Hydrophobic Cluster Analysis Research Articles

β Pore-forming Protein-based Evolutionary Divergence of Gnathostomata from Agnatha.

The first vertebrates were jawless fish, or Agnatha, whose evolution diverged into jawed fish, or Gnathostomes, around 550 million years ago. In this study, we investigated β PFT proteins' evolutionary divergence of lamprey immune protein from Agnatha, reportedly possessing anti-cancer activity, into Dln1 protein from Gnathostomes. Both proteins showed structural and functional divergence, and shared evolutionary origin. Primary, secondary and tertiary sequences were compared to discover functional domains and conserved motifs in order to study the evolution of these two proteins. The structural and functional information relevant to evolutionary divergence was revealed using hydrophobic cluster analysis. The findings demonstrate that two membrane proteins with only a small degree of sequence identity can have remarkably similar hydropathy profiles, pointing towards conserved and similar global structures. When facing the lipid bilayer or lining the pore lumen, the two proteins' aerolysin domains' corresponding residues displayed a similar and largely conserved pattern. Aerolysin-like proteins from different species can be identified using a fingerprint created by PIPSA analysis of the pore-forming protein. We were able to fully understand the mechanism of action during pore formation through structural studies of these proteins.

A sequence-based foldability score combined with AlphaFold2 predictions to disentangle the protein order/disorder continuum.

Order and disorder govern protein functions, but there is a great diversity in disorder, from regions that are-and stay-fully disordered to conditional order. This diversity is still difficult to decipher even though it is encoded in the amino acid sequences. Here, we developed an analytic Python package, named pyHCA, to estimate the foldability of a protein segment from the only information of its amino acid sequence and based on a measure of its density in regular secondary structures associated with hydrophobic clusters, as defined by the hydrophobic cluster analysis (HCA) approach. The tool was designed by optimizing the separation between foldable segments from databases of disorder (DisProt) and order (SCOPe [soluble domains] and OPM [transmembrane domains]). It allows to specify the ratio between order, embodied by regular secondary structures (either participating in the hydrophobic core of well-folded 3D structures or conditionally formed in intrinsically disordered regions) and disorder. We illustrated the relevance of pyHCA with several examples and applied it to the sequences of the proteomes of 21 species ranging from prokaryotes and archaea to unicellular and multicellular eukaryotes, for which structure models are provided in the AlphaFold protein structure database. Cases of low-confidence scores related to disorder were distinguished from those of sequences that we identified as foldable but are still excluded from accurate modeling by AlphaFold2 due to a lack of sequence homologs or to compositional biases. Overall, our approach is complementary to AlphaFold2, providing guides to map structural innovations through evolutionary processes, at proteome and gene scales.

IgE epitopes are within the hydrophobic domain of sesame oleosin Ses i 4

Aim of the studyOleosins are allergens implicated in severe anaphylactic reactions after sesame, peanut or hazelnut consumption. However, identifying oleosin allergenic epitopes is hindered by their hydrophobic properties that do not allow analysis in an aqueous solution. Therefore, the present study focused on determining IgE-binding domains of sesame oleosin Ses i 4 using immunoblotting. Patients and methodsWe relied on sera from ten patients with clinically established allergy to sesame. Immunoblotting coupled to Hydrophobic Cluster Analysis (HCA) approach using a series of recombinant full-length and truncated Ses i 4 variants enabled detection of IgE reactivity domains. Results and conclusionThe data showed that IgE-binding sequences were located in the hydrophobic domain of the Ses i 4. The three IgE-binding segments were in proximity but on the flanks of the central-most hydrophobic segment. Further, their amino acid sequences are highly conserved in Ses i 4-homologs from different allergenic sources. Therefore, unlike most clinical immunoassays, Western blotting may be helpful for in vitro detection of oleosin IgE reactivity.

Characterisation and comparative analysis of hydrophobin isolated from Pleurotus floridanus (PfH)

Hydrophobins are a class of small cysteine rich surface active proteins produced exclusively by filamentous fungi. It forms a nano layer in the cell-water interface, thereby protecting the emerging fungal hyphae from surrounding water. Even though hydrophobins have similar functions in fungi, they share less sequence similarity. In the current study, we made a comparative study of the hydrophobin produced by the mushroom Pleurotus floridanus (PfH). Mushroom P. floridanus was cultured in PD broth. The hydrophobin was purified by foam fractionation and characterized in terms of molecular weight, solubility and glycosylation. In the RP-HPLC analysis, the hydrophobin eluted at a retention time of 45.56 min. The molecular weight of the PfH was found to be 13.52 kDa by MALDI-TOF MS and the LC-MS/MS showed no similar sequence in MASCOT database. The hydrophobin gene of P. floridanus was amplified using custom-designed primers and the BLAST analysis showed 80% sequence similarity with the Vmh2-1 gene of Pleurotus ostreatus. The sequence was translated into protein using ExPASy, secondary and tertiary structure predictions were carried out using Jpred4 and Phyre2. The tertiary structure showed 91.5% similarity with the HYD1 hydrophobin of Schizophyllum commune. A comparative study of PfH with Vmh2-1 and HYD1 was performed using bioinformatics tools. Hydrophobic cluster analysis revealed that three of these proteins have uniformity in terms of amphiphilic and non-amphiphilic α-helices, whereas PfH has a unique proline clustering. Physicochemical analysis by ProtParam revealed that PfH shares similar properties with HYD1 and Vmh2-1, which can be correlated with its function.

UPLC characteristic fingerprint and chemical pattern recognition of Angong Niuhuang Pills

To establish the UPLC fingerprint of Zhongyi Angong Niuhuang Pills, in order to evaluate its quality by chemical pattern recognition. The method was developed on a column of Poroshell 120 EC-C_(18), with methanol-0.1% formic acid solution as the mobile phase for gradient elution at a flow rate of 0.4 mL·min~(-1). The column temperature was 30 ℃,and the detective wavelength was 254 nm. The similarity of 24 batches of Angong Niuhuang Pills was compared by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2004 A). Hydrophobic cluster analysis,principal components analysis and partial least squares discriminant analysis were conducted by using SIMCA 13.0 software to investigate different components among these products. The UPLC characteristic fingerprint was established in this study. And 17 common peaks were identified by standard reference and UPLC-MS. The similarity of 24 batches samples were above 0.980,which can be classified into three categories for pattern recognition. Baicalin,berberine,jatrorrhizine,wogonin and wogonoside were identified as the main markers that cause differences of various batches. The method is simple,rapid,accurate and reproducible,and can provide scientific basis for improving the quality standard of Zhongyi Angong Niuhuang Pills.

A topology-based investigation of protein interaction sites using Hydrophobic Cluster Analysis

Hydrophobic clusters, as defined by Hydrophobic Cluster Analysis (HCA), are conditioned binary patterns, made of hydrophobic and non-hydrophobic positions, whose limits fit well those of regular secondary structures. They were proved to be useful for predicting secondary structures in proteins from the only information of a single amino acid sequence and have permitted to assess, in a comprehensive way, the leading role of binary patterns in secondary structure preference towards a particular state. Here, we considered the available experimental 3D structures of protein globular domains to enlarge our previously reported hydrophobic cluster database (HCDB), almost doubling the number of hydrophobic cluster species (each species being defined by a unique binary pattern) that represent the most frequent structural bricks encountered within protein globular domains. We then used this updated HCDB to show that the hydrophobic amino acids of discordant clusters, i.e. those less abundant clusters for which the observed secondary structure is in disagreement with the binary pattern preference of the species to which they belong, are more exposed to solvent and are more involved in protein interfaces than the hydrophobic amino acids of concordant clusters. As amino acid composition differs between concordant/discordant clusters, considering binary patterns may be used to gain novel insights into key features of protein globular domain cores and surfaces. It can also provide useful information on possible conformational plasticity, including disorder to order transitions.

Microbial β-glucosidase: Source, production and applications

β-glucosidase is an enzyme that is ubiquitous in nature and is produced by bacteria, fungi, plants and animals, including humans. Although, microorganisms especially fungi are considered the best choice for enzyme production as in industrial utilization especially from Trichoderma reesei, the filamentous fungus Acremonium persicinum, Aspergillus oryzae and Thermoascus aurantiacus. Classification of β-glucosidase is based on substrate specificity, nucleotide sequences identity and hydrophobic cluster analysis. β-glucosidase is known to hydrolyse the glycosidic bond of a carbohydrate moiety to release non-reducing terminal glycosyl residues, glycoside and oligosaccharides. Microbial β-glucosidases are produced in low quantities, and are inhibited by glucose, which is their end product. This results in accumulation of cellobiose during cellulolysis, which in turn inhibits endo-glucanase and exo-glucanase thereby making β-glucosidase the key enzyme in determining the cellulase efficiency. β-glucosidases have diverse biotechnological applications in food, biofuel, and agricultural industries. The search for novel and improved β-glucosidase is still continued to fulfil demand of an industrially suitable enzyme with high productivity of β-glucosidase and high glucose tolerance, thermostability and catalytic efficiency. This review article, describes the classes of β-glucosidases, mode of actions, microbial sources of β-glucosidases, production methods, factors affecting their production and biotechnological application.

Cloning and molecular characterization of TaERF6, a gene encoding a bread wheat ethylene response factor.

Ethylene response factor proteins are important for regulating gene expression under different stresses. Different isoforms for ERF have previously isolated from bread wheat (Triticum aestivum L.) and related genera and called from TaERF1 to TaERF5. We isolated, cloned and molecular characterized a novel one based on TdERF1, an isoform in durum wheat (Triticum turgidum L.) and called TaERF6. Its cDNA was synthesized, sequenced and compared with genomic sequence to figure out intron and exon regions and determine coding sequence region. The length of TdERF1 gene was 1939 bp and cDNA was 1065 bp including two exons, the first one 259 bp and the second one 806 bp separated by a 874 bp intron with a 111 bp 5'-UTR (untranslated region) and 401 bp 3'-UTR. TaERF6 encodes a 353 amino acids protein with nearly 99% identity to TdERF1. Hydrophobic cluster analysis revealed an N-terminal hydrophobic domain contains a highly conserved motif with the consensus sequence of M [C/L/Y] [G/R] [G/R/P] [A/G/V/L/R] [I/L/R/S/P/Q] [L/I/R/H] and hydrophobic clusters in AP2/ERF domain of which tends to form -sheet. Three monopartite nuclear localization signals also identified in TaERF6 that play important role in getting back into the nucleus. The results showed several putative phosphorylation sites in TaERF6 that a motif from residues 246 to 266, the CMVII-4 motif, was predicted to phosphorylate by different kinase proteins and play important roles in TaERF6 function. Phylogenetic analysis showed 7 clusters (I to VII) and 10 subclusters according to their relatedness in Poaceae family.

Order in Disorder as Observed by the "Hydrophobic Cluster Analysis" of Protein Sequences.

Hydrophobic cluster analysis (HCA) is an original approach for protein sequence analysis, which provides access to the foldable repertoire of the protein universe, including yet unannotated protein segments (“dark proteome”). Foldable segments correspond to ordered regions, as well as to intrinsically disordered regions (IDRs) undergoing disorder to order transitions. In this review, how HCA can be used to give insight into this last category of foldable segments is illustrated, with examples matching known 3D structures. After reviewing the HCA principles, examples of short foldable segments are given, which often contain short linear motifs, typically matching hydrophobic clusters. These segments become ordered upon contact with partners, with secondary structure preferences generally corresponding to those observed in the 3D structures within the complexes. Such small foldable segments are sometimes larger than the segments of known 3D structures, including flanking hydrophobic clusters that may be critical for interaction specificity or regulation, as well as intervening sequences allowing fuzziness. Cases of larger conditionally disordered domains are also presented, with lower density in hydrophobic clusters than well‐folded globular domains or with exposed hydrophobic patches, which are stabilized by interaction with partners.

Origins and structural properties of novel and de novo protein domains during insect evolution.

Over long time scales, protein evolution is characterized by modular rearrangements of protein domains. Such rearrangements are mainly caused by gene duplication, fusion and terminal losses. To better understand domain emergence mechanisms we investigated 32 insect genomes covering a speciation gradient ranging from ~ 2 to ~ 390 mya. We use established domain models and foldable domains delineated by hydrophobic cluster analysis (HCA), which does not require homologous sequences, to also identify domains which have likely arisen de novo, that is, from previously noncoding DNA. Our results indicate that most novel domains emerge terminally as they originate from ORF extensions while fewer arise in middle arrangements, resulting from exonization of intronic or intergenic regions. Many novel domains rapidly migrate between terminal or middle positions and single- and multidomain arrangements. Young domains, such as most HCA-defined domains, are under strong selection pressure as they show signals of purifying selection. De novo domains, linked to ancient domains or defined by HCA, have higher degrees of intrinsic disorder and disorder-to-order transition upon binding than ancient domains. However, the corresponding DNA sequences of the novel domains of de novo origins could only rarely be found in sister genomes. We conclude that novel domains are often recruited by other proteins and undergo important structural modifications shortly after their emergence, but evolve too fast to be characterized by cross-species comparisons alone.

UPLC fingerprint and multi-components determination of three processed products of Rhizome of Curcuma wenyujin

To analyse the quality of three processed products of Rhizome of Curcuma wenyujin by establishing an ultra performance liquid chromatography (UPLC) method for simultaneously determining five sesquiterpene components in three processed products of rhizome of C. wenyujin and establishing UPLC fingerprints. Component determination was achieved on Waters ACQUITY UPLC BEH C₁₈ column（2.1 mm×50 mm, 1.7 μm), with acetonitrile-water as mobile phase for gradient elution. The flow rate was 0.3 mL·min⁻¹; column temperature was 30 °C; the detection wavelength was set at 214 nm and injection volume was 1 μL. The similarity was analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012.130723)", and hydrophobic cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted by using simca-p14.1 software to investigate the differences in components among these three kinds of processed products. The curzerene, curdione, curcumol, germacrone, furanodiene and β-elemene showed good linearity relationship with chromatographic peak area within the ranges of 10.8-320（r=0.999 9）, 10.36-259（r=0.998 1）, 10.54-263.5（r=0.999 3）, 30.2-755（r=0.999 6）and 34.38-862（r=0.999 9）mg·L⁻¹, respectively; their average recoveries were 98.75%, 98.69%, 98.63%, 99.76% and 99.57% respectively, with RSD of 2.67%, 1.47%, 1.29%, 2.54% and 0.87% respectively. The similarity of 30 batches of samples was larger than 0.9, indicating good consistency of the samples. The samples can be clearly classified into three categories for HCA, PCA and OPLS-DA pattern recognition, the differential chromatographic peak among three processed products was found respectively. The results showed that the pharmacology basis had changed obviously after processing of Rhizome of C. wenyujin, so it can provide the scientific basis for rational clinical application and establishing quality standards of three processed products of Rhizome of curcuma wenyujin.

The role of the jaw subdomain of peptidoglycan glycosyltransferases for lipid II polymerization

Bacterial peptidoglycan glycosyltransferases (PGT) catalyse the essential polymerization of lipid II into linear glycan chains required for peptidoglycan biosynthesis. The PGT domain is composed of a large head subdomain and a smaller jaw subdomain and can be potently inhibited by the antibiotic moenomycin A (MoeA). We present an X-ray structure of the MoeA-bound Staphylococcus aureus monofunctional PGT enzyme, revealing electron density for a second MoeA bound to the jaw subdomain as well as the PGT donor site. Isothermal titration calorimetry confirms two drug-binding sites with markedly different affinities and positive cooperativity. Hydrophobic cluster analysis suggests that the membrane-interacting surface of the jaw subdomain has structural and physicochemical properties similar to amphipathic cationic α-helical antimicrobial peptides for lipid II recognition and binding. Furthermore, molecular dynamics simulations of the drug-free and -bound forms of the enzyme demonstrate the importance of the jaw subdomain movement for lipid II selection and polymerization process and provide molecular-level insights into the mechanism of peptidoglycan biosynthesis by PGTs.

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting.

Lipocalin 2 (LCN2) is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

Members of Glycosyl-Hydrolase Family 17 of A. fumigatus Differentially Affect Morphogenesis.

Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3)glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17) of A. fumigatus, two β(1,3)glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p.

Exploring the dark foldable proteome by considering hydrophobic amino acids topology

The protein universe corresponds to the set of all proteins found in all organisms. A way to explore it is by taking into account the domain content of the proteins. However, some part of sequences and many entire sequences remain un-annotated despite a converging number of domain families. The un-annotated part of the protein universe is referred to as the dark proteome and remains poorly characterized. In this study, we quantify the amount of foldable domains within the dark proteome by using the hydrophobic cluster analysis methodology. These un-annotated foldable domains were grouped using a combination of remote homology searches and domain annotations, leading to define different levels of darkness. The dark foldable domains were analyzed to understand what make them different from domains stored in databases and thus difficult to annotate. The un-annotated domains of the dark proteome universe display specific features relative to database domains: shorter length, non-canonical content and particular topology in hydrophobic residues, higher propensity for disorder, and a higher energy. These features make them hard to relate to known families. Based on these observations, we emphasize that domain annotation methodologies can still be improved to fully apprehend and decipher the molecular evolution of the protein universe.

The respective roles of polar/nonpolar binary patterns and amino acid composition in protein regular secondary structures explored exhaustively using hydrophobic cluster analysis.

Several studies have highlighted the leading role of the sequence periodicity of polar and nonpolar amino acids (binary patterns) in the formation of regular secondary structures (RSS). However, these were based on the analysis of only a few simple cases, with no direct mean to correlate binary patterns with the limits of RSS. Here, HCA-derived hydrophobic clusters (HC) which are conditioned binary patterns whose positions fit well those of RSS, were considered. All the HC types, defined by unique binary patterns, which were commonly observed in three-dimensional (3D) structures of globular domains, were analyzed. The 180 HC types with preferences for either α-helices or β-strands distinctly contain basic binary units typical of these RSS. Therefore a general trend supporting the "binary pattern preference" assumption was observed. HC for which observed RSS are in disagreement with their expected behavior (discordant HC) were also examined. They were separated in HC types with moderate preferences for RSS, having "weak" binary patterns and versatile RSS and HC types with high preferences for RSS, having "strong" binary patterns and then displaying nonpolar amino acids at the protein surface. It was shown that in both cases, discordant HC could be distinguished from concordant ones by well-differentiated amino acid compositions. The obtained results could, thus, help to complement the currently available methods for the accurate prediction of secondary structures in proteins from the only information of a single amino acid sequence. This can be especially useful for characterizing orphan sequences and for assisting protein engineering and design.

Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009.

Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically. During 2009, 10 G10P[8] rotavirus strains were detected in the stools of children hospitalized with acute diarrhoea in several paediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P[8] strains analysed revealed nucleotide identities ranging from 94 to 99 %. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Amongst the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains. A hydrophobic cluster analysis (HCA) of the VP7 protein including aa 20-298 was performed for the G10 Italian sequences in comparison with the major human group A rotavirus G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.

Detection of orphan domains in Drosophila using "hydrophobic cluster analysis".

Comparative genomics has become an important strategy in life science research. While many genes, and the proteins they code for, can be well characterized by assigning orthologs, a significant amount of proteins or domains remain obscure "orphans". Some orphans are overlooked by current computational methods because they rapidly diverged, others emerged relatively recently (de novo). Recent research has demonstrated the importance of orphans, and of de novo proteins and domains for development of new phenotypic traits and adaptation. New approaches for detecting novel domains are thus of paramount importance. The hydrophobic cluster analysis (HCA) method delineates globular-like domains from the information of a protein sequence and thereby allows bypassing some of the established methods limitations based on conserved sequence similarity. In this study, HCA is tested for orphan domain detection on 12 Drosophila genomes. After their detection, the oprhan domains are classified into two categories, depending on their presence/absence in distantly related species. The two categories show significantly different physico-chemical properties when compared to previously characterized domains from the Pfam database. The newly detected domains have a higher degree of intrinsic disorder and a particular hydrophobic cluster composition. The older the domains are, the more similar their hydrophobic cluster content is to the cluster content of Pfam domains. The results suggest that, over time, newly created domains acquire a canonical set of hydrophobic clusters but conserve some features of intrinsically disordered regions. Our results agree with previous findings on orphan domains and suggest that the physico-chemical properties of domains change over evolutionary long time scale. The presented HCA-based method is able to detect domains with unusual properties without relying on prior knowledge, such as the availability of homologs. Therefore, the method has large potential for complementing existing strategies to annotate genomes, and for better understanding how molecular features emerge.

Analysis of acetohydroxyacid synthase1 gene in chickpea conferring resistance to imazamox herbicide.

Chickpea (Cicer arietinum L.) production in the Canadian prairies is challenging due to a lack of effective weed management mainly because of poor competition ability of the crop and limited registered herbicide options. Chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified. A point mutation in the acetohydroxyacid synthase1 (AHAS1) gene at C581 to T581, resulting in an amino acid substitution from Ala194 to Val194 (position 205, standardized to arabidopsis), confers the resistance to imazamox in chickpea. However, the molecular mechanism leading to the resistance is not fully understood. In many plant species, contrasting transcription levels of AHAS gene has been implicated in the resistant and susceptible genotypes in response to IMI. The objectives of this research were to compare the AHAS gene expression and AHAS enzyme activity in resistant and susceptible chickpea cultivars in response to imazamox herbicide treatment. Results from RT-qPCR indicated that there is no significant change in the transcript levels of AHAS1 between the susceptible and the resistant genotypes in response to imazamox treatment. Protein hydrophobic cluster analysis, protein-ligand docking analysis, and AHAS enzyme activity assay all indicated that the resistance to imazamox in chickpea is due to the alteration of interaction of the AHAS1 enzyme with the imazamox herbicide.

Searching for convergent evolution in manganese superoxidase dismutase using hydrophobic cluster analysis.

There are numerous examples of convergent evolution in nature. Major ecological adaptations such as flight, loss of limbs in vertebrates, pesticide resistance, adaptation to a parasitic way of life, etc., have all evolved more than once, as seen by their analogous functions in separate taxa. But what about protein evolution? Does the environment have a strong enough influence on intracellular processes that enzymes and other functional proteins play, to evolve similar functional roles separately in different organisms? Manganese Superoxide Dismutase (MnSOD) is a manganesedependant metallo-enzyme which plays a crucial role in protecting cells from anti-oxidative stress by eliminating reactive (superoxide) oxygen species. It is a ubiquitous housekeeping enzyme found in nearly all organisms. In this study we compare phylogenies based on MnSOD protein sequences to those based on scores from Hydrophobic Cluster Analysis (HCA). We calculated HCA similarity values for each pair of taxa to obtain a pair-wise distance matrix. A UPGMA tree based on the HCA distance matrix and a common tree based on the primary protein sequence for MnSOD was constructed. Differences between these two trees within animals, enterobacteriaceae, planctomycetes and cyanobacteria are presented and cited as possible examples of convergence. We note that several residue changes result in changes in hydrophobicity at positions which apparently are under the effect of positive selection.