Combination Of Amino Acid Residues Research Articles

Effect of Isomerization of TX-2036 Derivatives on the Interaction With Tyrosine Kinase Domain of EGF Receptor.

From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC50 value of TX-2036 was 1.8 μM. R-configured TXs interacted with Lys721 and Thr766 of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile765 and Thr766 (TX-2044), Ser696, Thr766, and Thr830 (TX-2031), Gly772, Cys773, and Thr830 (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.

Cytochrome P450 2B diversity in a dietary specialist—the red tree vole (Arborimus longicaudus)

AbstractAlthough herbivores rely on liver enzymes to biotransform plant secondary metabolites ingested in plant-based diets, only a few enzymes from a handful of species have been characterized at the genomic level. In this study, we examined cytochrome P450 2B (CYP2B) sequence diversity and gene copy number in a conifer specialist, the red tree vole (Arborimus longicaudus). We fed captive individuals exclusively Douglas-fir (Pseudotsuga menziesii) foliage, cloned and sequenced their liver CYP2B cDNA, and estimated CYP2B gene copy number. We identified 21 unique CYP2B nucleotide sequences, and 20 unique CYP2B amino acid sequences. Gene copy number of CYP2B was estimated at 7.7 copies per haploid genome. We compared red tree vole CYP2B with CYP2B sequences of a generalist, the prairie vole (Microtus ochrogaster), found in GenBank. Our study revealed that the CYP2B enzymes of red tree voles possess unique sequences compared to CYP2B enzymes of other herbivorous species. The unique combination of amino acid residues at key substrate recognition sites of CYP2B enzymes may underlie the ability of the red tree vole to specialize on a highly toxic diet of Douglas-fir.

Cytochrome P450 2B Diversity and Dietary Novelty in the Herbivorous, Desert Woodrat (Neotoma lepida)

Detoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet.

Supramolecular hydrogels based on short peptides linked with conformational switch

Short peptides appropriately linked with an azobenzene conformational switch were found to be motif and pH dependant supramolecular hydrogelators. The hydrogelation properties of the short peptides linked with the conformational switch were studied in detail with respect to dependence on amino acid residue, pH and salt effect. The presence of amino acids with aromatic side chains such as Phe and Tyr was found to be favorable for the short peptides to gel water at an appropriate pH range. Cationic amino acid residues such as Arg and Lys in the short peptides were found to be unfavorable for hydrogelation. pH and salt effect were also found to be important factors for the hydrogelation properties of the short peptides. A series of short peptides with bioactive sequences were linked with the conformational switch and their hydrogelation properties were investigated. Photoresponsive supramolecular hydrogels were realized based on the E-/Z- transition of the conformational switch upon light irradiation. Proper combination of amino acid residues in the short peptides resulted in smart supramolecular hydrogels with responses to multiple stimuli.

New Primers for the Avian SWS1 Pigment Opsin Gene Reveal New Amino Acid Configurations in Spectral Sensitivity Tuning Sites

Recently, polymerase chain reaction-based estimates of visual pigment spectral tuning from genomic DNA have offered an alternative to the authoritative but rather slow and complicated retinal microspectrophotometry method. The genomic DNA method involves sequencing a fragment of the short-wavelength sensitive pigment, type 1 (SWS1) opsin gene covering amino acid positions 86, 90, and 93 and has been utilized in a wide range of avian species. Other key tuning sites have been proposed but not sequenced in the genomic DNA-based spectral sensitivity studies. We have designed 5 new primers for sequencing gene fragments of the ultraviolet-/violet-tuned SWS1 opsin gene containing the first, second and third, and sixth and seventh alpha-helical transmembrane regions and the spectral tuning sites 49, 86, 90, 93, 116, 118 and 298. Testing these primers on various bird species reveals some novel combinations of amino acid residues at the tuning sites. The potential significance of these on spectral tuning is discussed.

Stereochemical Preference in the Reactions of N-Protected L-Amino Acid 1-Hydroxybenzotriazole Esters with Racemic Amino Acid Derivatives

Stereochemical preference for homochiral or heterochiral couplings was investigated in the reactions of N-protected L-amino acid 1-hydroxybenzotriazole esters with racemic amino acid derivatives. It was found to be dependent on the combination of amino acid residues as the carboxyl and amino components and the protecting groups of the amino acid residues, especially the N-protecting groups. Very high diastereomeric ratios were observed when t-leucine was employed as the carboxyl component and/or proline as the amino component and when the N-protecting group was the phthaloyl group.

Shape-Shifting Chemokines

Lymphotactin (Ltn) is the odd-man-out amongst the chemokines. Whereas all the others have two disulfide bridges, Ltn has only one, a distinctive feature that may explain its unique structural properties. In vitro Ltn adopts two conformations: Ltn10, which predominates in high-salt conditions at low temperatures (10°C), and Ltn40, a high-temperature, low-salt (40°C) form. The two forms are in equilibrium under physiological conditions. Tuinstra et al . addressed the functional implications of this conformational variability. Nuclear magnetic resonance studies showed that Ltn10 and Ltn40 corresponded to distinct structures that used different combinations of amino acid residues for internal hydrogen bonding. The authors engineered recombinant Ltn10 and Ltn40 proteins by making amino acid substitutions that trapped the protein in one conformation or the other. Like other chemokines, Ltn binds to both its receptor (XCR1) and extracellular matrix glycosaminoglycans such as heparin. However, recombinant Ltn10 bound with high affinity to XCR1, but not to heparin, whereas recombinant Ltn40 bound with high affinity to heparin, but not XCR1. Neither protein mimicked the activity of Ltn in stimulating chemotaxis of human T cells in vitro. Furthermore, the authors found that Ltn could not bind to XCR1 and heparin simultaneously and suggested that Ltn must release the extracellular matrix component before it binds to and activates its receptor. This study suggests that altering the equilibrium between two conformational states of a protein by changing physiological conditions such as temperature (for example, during inflammation) may constitute a previously unrecognized means of affecting signaling pathway dynamics. R. L. Tuinstra, F. C. Peterson, S. Kutlesa, E. S. Elgin, M. A. Kron, B. F. Volkman, Interconversion between two unrelated protein folds in the lymphotactin native state. Proc. Natl. Acad. Sci. U.S.A. 105 , 5057-5062 (2008). [Abstract] [Full Text]

The Carboxylate Shift in Zinc Enzymes: A Computational Study

Zinc is the second most abundant transition element in biology and the only metal known to be represented in enzymes from each one of the six classes established by the International Union of Biochemistry. The flexible coordination geometry, the fast ligand exchange, the lack of redox activity, and its role as Lewis acid are just some of the features that make zinc an invaluable element in biological catalysis. In this study, we have analyzed the importance in mononuclear Zn enzymes of an interesting mechanistic phenomenon known as carboxylate shift, which is characterized by a change in the coordination mode of a carboxylate group (mono to bidentate or vice versa) with both ligand entrance or exit from the metal coordination sphere. Using B3LYP calculations, we were able to unveil in detail patterns relating the intrinsic characteristics of a given Zn coordination sphere with the existence or not of a carboxylate-shift mechanism and the additional energy stabilization arising from it. In particular, a specific Zn coordination sphere containing a carboxylate ligand (Asp or Glu), a cysteine, and a histidine has been shown to have the most favorable combination of amino acid residues that ensures a fast ligand exchange.

Meta-analysis of the Association between HLA-DRB1 Allele and Rheumatoid Arthritis Susceptibility in Asian Populations

The aims of this study were to summarize results on the association of HLA-DRB1 with rheumatoid arthritis (RA) in Asians and to determine if the shared epitope (SE) hypothesis could explain the meta-analysis results. Among the papers published between January 1987 and July 2006 on RA susceptibility in Asian-Mongoloid populations (Korean, Japanese, Chinese, and Thai), 12 were selected for the meta-analysis. Mongoloid-Asian patients with RA had significantly higher frequencies of HLA-DRB1*0101, *0401, *0410, and *1001 than controls (OR 1.5-2.1, p<0.05 for association). When analyses were restricted to more ethnically homogeneous populations, HLA-DRB1*0405 showed a significant susceptibility to RA in Koreans (OR 5.65, 95% CI 4.32-7.39), whereas the HLA-DRB1*0301, *0403, *0406, *0701, *1301, and *1405 alleles showed protective association with RA (OR 0.32-0.70, p<0.05 for association). In conclusion, it was found that HLA-DRB1 *0101, *0401, *0405, *0410, and *1001 are susceptible, while HLA-DRB1*0301, *0403, *0406, *0701, *1301, and *1405 are protective in Asian-Mongoloids. All the RA-associated alleles except DRB1*0301 could be explained by the structural model supporting the SE hypothesis that RA susceptibility is determined by the combination of amino acid residues at HLA-DR β71 and β74, not by β71 alone.

Recognition Site for the Side Chain of 2-Ketoacid Substrate in D-Lactate Dehydrogenase

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.

Synthesis and anti‐Helicobacter pylori Activity of Pyloricidin Derivatives. Part 2. The Combination of Amino Acid Residues in the Dipeptidic Moiety and Its Effect on the anti‐Helicobacter pylori Activity.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Directed evolution of barnase stability using proteolytic selection.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Synthesis and anti-Helicobacter pylori activity of pyloricidin derivatives II. The combination of amino acid residues in the dipeptidic moiety and its effect on the anti-Helicobacter pylori activity.

The novel natural antibiotics pyloricidin A, B and C, consisting of a common (2S,3R,4R,5S)-5-amino-2,3,4,6-tetrahydroxyhexanoyl-beta-D-phenylalanine moiety and a terminal peptidic moiety (pyloricidin A: L-valine-L-valine-L-leucine; pyloricidin B: L-valine-L-leucine; pyloricidin C: L-leucine), exhibit potent and highly selective anti-Helicobacter pylori activity. In order to develop more potent compounds and to investigate structure activity relationships for the peptidic moiety with regard to the combination of amino acids, a series of derivatives with various dipeptidic moieties were prepared and evaluated for their anti-H. pylori activity. The combination of the two amino acids in the moiety was found to have a significant effect on the activity; the compound with Nva-Abu showed excellent anti-H. pylori activity with an MIC value of 0.013 microg/ml against H. pylori TN2. In addition, this compound was found to show 60% clearance of H. pylori from infected Mongolian gerbils upon repetitive oral administration (10 mg/kg, b. i. d. for 7 days).

Aspergillus melleus protease-catalyzed peptide synthesis using the carbamoylmethyl ester as an acyl donor in 1,1,1,3,3,3-hexafluoro- 2-propanol/ N , N -dimethylformamide

The coupling between the carbamoylmethyl ester of an N-protected amino acid or dipeptide (at 25 mM) and an amino acid amide (at 100 mM) was achieved using Aspergillus melleus protease in 1,1,1,3,3,3-hexafluoro-2-propanol/N,N-dimethylformamide (1:1, v/v); the coupling efficiencies were dependent largely on the combination of amino acid residues: e.g. the dipeptide yields after 48 h were for l-Ala + Gly, ∼100% and for l-Leu + l-Leu, 16%.

Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP IAI

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1.

Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.

Vertebrate serpins: construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses.

A combination of three independent biological features, genomic organization, diagnostic amino acid sites, and rare indels, was used to elucidate the phylogeny of the vertebrate serpin (serine protease inhibitor) superfamily. A strong correlation between serpin gene families displaying (1) a conserved exon-intron pattern and (2) family-specific combinations of amino acid residues at specific sites suggests that present-day vertebrates encompass six serpin gene families which evolved from primordial genes by massive intron insertion before or during early vertebrate radiation. Introns placed at homologous positions in the gene sequences in combination with diagnostic sequence characters may also constitute a reliable kinship indicator for other protein superfamilies.

FASTA-SWAP and FASTA-PAT: Pattern Database Searches Using Combinations of Aligned Amino Acids, and a Novel Scoring Theory

We introduce two new pattern database search tools that utilize statistical significance and information theory to improve protein function identification. Both the general pattern scoring theory with the specific matrices introduced here and the low redundancy of pattern databases increase search sensitivity and selectivity. Pattern scoring preferentially rewards matches at conserved positions in a pattern with higher scores than matches at variable positions, and assigns more negative scores to mismatches at conserved positions than to mismatches at variable positions. The theory of pattern scoring can be used to create log-odds pattern scores for patterns derived from any set of multiple alignments. This theoretical framework can be used to adapt existing sequence database search tools to pattern analysis. Our FASTA-SWAP and FASTA-PAT tools are extensions of the FASTA program that search a sequence query against a pattern database. In the first step, FASTA-SWAP searches the diagonals of the query sequence and the library pattern for high-scoring segments, while FASTA-PAT performs an extended version of hashing. In the second step, both methods refine the alignments and the scores using dynamic programming. The tools utilize an extremely compact binary representation of all possible combinations of amino acid residues in aligned positions. Our FASTA-SWAP and FASTA-PAT tools are well suited for functional identification of distant relatives that may be missed by sequence database search methods. FASTA-SWAP and FASTA-PAT searches can be performed using out World-Wide Web Server (http://dot.imgen.bcm.tmc.edu:9331/seq-search/Options/fastapat.html).

HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides.

Eight nonamer peptides that comply with the major anchor residue motifs (the combination of amino acid residues at positions 2 and 9), R - K and R - R, of HLA-B27 (B*2705)-binding peptides were synthesized and tested for their direct binding to HLA class I alpha chains by the HLA class I alpha chain refolding assay previously described. One was a known B27 (B*2705)-binding heat shock protein peptide, HSP89 alpha (201-209), and the other seven were derived from the sequence of wild-type P53, a human tumor suppressor protein. A total of 36 HLA class I allospecificities were tested. HSP89 alpha (201-209) and two P53 peptides, P53 (362-370) and P53 (378-386), all possessing the motif R - K, bound strongly to B27 (B*2705) alpha chains. A weak binding was seen for P53 (272-280) and P53 (334-342), both showing the motif R - R. Most of these B27-binding peptides were found to bind to A3 alpha chains as well. In addition, P53 (173-181) and P53 (334-342), both with the R - R motif, showed substantial binding with A31 alpha chains. All the peptides carrying the motif R - K also showed weak binding with A31 alpha chains. The remaining two peptides, P53 (201-209) and P53 (282-290), with the motif R - R, did not show significant binding with any of the alpha chains tested. This study demonstrates both the specificity of peptide binding to a given HLA allelic product and the occurrence of cross-peptide-binding between the allelic products of different HLA loci.

Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.