Hydropathy Profile Research Articles

Nanoscale Topography Dictates Residue Hydropathy in Proteins.

Proteins exhibit diverse structures, including pockets, cavities, channels, and bumps, which are crucial in determining their functions. This diversity in topography also introduces significant chemical heterogeneity, with polar and charged domains often juxtaposed with nonpolar domains in proximity. Consequently, accurately assessing the hydropathy of amino acid residues within the intricate nanoscale topology of proteins is essential. This study presents quantitative hydropathy data for 277,877 amino acid residues, computed using the Protocol for Assigning a Residue's Character on a Hydropathy (PARCH) scale. Leveraging this data set comprising 1000 structurally diverse proteins sourced from the Protein Data Bank, we examined residues situated in various nanoscale environments and analyzed hydropathy in relation to protein topography. Our findings indicate that the hydropathy of a residue is intricately linked to both its individual characteristics and the geometric features of its neighboring residues in response to water. Changes in the number and chemical identity of the neighbors, as well as the nanoscale topography surrounding a residue, are mirrored in its hydropathy profile. Our calculations reveal the intricate interplay of hydrophilic, hydroneutral, and hydrophobic residues distributed across the surface and core of proteins. Notably, we observe that protein surfaces can be ten times more hydrophilic than their cores.

Molecular characterization and expression profiling of 3 beta-hydroxysteroid dehydrogenase and hydroxysteroid 17-beta dehydrogenase in response to HCG stimulation in striped murrel, Channa striata (Bloch, 1793)

Striped murrel (Channa striata) is highly valued in the Indian subcontinent and Southeast Asia for its nutritional and medicinal benefits. However, the limited availability of high-quality seeds of murrel and the asynchronous maturation of brooders, particularly during peak spawning seasons creates problems in aquaculture. Beta-hydroxysteroid dehydrogenases (β-HSDs) are steroidogenic enzymes involved in the biosynthesis of active steroids in gametogenesis and steroidogenesis. This study aimed to clone and characterize the full-length cDNA sequences of two crucial enzymes, 3-beta hydroxysteroid dehydrogenase (Hsd3β) and 17-beta hydroxysteroid dehydrogenase (Hsd17β). It resulted in full-length cDNA sequences for Hsd3β and Hsd17β with 1101bp and 771bp length, respectively, encoding 366 and 256 amino acids. Signal peptide analysis indicated that both proteins are secretory, and hydropathy profiles suggested their hydrophilic nature. Notably, Rossmann-fold NAD(P)(+)-binding domains characteristic of Short-chain dehydrogenases/reductases (SDR) family genes were identified in Hsd3β (between 9-355 aa) and in Hsd17β (between 10-254 aa). Gene expression analyses were performed in testes and ovaries using qPCR at three key stages: preparatory, mature, and 16 h post human chorionic gonadotropin (hCG) injection (16 hpi). Results demonstrated a significant 2-fold upregulation of Hsd17β in mature and 16 hpi testes, while Hsd3β showed a significant 10-fold upregulation in mature testes compared to premature and 16 hpi stages. Ovarian expression of Hsd3β and Hsd17β showed minimal expression upon hCG injection (P < 0.05). These findings contribute to our understanding of Beta-hydroxysteroid dehydrogenases (β-HSDs), providing insights into regulating sex steroid hormone synthesis during the gonadal development of striped murrel.

The Pentameric Ligand-Gated Ion Channel Family: A New Member of the Voltage Gated Ion Channel Superfamily?

In this report we present seven lines of bioinformatic evidence supporting the conclusion that the Pentameric Ligand-gated Ion Channel (pLIC) Family is a member of the Voltage-gated Ion Channel (VIC) Superfamily. In our approach, we used the Transporter Classification Database (TCDB) as a reference and applied a series of bioinformatic methods to search for similarities between the pLIC family and members of the VIC superfamily. These include: (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, (5) similarity of Hidden Markov Model profiles between families, (6) common 3D structural folds, and (7) clustering analysis of all families. Furthermore, sequence and structural comparisons as well as the identification of a 3-TMS repeat unit in the VIC superfamily suggests that the sixth transmembrane segment evolved into a re-entrant loop. This evidence suggests that the voltage-sensor domain and the channel domain have a common origin. The classification of the pLIC family within the VIC superfamily sheds light onto the topological origins of this family and its evolution, which will facilitate experimental verification and further research into this superfamily by the scientific community.

In vitro synthesis and biochemical characterization of acyl-homoserine lactone synthase and its deletion mutant.

Quorum sensing can induce density-dependent gene expressions that cause various problems. For quorum-sensing inhibition, fundamental solutions such as gene manipulation are required, and acyl-homoserine lactone synthase (AHL synthase), which synthesizes the universal quorum-sensing signal of gram-negative bacteria, can be used as a target. In this study, researchers synthesized His-tagged AHL synthase and its deletion mutant that lacks the active site and compared their biochemical characteristics. His-YpeI, the 6x His-tagged AHL synthase of Serratia fonticola, and His-ΔYpeI, its deletion mutant, were designed, and their property conservation were examined using in silico projection tools. For in vitro synthesis of enzymes, the His-YpeI CFPS template was synthesized by in vitro gene synthesis, and the His-ΔYpeI CFPS template was obtained by deletion PCR. CFPS was performed and the products were purified with the 6x His-tag. The enzymes' properties were compared using an enzymatic assay. The bioinformatic analysis confirmed the conservation of biochemical properties between 6x His-tagged and untagged enzymes, including helix-turn-helix interactions, hydropathy profiles, and tertiary structure between His-YpeI and YpeI and between His-ΔYpeI and ΔYpeI. His-YpeI and His-ΔYpeI synthesized by CFPS were found to have the expected molecular weights and demonstrated distinct differences in enzyme activity. The analyzed enzymatic constants supported a significant decrease in substrate affinity and reaction rate as a result of YpeI's enzyme active site deletion. This result showed that CFPS could be used for in vitro protein synthesis, and quorum sensing could be inhibited at the enzymatic level due to the enzyme active site's deletion mutation.

Cloning, computational analysis and expression profiling of steroid 5 alpha-reductase 1 (SRD5A1) gene during reproductive phases and ovatide stimulation in endangered catfish, Clarias magur

The cloning and characterization of the complete coding sequence of the Clarias magur SRD5A1 (CmSRD5A1) gene, which encodes an enzyme responsible for regulating steroid levels by converting testosterone into 5α-dihydrotestosterone (DHT), have been successfully achieved. DHT plays a vital role in enabling the complete expression of testosterone's actions in neuroendocrine tissues. The ORF of the full-length cDNA sequence of SRD5A1 was 795 bp, translating into 265 amino acids, with a total length of 836 bp including UTRs. Like other vertebrates, the signal peptide analysis revealed that SRD5A1 is a non-secretory protein, and hydropathy profiles indicated that it is hydrophobic in nature. The 3D structure of CmSRD5A1 sequence generated above was predicted using highly accurate AlphaFold 2 in Google Colab online platform. CmSRD5A1 contains seven transmembrane helices connected by six loops, with the N-termini located on the periplasmic side and C-termini on the cytosolic side. Structural superimposition with known bacterial and human SRD5As showed very high structural similarity. The electrostatic potential calculation and surface analysis of CmSRD5A1 revealed the presence of a large cavity with two openings one highly electropositive towards the cytosolic side and another relatively neutral towards the transmembrane region. The structural comparison revealed that the electropositive side of the cavity should bind to NADPH and the steroid hormone in the hydrophobic environment. Polar residues binding to NADPH are highly conserved and the same as known strictures. The conserved residues involved in hydrogen bonding with the ketone group at C-3 in the steroids hence fevering Δ4 double-bond reduction are identified as E66 and Y101. Our findings showed that SRD5A1 expression was lower during the spawning phase than the preparatory phase in female fish, while the administration of Ovatide (a GnRH analogue) resulted in up-regulation of expression after 6 h of injection in the ovary. In males, the lowest expression was observed during the preparatory phase and peaked at 16 h post- Ovatide injection in the testis. The expression of SRD5A1 in the brain of female fish was slightly higher during the Ovatide stimulation phase than the spawning phase. This study represents the first report on the cloning and characterization of the full-length cDNA of SRD5A1 in Indian catfish.

Molecular characterization of gonadotropin-releasing hormone receptor and its expression profiling in response to HCG stimulation in striped murrel, Channa striata (Bloch, 1793)

The gonadotropin releasing hormone receptors (GnRHR) play an important role in the activation of GnRH and elicit the release of gonadotropins that plays an important role in maturation and spawning. In the present work, molecular characterization of the GnRHR gene of snakehead murrel, Channa striata was carried out. The full-length cDNA of GnRH receptors of the 1581 bp sequence, which encodes 440 amino acids was identified by RACE-PCR. Signal peptide analysis confirmed that GnRHR is a non-secretory protein, and hydropathy profiles indicated its hydrophobic nature. The deduced amino acid sequences of the GnRHR showed the expected conserved domains, which include seven membrane-spanning domains, two potential N-linked glycosylation sites (631 Asp, 561 Asp), and 32 sites phosphorylation sites of serine and threonine residues. A homology search of GnRHR cDNA sequence revealed 88 % identity with Channa argus, followed by 85 % and 83 % sequence identities with Lates calcarifer, and Anabas testudineus at the protein level. Phylogenetic analysis depicted a close association with Channa argus and other fish species. Our findings showed differential GnRHR expression during different reproductive stages, with higher expression after HCG administration. Expression profiling revealed that GnRHR is highly expressed in the brain and testis at 16 h post-HCG injection, while it was neutrally regulated in female ovarian tissues. This study provides full-length coding sequence information of the GnRHR gene and will help to understand the reproductive biology of murrel using expression studies and devise strategies for induced breeding.

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.

Mitochondrial genomic analyses provide new insights into the “missing” atp8 and adaptive evolution of Mytilidae

BackgroundMytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length.ResultsIn this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater.ConclusionsOur analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments.

Alternative CUG Codon Usage in the Halotolerant Yeast Debaryomyces hansenii: Gene Expression Profiles Provide New Insights into Ambiguous Translation

The halotolerant yeast Debaryomyces hansenii belongs to the CTG-Ser1 clade of fungal species that use the CUG codon to translate as leucine or serine. The ambiguous decoding of the CUG codon is relevant for expanding protein diversity, but little is known about the role of leucine–serine ambiguity in cellular adaptations to extreme environments. Here, we examine sequences and structures of tRNACAG from the CTG-Ser1 clade yeasts, finding that D. hansenii conserves the elements to translate ambiguously. Then, we show that D. hansenii has tolerance to conditions of salinity, acidity, alkalinity, and oxidative stress associated with phenotypic and ultrastructural changes. In these conditions, we found differential expression in both the logarithmic and stationary growth phases of tRNASer, tRNALeu, tRNACAG, LeuRS, and SerRS genes that could be involved in the adaptive process of this yeast. Finally, we compare the proteomic isoelectric points and hydropathy profiles, detecting that the most important variations among the physicochemical characteristics of D. hansenii proteins are in their hydrophobic and hydrophilic interactions with the medium. We propose that the ambiguous translation, i.e., leucylation or serynation, on translation of the CUG-encoded residues, could be linked to adaptation processes in extreme environments.

AlignMe: an update of the web server for alignment of membrane protein sequences.

The AlignMe web server is dedicated to accurately aligning sequences of membrane proteins, a particularly challenging task due to the strong evolutionary divergence and the low compositional complexity of hydrophobic membrane-spanning proteins. AlignMe can create pairwise alignments of either two primary amino acid sequences or two hydropathy profiles. The web server for AlignMe has been continuously available for>10 years, supporting 1000s of users per year. Recent improvements include anchoring, multiple submissions, and structure visualization. Anchoring is the ability to constrain a position in an alignment, which allows expert information about related residues in proteins to be incorporated into an alignment without manual modification. The original web interface to the server limited the user to one alignment per submission, hindering larger scale studies. Now,batches of alignmentscan be initiatedwith a single submission. Finally, to provide structural context for the relationship between proteins, sequence similarity can now be mapped onto one or more structures (or structural models) of the proteins being aligned, by links to MutationExplorer, a web-based visualization tool. Together with a refreshed user interface, these features further enhance an important resource in the membrane protein community. The AlignMe web server is freely available at https://www.bioinfo.mpg.de/AlignMe/.

Discovery and Characterization of the Phospholemman/SIMP/Viroporin Superfamily

Using bioinformatic approaches, we present evidence of distant relatedness among the Ephemerovirus Viroporin family, the Rhabdoviridae Putative Viroporin U5 family, the Phospholemman family, and the Small Integral Membrane Protein family. Our approach is based on the transitivity property of homology complemented with five validation criteria: (1) significant sequence similarity and alignment coverage, (2) compatibility of topology of transmembrane segments, (3) overlap of hydropathy profiles, (4) conservation of protein domains, and (5) conservation of sequence motifs. Our results indicate that Pfam protein domains PF02038 and PF15831 can be found in or projected onto members of all four families. In addition, we identified a 26-residue motif conserved across the superfamily. This motif is characterized by hydrophobic residues that help anchor the protein to the membrane and charged residues that constitute phosphorylation sites. In addition, all members of the four families with annotated function are either responsible for or affect the transport of ions into and/or out of the cell. Taken together, these results justify the creation of the novel Phospholemman/SIMP/Viroporin superfamily. Given that transport proteins can be found not just in cells, but also in viruses, the ability to relate viroporin protein families with their eukaryotic and bacterial counterparts is an important development in this superfamily.

Characterizing Hydropathy of Amino Acid Side Chain in a Protein Environment by Investigating the Structural Changes of Water Molecules Network.

Assessing the hydropathy properties of molecules, like proteins and chemical compounds, has a crucial role in many fields of computational biology, such as drug design, biomolecular interaction, and folding prediction. Over the past decades, many descriptors were devised to evaluate the hydrophobicity of side chains. In this field, recently we likewise have developed a computational method, based on molecular dynamics data, for the investigation of the hydrophilicity and hydrophobicity features of the 20 natural amino acids, analyzing the changes occurring in the hydrogen bond network of water molecules surrounding each given compound. The local environment of each residue is complex and depends on the chemical nature of the side chain and the location in the protein. Here, we characterize the solvation properties of each amino acid side chain in the protein environment by considering its spatial reorganization in the protein local structure, so that the computational evaluation of differences in terms of hydropathy profiles in different structural and dynamical conditions can be brought to bear. A set of atomistic molecular dynamics simulations have been used to characterize the dynamic hydrogen bond network at the interface between protein and solvent, from which we map out the local hydrophobicity and hydrophilicity of amino acid residues.

Pseudomonas putida Δ9-fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress.

Pseudomonas putida counteract the fluidizing effect of cationic surfactants decreasing the content of membrane unsaturated fatty acid (UFA). A Δ9-fatty acid desaturase gene (desA) from P. putida was isolated, cloned, and successfully expressed in Escherichia coli, a Δ9 desaturase deficient organism. desA consists of 1185 bp and codes for 394 amino acids. The deduced amino acid sequence reveals three histidine clusters and a hydropathy profile, typical of membrane-bound desaturases. Validating desA expression in E. coli cells, the amount of palmitoleic acid increased from 2.05 to 7.36%, with the concomitant increase in membrane fluidity (fluorescence polarization value decrease from 0.13 ± 0.03 to 0.09 ± 0.02). Also, when DesA activity was assayed in vivo, the percentage of UFA obtained from exogenous palmitic acid [1-14 C] increased 10-fold. In contrast, when cells expressing desA were exposed 15 min at sublethal concentration of cationic surfactants, the amount of UFA was 82% lower than that detected in cells non-exposed. Thus, the decrease in UFA content to counteract the fluidizing effect of cationic surfactants can be correlated with reduction of DesA activity.

Molecular characterization and expression profiling of 17-beta-hydroxysteroid dehydrogenase 2 and spermatogenesis associated protein 2 genes in endangered catfish, Clarias magur (Hamilton, 1822)

The 17-beta-hydroxysteroid dehydrogenase 2 (17β-HSD2) enzyme regulates steroid levels by the inactivation of estrogen and androgens. Spermatogenesis associated protein 2 (SPATA2) plays a vital role in spermatogenesis in vertebrates including fish. We report cloning and characterization of full cds of 17β-HSD2 and SPATA2 genes in Clarias magur. The full-length cDNA sequences of 17β-HSD2 and SPATA2 were 1187 bp (ORF 1125 bp) and 1806 bp (ORF 1524 bp) encoding 375 and 508 amino acids, respectively. Signal peptide analysis revealed SPATA2 is nonsecretory, while 17β-HSD2 is a secretory protein. Hydropathy profiles showed both proteins are hydrophilic in nature. Tissue distribution of both the genes revealed high mRNA level of SPATA2 in all tissues examined indicating its wide range of expression. 17β-HSD2 indicated higher expression in preparatory phase compared to spawning phase in ovary while it was opposite in case of testis. SPATA2 showed significantly higher expression in preparatory phase compared to spawning phase in both ovary and testis. Administration of OvatideTM (GnRH analog) resulted in upregulation of SPATA2 expression at 6 and 16 h post-injection while 17β-HSD2 showed upregulation only at 6 h post-injection. To the best of our knowledge, this is a first report on characterization of 17β-HSD2 and SPATA2 full-length cDNA in catfish.

Identification and quantification of proteins at adsorption layer of emulsion stabilized by pea protein isolates.

This study adopted the method of quantitative proteomics to analyze the adsorbed proteins in oil-in-water emulsions stabilized by pea protein isolate (PPI). Adsorbed proteins were precipitated by an optimized precipitation method and precipitates were labeled and subjected to a reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry (RPLC-ESI-MS/MS) for protein identification and quantification. In total, 77 proteins were identified, of which 49 proteins with significant differences were observed. There were 25 upregulated proteins (fold change > 1) and 24 downregulated proteins (fold change < 1). The interfacial adsorption abilities of these proteins were compared according to the classification of protein families. The results showed that all isoforms of vicilins exhibited high adsorption abilities at the oil-water interface. Compared with vicilin, convicilin showed opposite adsorption capacity. Different legumin families showed significantly different affinities on the oil-water interface. In contrast to albumin-1, albumin-2 was preferentially adsorbed to the interface. The amino acid sequence alignment and hydropathy profile analysis of these proteins showed that the proteins well-balanced between hydrophobic and hydrophilic amino acid groups displayed high interfacial activity. In contrast, a long hydrophilic or hydrophobic fragment could adversely influence protein interfacial activity. This study provides an insight into the interfacial behaviors of proteins by supplying detailed quantitative information of interfacial layer.

A Structural and Functional Role for Disulfide Bonds in a Class II Hydrophobin.

Hydrophobins are multifunctional, highly surface active proteins produced in filamentous fungi and can be identified by eight conserved cysteine residues, which form four disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide and then examining the protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in the level of random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements performed on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster resonance energy transfer (FRET) was used to assess the role of disulfides in self-assembly in solution, and near complete loss of the FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this, we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution self-assembly.

Molecular cloning and mRNA expression pattern of Sox4 in Paramisgurnus dabryanus

Sox4 belonged to the SoxC subfamily of the Sox family, which play important roles in the development of the vertebrate gonad and nervous system. A Sox4 homologue was cloned from brain of Paramisgurnus dabryanus by using homologous cloning and rapid amplification of cDNA ends (RACE), designated as PdSox4. The full-length cDNA was 2163bp, containing the 759bp 5'-untranslated region, 267bp 3'-untranslated region and encoding a putative protein of 378 amino acids with a characteristic high mobility group box (HMG-box) DNA-binding domain of 79 amino acids with the specific motif (RPMNAFMVW). Alignment and phylogenetic analyses indicated that PdSox4 shares highly identical sequence with Sox4 homologues from different species. The signal peptide analysis predicted that PdSox4 is a non-secretory protein. The hydropathy profile of PdSox4 protein revealed that this protein is hydrophilic in nature. The expression profiles of PdSox4 in different developmental stages and various adult tissues of sexs were analyzed by quantitative real-time RT-PCR (qRT-PCR) and In situ hybridization (ISH). The results showed that PdSox4 was ubiquitously expressed during embryogenesis and various adult tissues, especially in central nervous system. Tissue distribution analyses revealed that PdSox4 was expression in developing germ cells. Taken together, these preliminary findings suggested that PdSox4 is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, neurogenesis and gonad development.

Sequence editing strategy for improving performance of β-glucuronidase from Aspergillus terreus

β-Glucuronidase from Aspergillus terreus Li-20 (AtGUS) can hydrolyze the glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) and glycyrrhetinic acid (GA). However, its low catalytic efficiency and thermal instability limit its application at industrial scale biotransformation of GL. In this work, sequence editing strategy was employed to improve the performance of AtGUS. Herein, three mutants, AtGUS-t1, AtGUS-t2 and AtGUS-t3 were constructed through partial truncation of the C-terminal non-conserved sequence of wild-type enzyme AtGUS based on the hydropathy profile and secondary structure of the C-terminal non-conserved. In addition, two more mutants were constructed by splicing of the C-terminal non-conserved sequence. The splicing mutant AtGUS(P) has a C-terminus consisting of merely polar amino acid residues with all non-polar amino acid residues knocked out from the C-terminal non-conserved sequence, consequently a highly hydrophilic C-terminus generated. Conversely, the other splicing mutant AtGUS(NP) has a C-terminus composed of merely non-polar amino acid residues, presenting a highly hydrophobic C-terminus. Among the truncated mutants, only the mutant AtGUS-t3 deleted a C-terminal hydrophilic coil peptide increased 3.8-fold catalytic efficiency with kcat/Km value of 8.31mM−1·s−1. The splicing mutant AtGUS(P) expanded the optimal pH range compared to the wild-type AtGUS and retained more than 80% relative activity in alkaline pH (pH 8.0). The another splicing mutant AtGUS(NP) displayed an increased thermal stability and conserved more than 50% relative activity at 65°C for 30min compared to AtGUS, which only held less than 20% relative activity under the same treatment condition. The results of this study demonstrate that sequence editing in truncation/splicing of C-terminal non-conserved region/residues could improve enzyme performance and is a potentially simple and efficient protein engineering strategy for the important industrial enzymes.

Heme Assimilation in Schizosaccharomyces pombe Requires Cell-surface-anchored Protein Shu1 and Vacuolar Transporter Abc3

The Schizosaccharomyces pombe shu1+ gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol.

Advanced Protein Alignments Based on Sequence, Structure and Hydropathy Profiles; The Paradigm of the Viral Polymerase Enzyme

One of the major drawbacks of modern bioinformatics is the fact that protein similarity and blast searches are still based on primary amino acid sequence rather than structural data. Primary sequence searches are inadequate, as they fail to provide a realistic fingerprint for the query protein. Protein function is much more related to protein structure rather than to its amino acid sequence. After all structure is much more conserved than sequence in nature. In this direction and in an effort to bridge this flaw, a novel platform has been developed, which is capable of performing fast similarity searches using protein primary and secondary structural information. The protein secondary structure profile (PSSP) tool is capable of performing conventional blast searches, based on protein sequences, as well as alignments based on a custom made hydropathy substitution matrix that takes into account the physicochemical profile of the amino acids that compose the query protein. Moreover, PSSP is capable of efficiently exploiting protein secondary structural information from the PDB database when available. If the query protein is not indexed in the RCSB PDB database, it will automatically determine the secondary elements of the given protein by performing an ‘on the fly’ secondary structure prediction. All query proteins are then blasted against the RCSB PDB secondary elements database. Hits are scored, ranked and returned to the user via a well-organized and user friendly graphical interface.