Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Synthesis, characterisation and thermal analysis of copper (II) and chromium (II, III) hydrazine carboxylates

Copper(II) hydrazine carboxylate monohydrate, Cu(N2H3COO)2·H2O and chromium (II, III) hydrazine carboxylate hydrates, Cu(N2H3COO)2·H2O and Cu(N2H3COO)2·3H2O have been prepared and characterised by chemical analysis, IR, visible spectra and magnetic measurements. Thermal analysis of the copper complex yields a mixture of copper metal and copper oxide. Chromium complexes on thermal decomposition yield Cr2O3 as residue. Decomposition of chromium(HI) complex under hydrothermal conditions yield CrOOH, a precursor to CrO2.

Diproline Templates as Folding Nuclei in Designed Peptides. Conformational Analysis of Synthetic Peptide Helices Containing Amino Terminal Pro-Pro Segments

The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (2). The structure of 1 in crystals, determined by X-ray diffraction, reveals a helical (RR) conformation for the segment residues 2 to 5, stabilized by one 4 -> 1 hydrogen bond and two 5 -> 1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (P-II) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl3, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an alpha(R)-alpha(R) conformation, whereas in the latter, a P-II-alpha(R) structure is established. The conformational equilibrium shifts in favor of the P-II-alpha(R) structure in solvents like methanol and DMSO. A significant population of the Pro(1)- Pro(2) cis conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro- Pro segment: trans alpha(R)-alpha(R), trans P-II-alpha(R) and cis P-II-alpha(R). Of these, the two trans conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between cis and trans form is slow. Similar results are obtained with peptide 2. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the alpha(R)-alpha(R) conformation followed by a helix. Modeling and energy minimization studies suggest that both P-II-alpha(R) and alpha(R)-alpha(R) conformations have very similar energies in the model hexapeptide 1

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.

Analysis of the effects of amino-acid-sequence on the structure of proteins

The conformation of amino acid side chains as observed in well-determined structures of globular proteins has earlier been extensively investigated. In contrast, the structural features of the polypeptide backbone that result from the occurrence of specific amino acids along the polypeptide have not been analysed. In this article, we present the statistically significant features in the backbone geometry that appear to be a consequence of the occurrence of rotamers of different amino acid side chains by analysing 102 well-refined structures that form a random collection of proteins. It is found that the persistence of helical segments around each residue is influenced by the residue type. Several residues exert asymmetrical influence between the carboxyl and amino terminal polypeptide segments. The degree to which secondary structures depart from an average geometry also appears to depend on residue type. These departures are correlated to the corresponding Chou and Fasman parameters of amino acid residues. The frequency distribution of the side chain rotamers is influenced by polypeptide secondary structure. In turn, the rotamer conformation of side chain affects the extension of the secondary structure of the backbone. The strongest correlation is found between the occurrence of g+ conformation and helix propagation on the carboxyl side of many residues.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues-K12, H95, E97 and E165-are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three-dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000-fold reduction in k(cat) is observed for E97Q, whereas the E97D mutant shows a 100-fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000-fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 angstrom resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans-to-gauche transition about the C gamma-C delta (chi(3)) bond. In the E97D mutant, the K12 side chain maintains the wild-type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.

A transient detection algorithm for audio using iterative analysis of STFT

We propose an iterative algorithm to detect transient segments in audio signals. Short time Fourier transform(STFT) is used to detect rapid local changes in the audio signal. The algorithm has two steps that iteratively - (a) calculate a function of the STFT and (b) build a transient signal. A dynamic thresholding scheme is used to locate the potential positions of transients in the signal. The iterative procedure ensures that genuine transients are built up while the localised spectral noise are suppressed by using an energy criterion. The extracted transient signal is later compared to a ground truth dataset. The algorithm performed well on two databases. On the EBU-SQAM database of monophonic sounds, the algorithm achieved an F-measure of 90% while on our database of polyphonic audio an F-measure of 91% was achieved. This technique is being used as a preprocessing step for a tempo analysis algorithm and a TSR (Transients + Sines + Residue) decomposition scheme.

Crystal-structure analysis of cis-X-pro-containing peptidomimetics: understanding the steric interactions at cis X-pro amide bonds

Catch the twist: The cis Piv-Pro conformer (Piv=pivaloyl) of peptides is no longer inaccessible. Any cis X-Pro tertiary-amide-bond conformer can be stabilized in crystals of peptides by accommodating the unavoidable distortion of the dihedral angle of the peptide bond to the carbonyl group of the Pro residue (see picture), in this case through ni−1→πi* interactions. Steric clashes were not observed in the cis Piv-Pro rotamers studied.

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (phi, ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (phi, ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (phi, ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.

PROTEIN DISULFIDE ANALYSIS AND DESIGN

The significant contribution of naturally occurring disulfide bonds to protein stability has encouraged development of methods to engineer non-native disulfides in proteins. These have yielded mixed results. We summarize applications of the program MODIP for disulfide engineering. The program predicts sites in proteins where disulfides can be stably introduced. The program has also been used as an aid in conformational analysis of naturally occurring disulfides in a-helices, antiparallel and parallel beta-strands. Disulfides in a-helices occur only at N-termini, where the first cysteine residue is the N-cap residue of the helix. The disulfide occurs as a CXXC motif and can possess redox activity. In antiparallel beta-strands, disulfides occur exclusively at non-hydrogen bonded (NHB) registered pairs of antiparallel beta-sheets with only 1 known natural example occurring at a hydrogen bonded (HB) registered pair. Conformational analysis suggests that disulfides between HB residue pairs are under torsional strain. A similar analysis to characterize disulfides in parallel beta-strands was carried out. We observed that only 9 instances of cross-strand disulfides exist in a non-redundant dataset. Stereochemical analysis shows that while tbe chi(square) angles are similar to those of other disulfides, the chi(1) and chi(2) angles show more variation and that one of tbe strands is generally an edge strand.

Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

The isolation and partial biochemical analysis of Sindbis virus proteins

Recently, the amino acid sequences have been reported for several proteins, including the envelope glycoproteins of Sindbis virus, which all probably span the plasma membrane with a common topology: a large N-terminal, extracellular portion, a short region buried in the bilayer, and a short C-terminal intracellular segment. The regions of these proteins buried in the bilayer correspond to portions of the protein sequences which contain a stretch of hydrophobic amino acids and which have other common characteristics, as discussed. Reasons are also described for uncertainty, in some proteins more than others, as to the precise location of some parts of the sequence relative to the membrane.

The signal hypothesis for the transmembrane translocation of proteins is briefly described and its general applicability is reviewed. There are many proteins whose translocation is accurately described by this hypothesis, but some proteins are translocated in a different manner.

The transmembraneous glycoproteins E1 and E2 of Sindbis virus, as well as the only other virion protein, the capsid protein, were purified in amounts sufficient for biochemical analysis using sensitive techniques. The amino acid composition of each protein was determined, and extensive N-terminal sequences were obtained for E1 and E2. By these techniques E1 and E2 are indistinguishable from most water soluble proteins, as they do not contain an obvious excess of hydrophobic amino acids in their N-terminal regions or in the intact molecule.

The capsid protein was found to be blocked, and so its N-terminus could not be sequenced by the usual methods. However, with the use of a special labeling technique, it was possible to incorporate tritiated acetate into the N-terminus of the protein with good specificity, which was useful in the purification of peptides from which the first amino acids in the N-terminal sequence could be identified.

Nanomole amounts of PE2, the intracellular precursor of E2, were purified by an immuno-affinity technique, and its N-terminus was analyzed. Together with other work, these results showed that PE2 is not synthesized with an N-terminal extension, and the signal sequence for translocation is probably the N-terminal amino acid sequence of the protein. This N-terminus was found to be 80-90% blocked, also by Nacetylation, and this acetylation did not affect its function as a signal sequence. The putative signal sequence was also found to contain a glycosylated asparagine residue, but the inhibition of this glycosylation did not lead to the cleavage of the sequence.

Stability analysis of RF amplifiers based on MIMO pole-zero identification

Spurious oscillations are one of the principal issues faced by microwave and RF circuit designers. The rigorous detection of instabilities or the characterization of measured spurious oscillations is still an ongoing challenge. This project aims to create a new stability analysis CAD program that tackles this chal- lenge. Multiple Input Multiple Output (MIMO) pole-zero identification analysis is introduced on the program as a way to create new methods to automate the stability analysis process and to help designers comprehend the obtained results and prevent incorrect interpretations. The MIMO nature of the analysis contributes to eliminate possible controllability and observability losses and helps differentiate mathematical and physical quasi-cancellations, products of overmodeling. The created program reads Single Input Single Output (SISO) or MIMO frequency response data, and determines the corresponding continuous transfer functions with Vector Fitting. Once the transfer function is calculated, the corresponding pole/zero diagram is mapped enabling the designers to analyze the stability of an amplifier. Three data processing methods are introduced, two of which consist of pole/zero elimina- tions and the latter one on determining the critical nodes of an amplifier. The first pole/zero elimination method is based on eliminating non resonant poles, whilst the second method eliminates the poles with small residue by assuming that their effect on the dynamics of a system is small or non-existent. The critical node detection is also based on the residues; the node at which the effect of a pole on the dynamics is highest is defined as the critical node. In order to evaluate and check the efficiency of the created program, it is compared via examples with another existing commercial stability analysis tool (STAN tool). In this report, the newly created tool is proved to be as rigorous as STAN for detecting instabilities. Additionally, it is determined that the MIMO analysis is a very profitable addition to stability analysis, since it helps to eliminate possible problems of loss of controllability, observability and overmodeling.