Insofar as the prediction of the structure of protein III is reasonable, it appears that some sequences of residues produce greater constraints on their conformations than others, if one considers only the hydrophobic and hydrophilic nature of the residues. These results imply that, in the folding of real proteins in three dimensions, the competition for hydrophobic (and hydrophilic) residues for inside (outside) positions in the molecule probably constitutes a necessary but not a sufficient condition to form and stabilize the native structure. The failure to predict the structure of protein II, and part of that of protein I, suggests that there are two types of long-range interactions. One (which we considered here) is nonspecific (i.e., is defined only in terms of contacts between residues of the same or different polarity) and acts at any stage of protein folding; the other (which we did not consider here) is a specific interaction between residues in pairs and contributes only when the residues in the specific pair take on the native conformation. Presumably, incorporation of such specific long-range interactions, together with the nonspecific ones, is necessary for successful protein folding, using the distance constraint approach.     

Statistical mechanical treatment of α-helices and extended structures in proteins with inclusion of short- and medium-range interactions

A statistical mechanical model of protein conformation with medium-range interactions between theith and (i+k)^th residues (k<-4) is presented. Two two-state models, an α-helix-coil and an extended-structure-coil model, are formulated using the same form of the partition function, but the two models are applied independently to predict the locations of α-helical, extended, and coil segments; in the relatively few cases (<2%) where the predictions from the two models are in conflict, the prediction is scored as an incorrect one. Two independent sets of statistical weights (one set for each model) are derived to describe the interactions between the 20 amino acid residues for each range of interactionk; they are evaluated by minimizing an objective function so that the probability profiles for the α-helix or extended structure, respectively, in proteins computed from these statistical weights correlate optimally with the experimentally observed native conformations of these proteins. Examination of the resulting statistical weights shows that those for the interactions between hydrophobic residues and between a hydrophobic and a hydrophilic residue have reasonable magnitudes compared to what would be expected from the spatial arrangements of the side chains in the α-helix and the extended structure, and that those for the α-helix-coil model correlate well with experimentally determined values of the Zimm-Bragg parameterss and σ of the helix-coil transition theory. From the point of view of a method to predict the conformational states (i.e., α-helix, extended structure, and coil) of each residue, the statistical weights (as inall empirical prediction schemes) depend very much on the proteins used for the data base, since the presently available set of proteins of known structure is still too small for very high predictability; as a result, the correctness of the prediction is not very good for proteins not included in the data base. However, the correctness of the prediction, at least for the 37 proteins utilized as the data base in this study, is 91% and 87% for the α-helix-coil and the extended-structure-coil models, respectively; further, 79% of all the residues are predicted correctly when both the α-helix-coil and extended-structure-coil models are applied independently.     

Analysis of structural motifs of proteins using sets of codes, describing local structures]

An amino acid sequence pattern conserved among a family of proteins is called motif. It is usually related to the specific function of the family. On the other hand, functions of proteins are achieved by their 3D structures. Specific local structures, called structural motifs, are considered related to their functions. However, searching for common structural motifs in different proteins is much more difficult than for common sequence motifs. We are attempting in this study to convert the information about the structural motifs into a set of one-dimensional digital strings, i.e., a set of codes, to compare them more easily by computer and to investigate their relationship to functions more quantitatively. By applying the Delaunay tessellation to a 3D structure of a protein, we can assign each local structure to a unique code that is defined so as to reflect its structural feature. Since a structural motif is defined as a set of the local structures in this paper, the structural motif is represented by a set of the codes. In order to examine the ability of the set of the codes to distinguish differences among the sets of local structures with a given PROSITE pattern that contain both true and false positives, we clustered them by introducing a similarity measure among the set of the codes. The obtained clustering shows a good agreement with other results by direct structural comparison methods such as a superposition method. The structural motifs in homologous proteins are also properly clustered according to their sources. These results suggest that the structural motifs can be well characterized by these sets of the codes, and that the method can be utilized in comparing structural motifs and relating them with function.     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

Evaluation of trichloroethylene degradation by E. coli transformed with dimethyl sulfide monooxygenase genes and/or cumene dioxygenase genes

Pseudomonas fluorescens IP01 grown on isopropylbenzene (cumene) and Acinetobacter sp. 20B grown on dimethyl sulfide (DMS) degraded up to 90% and 25% of 1.5 mg trichloroethylene (TCE)/l, respectively. Escherichia coli harboring the DMS monooxygenase genes from strain 20B, the cumene dioxygenase genes from strain IP01 and both oxygenase genes, degraded up to 50%, 75% and 88% of 75 mg TCE/l, respectively. The growth rates of the E. coli recombinants remained nearly unaffected by TCE at 15 150 mg/l. Thus, the E. coli recombinants were indicated to degrade high concentrations of TCE efficiently at least up to 150 mg l–1.     

Lysyl oxidase coupled with catalase in egg shell membrane.

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).     

Unique DNA plasmid pRS64 associated with chromosomal DNAs of the plant pathogenic fungus Rhizoctonia solani.

Unique DNA sequences homologous to the linear DNA plasmid pRS64 were investigated in chromosomal DNAs of isolates belonging to anastomosis group 4 (AG-4) of the plant pathogenic fungus Rhizoctonia solani. Chromosome-sized DNAs of isolates RI-64 and 1271 of AG-4 were separated into six bands by orthogonal-field-alternation gel electrophoresis and hybridized to a cloned segment of pRS64. A small chromosome-sized DNA band of approximately 1.1 Mb carried the sequences homologous to pRS64 DNA. Sequences homologous to pRS64 were also maintained within the chromosomal DNA of isolate 127.1 of AG-4 which does not possess the plasmid. The plasmid showed no homology to the mitochondrial DNA of isolate 1271. The possibility that the linear plasmid pRS64 may act as a transposable genetic element is discussed.     

Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.