The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides.

A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.     

Prediction of contact numbers of amino acid residues using a neural network regression algorithm

The profile of contact numbers of amino acid residues in proteins contains important information about the protein structure and is connected with the accessibility of residues to solvent. Here we propose a method for predicting the profile of contact numbers of residues in protein from its amino acid sequence. The method is based on regression using a neural network algorithm. The algorithm predicts two types of profiles, namely, the total number of contacts and the number of close contacts with the neighbors in the chain. The Pearson coefficient of correlation between the actual and predicted values of total contact numbers amounted to 0.526–0.703. As for the number of close contacts, this coefficient was higher (0.662–0.743) for all the considered threshold contact distances (6, 8, 10, and 12 Å). The program for prediction of contact numbers CONNP is available at http://wwwmgs2.bionet.nsc.ru/reloaded.

     

Protein subunit interfaces: a statistical analysis of hot spots in Sm proteins

The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the properties of Sm protein interfaces with those of a test set, the Binding Interface Database (BID). The comparison revealed that the main differences between the interfaces of Sm proteins and those of the BID set are the content of charged residues, the coordination numbers of the residues, knowledge-based pair potentials, and the conservation scores of hot spots. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surfaces, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Hot spots are residues that make up a small fraction of the interfaces, but they contribute most of the binding energy. These residues are critical to protein–protein interactions. Analyses of knowledge-based pair potentials of hot spot and non-hot spot residues in Sm proteins show that they are significantly different; their mean values are 31.5 and 11.3, respectively. In the BID set, this difference is smaller; in this case, the mean values for hot spot and non-hot spot residues are 20.7 and 12.4, respectively. Hence, the pair potentials of hot spots differ significantly for the Sm and BID data sets. In the interfaces of Sm proteins, the amino acids are tightly packed, and the coordination numbers are larger in Sm proteins than in the BID set for both hot spots and non-hot spots. At the same time, the coordination numbers are higher for hot spots; the average coordination number of the hot spot residues in Sm proteins is 7.7, while it is 6.1 for the non-hot spot residues. The difference in the calculated average conservation score for hot spots and non-hot spots in Sm proteins is significantly larger than it is in the BID set. In Sm proteins, the average conservation score for the hot spots is 7.4. Hot spots are surrounded by residues that are moderately conserved (5.9). The average conservation score for the other interface residues is 5.6. The conservation scores in the BID set do not show a significant distinction between hot and non-hot spots: the mean values for hot and non-hot spot residues are 5.5 and 5.2, respectively. These data show that structurally conserved residues and hot spots are significantly correlated in Sm proteins.     

Prediction of contact numbers of amino acid residues using a neural network regression algorithm

The profile of contact numbers of amino acid residues in proteins contains important information about the protein structure and is connected with the accessibility of residues to solvent. Here we propose a method for predicting the profile of contact numbers of residues in protein from its amino acid sequence. The method is based on regression using a neural network algorithm. The algorithm predicts two types of profiles, namely, the total number of contacts and the number of close contacts with the neighbors in the chain. The Pearson coefficient of correlation between the actual and predicted values of total contact numbers amounted to 0.526–0.703. As for the number of close contacts, this coefficient was higher (0.662–0.743) for all the considered threshold contact distances (6, 8, 10, and 12 Å). The program for prediction of contact numbers CONNP is available at http://wwwmgs2.bionet.nsc.ru/reloaded.     

Effects of Jet Fuel Spills on the Microbial Community of Soil

Hydrocarbon residues, microbial numbers, and microbial activity were measured and correlated in loam soil contaminated by jet fuel spills resulting in 50 and 135 mg of hydrocarbon g of soil⁻¹. Contaminated soil was incubated at 27°C either as well-aerated surface soil or as poorly aerated subsurface soil. In the former case, the effects of bioremediation treatment on residues, microbial numbers, and microbial activity were also assessed. Hydrocarbon residues were measured by quantitative gas chromatography. Enumerations included direct counts of metabolically active bacteria, measurement of mycelial length, plate counts of aerobic heterotrophs, and most probable numbers of hydrocarbon degraders. Activity was assessed by fluorescein diacetate (FDA) hydrolysis. Jet fuel disappeared much more rapidly from surface soil than it did from subsurface soil. In surface soil, microbial numbers and mycelial length were increased by 2 to 2.5 orders of magnitude as a result of jet fuel contamination alone and by 3 to 4 orders of magnitude as a result of the combination of jet fuel contamination and bioremediation. FDA hydrolysis was stimulated by jet fuel and bioremediation, but was inhibited by jet fuel alone. The latter was traced to an inhibition of the FDA assay by jet fuel biodegradation products. In subsurface soil, oxygen limitation strongly attenuated microbial responses to jet fuel. An increase in the most probable numbers of hydrocarbon degraders was accompanied by a decline in other aerobic heterotrophs, so that total plate counts changed little. The correlations between hydrocarbon residues, microbial numbers, and microbial activity help in elucidating microbial contributions to jet fuel elimination from soil.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

Topography of all tyrosine residues in subtilisin DY

The extracellular alkaline proteinase subtilisin DY was nitrated with increasing amounts of tetranitromethane. At 2-fold molar excess of the reagent with respect to the tyrosine residues in the enzyme, when 1.3 residues were modified, a peak of the caseinolytic activity (13% increase) was observed. Evidence is provided that the diminishing of the pK of the phenolic hydroxyl group in Tyr(3NO2)104 causes this phenomenon. The products obtained after nitration of the enzyme with 5-fold and 200-fold molar excess of tetranitromethane were cleaved by trypsin and cyanogen bromide and the peptides obtained were studied by analysis with respect to the tyrosine and 3-nitrotyrosine residues. Their degree of substitution was established. Tyrosine-104 was the first modified residue, then follow the residues with numbers 57, 143, 206, 262 and somewhat later 21, 209, 263, all fully modified by 200-fold molar excess of the reagent. Partial modification was observed at numbers 91, 167, 214, 238 and no modification at numbers 6 and 171. It has been established that the nonmodified residues are buried inside the molecule and the partially modified residues are screened by the side chains of lysine, valine, leucine, and tryptophan as seen on a working video three-dimensional model of subtilisin Carlsberg. The approach for characterization of tyrosyl groups in proteins based on peptide sequencing and HPLC quantitation of the phenylthiohydantoin derivatives of tyrosine and 3-nitrotyrosine was further developed with respect to the quantitation of the HPLC-separated peptides using fragments of the protein studied.     

Comparison of Communities of Stored Product Mites in Grain Mass and Grain Residues in the Czech Republic

In storage facilities one can find grain either in stored grain mass or in grain residues in the store corners or machinery. Although it is claimed that grain residues are serious pest reservoirs since they harbor numbers of stored product arthropods and are connected via continuous emigration with grain mass, the documentation for this is not convincing. Therefore in 78 selected grain stores, we simultaneously sampled the grain mass and residues in order to compare concurrent mite communities in these two different habitats. We found 30 species in about 614 000 individuals in residues and 23 species in about 20 000 individuals in grain mass. Canonical correspondence analysis (CCA) of transformed abundance data showed differences in the communities of mites in grain mass and residues: (i) species associated to grain residues (e.g. Tyrophagus longior, Tydeus interruptus, Acarus farris and Cheyletus eruditus) and (ii) species associated to both grain mass and grain residues (e.g. Tarsonemus granarius, Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor and Cheyletus malaccensis). Although the residue samples had more mites and higher species diversity than the stored grain mass, no correlation in mite abundance and species numbers between samples from grain residues and grain mass was found, thereby indicating low connectivity of these two habitats.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

Studies on Tryptophane Residues in CF1-ATPase and the Enzymatic Property of CF1-ATPase Modified by NBS and Photooxidation

The numbers of tryptophane residues in spinach CF1-ATPase were measured by means of chemical modification with N-bromosuccinimide (NBS) and photooxidation. There are 3.5 tryptophane residues in CF1-ATPase, among which two are essential for the enzyme activity. Photooxidation of CF1-ATPase led to increased O2 uptake of the reaction system and loss in activity of CF1-ATPase . Immunological property of CF1-ATPase has been altered by chemical modification with NBS and photooxidation. The resuits show that tryptophane residues seen to be essential for activity and antigenic properties of CF1-ATPase.     

The HC-pro protein of potato virus Y interacts with NtMinD of tobacco

Potato virus Y (PVY) infections often lead to altered numbers of host plant chloroplasts, as well as changes in morphology and inhibited photosynthesis. The multifunctional protein helper component-proteinase, HC-Pro, has been identified in PVY-infected leaf chloroplasts. We used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that HC-Pro can interact with the chloroplast division-related factor NtMinD in yeast and tobacco cells, respectively. In addition, we confirmed that residues 271 to 314 in NtMinD are necessary for its interaction with PVY HC-Pro in a yeast two-hybrid analysis using four NtMinD deletion mutants. These residues are necessary for the dimerization of NtMinD, which plays a vital role in chloroplast division. Thus, PVY HC-Pro may affect NtMinD activity by inhibiting the formation of NtMinD homodimers, and this may interfere with chloroplast division and contribute to changes in the numbers of chloroplast per cell observed in PVY-infected plants.     

RankViaContact: ranking and visualization of amino acid contacts

SUMMARY: RankViaContact is a web service for calculation of residue-residue contact energies in proteins based on a coarse-grained model, and for visualization of interactions. The service provides information about ranked contact energies of residues, coordination numbers and the relative solvent accessibility of selected residues, as well as sequence and structure information. The program can be used to design stabilizing mutations, to analyze residue-residue contacts and to study the consequences of mutations. AVAILABILITY: http://bioinf.uta.fi/Rank.htm.     

SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges.

SP-40,40 is a two-chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP-40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP-40,40 and other multi-chain proteins containing disulphide bonds.     

Symmetric key structural residues in symmetric proteins with beta-trefoil fold

To understand how symmetric structures of many proteins are formed from asymmetric sequences, the proteins with two repeated beta-trefoil domains in Plant Cytotoxin B-chain family and all presently known beta-trefoil proteins are analyzed by structure-based multi-sequence alignments. The results show that all these proteins have similar key structural residues that are distributed symmetrically in their structures. These symmetric key structural residues are further analyzed in terms of inter-residues interaction numbers and B-factors. It is found that they can be distinguished from other residues and have significant propensities for structural framework. This indicates that these key structural residues may conduct the formation of symmetric structures although the sequences are asymmetric.     

Arginine residues involved in strong histone--DNA interactions to fold DNA into the nucleosome core particle.

The numbers of the arginine residues involved in strong histone-DNA interactions to fold DNA into a nucleosome core particle were determined for each of the four core histones, by kinetic studies of chemical modification of the residues in the nucleosome core particle. It was suggested that the arginines in the globular region of H3 histone make major contributions to the strong binding of the octameric histones to the core DNA.     

Neuraminidase treatment modifies the function of electroplax sodium channels in planar lipid bilayers

Sodium channels from several sources are covalently modified by unusually large numbers of negatively charged sialic acid residues. In the present studies, purified electroplax sodium channels were treated with neuraminidase to remove sialic acid residues and then examined for functional changes in planar lipid bilayers. Neuraminidase treatment resulted in a large depolarizing shift in the average potential required for channel activation. Additionally, desialidated channels showed a striking increase in the frequency of reversible transitions to subconductance states. Thus it appears that sialic acid residues play a significant role in the function of sodium channels, possibly through their influence on the local electric field and/or conformational stability of the channel molecule.     

Enhanced antitumor activity and selectivity of lactoferrin-derived peptides.

A number of peptide analogs derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT-29 and MT-1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure-activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.     

Computer methods for locating kinetoplastid cryptogenes.

RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.     

Trypanosoma (Nannomonas) congolense: analysis by fluorescein-conjugated plant lectins of surface saccharides of cloned variant antigen types differing in infectivity for mice

Surface saccharides of 4 cloned VATs (variant antigen types) of Trypanosoma (Nannomonas) congolense, AmNats (Amherst Nannomonas antigen types) 1.1, 1.2, 2.1, and 3.1, derived from 3 different stocks, were compared by fluorescein-conjugated, plant lectins using a quantitative fluorescence method. It was ascertained by the ID63 assay that the 4 AmNats differed in their infectivity for mice. The lectins employed for AmNats 1.1, 2.1, and 3.1 were concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), garden pea agglutinin (GPA), and gorse seed (Ulex europaeus) agglutinin (UEA). In view of the results obtained with these 3 AmNats, only Con A, WGA, and GPA were used with AmNat 1.2, which was isolated after the lectin analyses of the other cloned VATs were completed. On the basis of experimental results, we concluded that the amounts of saccharide residues binding the several lectins differed among the 4 AmNats. In each instance, the reaction specificity was controlled by inclusion of an appropriate sugar in the incubation mixture. Although the actual numbers of various specific lectin-binding sites differed among the AmNats 1.1, 2.1, and 3.1, all of them were found to have the following sugars on their surfaces: alpha-D-mannose, N-acetyl-D-glucosamine, D-galactose, alpha-D-glucose, and alpha-L-fucose. AmNat 1.2 treated with Con A, WGA, and GPA only had the first 2 sugars named above and alpha-D-glucose residues. The results of the ID63 assay indicated AmNats 1.1 and 2.1 to be significantly more infective for mice than AmNats 1.2 and 3.1. The lectin analysis revealed that the 2, more infective, cloned VATs incubated with Con A or WGA emitted significantly (approximately 39% to approximately 62%) more fluorescence than the less infective ones. Thus there were significantly more numerous Con A and WGA binding sites on the more infective AmNats. The situation was reversed with regard to GPA. Upon treatment with this lectin, fluorescence emitted by AmNats 1.1 and 2.1 was significantly (approximately 56% to approximately 81%) lower than that recorded for the less infective AmNats 1.2 and 3.1. In light of our results, infectivity of T. congolense cloned VATs was correlated with the presence of higher numbers of alpha-D-mannose and N-acetyl-D-glucosamine residues and of lower numbers of alpha-D-glucose residues on the surface of the bloodstream trypanosomes. There appeared to be no correlation between infectivity and the numbers of D-galactose and alpha-L-fucose residues present on these parasites.     

Relative hydrophobicity of amino acid residues in homooligopeptides as measured by aqueous two-phase partitioning.

Partitioning of 17 amino acids and their homooligopeptides of different lengths in an aqueous dextran-PEG two-phase system containing 0.15 m NaCl in 0.01 M sodium phosphate buffer, pH 7.4 and 0.11 m sodium phosphate buffer, pH 7.4 was examined. The relative hydrophobicity of the amino acid residues was estimated and expressed in equivalent numbers of methylene units. Analysis of the data shows that the additivity principle does hold for the hydrophobicity of homooligopeptides. The relative hydrophobicity of essentially all amino acid residues is noticeably affected by the ionic composition of aqueous media.