Sequence Of Amino Acid Residues Research Articles

Cloning and Expression of a New Gene from Shark Liver and its Inhibitory Effects on Hepatoma Cells

Aim To clone and express a new gene psl12 from shark liver, purify the expression product, and study its inhibitory effects on hepatoma cells. Methods and Results We have previously reported the discovery of Hepatocyte regenerational stimulatory factor from shark liver (sHRSF). According to the sequence of the N-terminal amino acid residues of sHRSF and using RT-PCR method, we obtained 350 bp cDNA fragment from the total RNA extracted from regenerated hepatic tissues. The cDNA has an ORF of 333 nucleotides and encodes a peptide of 111 amino acid residues. The front 7 N-terminal amino acid residues agree with those of sHRSF. We named this peptide PSL12 (peptide from shark liver with a molecular mass of about 12 kDa). The cDNA was ligated into plasmid pGEM-T-Easy and obtained recombinant plasmid pGEM-T-psl12. Based on the sequence of psl12 gene and multiple cloning sites ( BamH I and Sal I restriction sites) of expression plasmid pET-32a, the specific primers for PCR amplifying psl12 gene were designed and synthesized. The PCR was operated using plasmid pGEM-T- psl12 as template. The expression plasmid pET-32a- psl12 was constructed by inserting the PCR product into pET-32a and was transformed into an E. coli host cell BL21. After inducing with IPTG, the fusion protein with his-tag was expressed to about 40% and was purified using metal chelation chromatography on His-Bind resin. After the fusion protein was cleaved with FXa, PSL12 was purified using Resource Q and Mono Q column chromatography. The purified PSL12 could inhibit proliferation of the hepatoma cell lines SMMC-7721 and HepG 2. Conclusions A new gene psl12 was obtained from shark liver. The expression product PSL12 of the gene could inhibit proliferation of the hepatoma cells cultured in vitro.

Structural characterization and comparative modeling of PD-Ls 1–3, type 1 ribosome-inactivating proteins from summer leaves of Phytolacca dioica L.

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1–2 and PD-Ls 3–4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1–3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man) 3 (GlcNAc) 2 (Fuc) 1 (Xyl) 1 linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.

Two-dimensional Self-assembly of a Designed Amphiphilic Peptide at Air/Water Interface

A peptide having alternate sequence of hydrophobic and hydrophilic amino acid residues, (QAQL)4 (CH3CO-(Gln-Ala-Gln-Leu)4-NH2), was designed to form a β-sheet monolayer at the air/water interface. According to the designing of the peptide having charge free amino acid as hydrophilic residues, the peptide showed stable monolayer at wide range pH. The peptide monolayer prepared by Langmuir-Blodgett (LB) method showed an arranged nano-fibrous object. (QAQL)4-PEG was also an attractive building block for construction of nano-architecture by self-assembly at the air/water interface. PEG attached on the peptide segment showed a conformational transition from a flattened state to brush one, dependent on the compressing degree of the monolayer on water subphase. The conformational transition was simultaneously induced a morphological change of the nano-architecture from disk-like structure to fibrous array.

Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel 2

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca2+ and Mg2+ permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg2+ permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca2+ permeability along with concentration-dependent inhibition of Na+-carrying currents by Ca2+. Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca2+-permeable TRPM channels, substantially increased Ca2+ permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca2+ permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

Quantum chemical 13 C α chemical shift calculations for protein NMR structure determination, refinement, and validation

A recently determined set of 20 NMR-derived conformations of a 48-residue all-alpha-helical protein, (PDB ID code 2JVD), is validated here by comparing the observed (13)C(alpha) chemical shifts with those computed at the density functional level of theory. In addition, a recently introduced physics-based method, aimed at determining protein structures by using NOE-derived distance constraints together with observed and computed (13)C(alpha) chemical shifts, was applied to determine a new set of 10 conformations, (Set-bt), as a blind test for the same protein. A cross-validation of these two sets of conformations in terms of the agreement between computed and observed (13)C(alpha) chemical shifts, several stereochemical quality factors, and some NMR quality assessment scores reveals the good quality of both sets of structures. We also carried out an analysis of the agreement between the observed and computed (13)C(alpha) chemical shifts for a slightly longer construct of the protein solved by x-ray crystallography at 2.0-A resolution (PDB ID code 3BHP) with an identical amino acid residue sequence to the 2JVD structure for the first 46 residues. Our results reveal that both of the NMR-derived sets, namely 2JVD and Set-bt, are somewhat better representations of the observed (13)C(alpha) chemical shifts in solution than the 3BHP crystal structure. In addition, the (13)C(alpha)-based validation analysis appears to be more sensitive to subtle structural differences across the three sets of structures than any other NMR quality-assessment scores used here, and, although it is computationally intensive, this analysis has potential value as a standard procedure to determine, refine, and validate protein structures.

Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 °C) and in infected tissues as a yeast form (36 °C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

An Adenoviral Platform for Selective Self‐Assembly and Targeted Delivery of Nanoparticles

Metallic nanoparticles (NPs) can be used for the diagnosis, imaging, and therapy of tumors and cardiovascular disease. However, targeted delivery of NPs to specific cells remains a major limitation for clinical realization of these potential treatment options. Herein, a novel strategy for the specific coupling of NPs to a targeted adenoviral (Ad) platform to deliver NPs to specific cells is defined. Genetic manipulation of the gene-therapy vector is combined with a specific chemical coupling strategy. In particular, a high-affinity interaction between a sequence of six-histidine amino acid residues genetically incorporated into Ad capsid proteins and nickel(II) nitrilotriacetic acid on the surface of gold NPs is employed. The selective self-assembly of gold NPs and Ad vectors into multifunctional platforms does not negatively affect the targeting of Ad to specific cells. This opens the possibility of using Ad vectors for targeted NP delivery, thereby providing a new type of combinatorial approach for the treatment of diseases that involves both nanotechnology and gene therapy.

Construction of an arranged nano-fibrous structure by selforganization of a designed amphiphilic peptide based on β- strand

AbstractAn amphiphilic peptide having alternate sequence of hydrophobic and charged amino acid residues, RFDF16 (CH3CO-RFDFRFDFRFDFRFDF-NH2), was designed to form a β-sheet monolayer at the air/water interface. The peptide monolayer was prepared by Langmuir-Blodgett (LB) method. The LB films were transferred onto mica substrates without the compressing process at various pH values in order to investigate a self-organized structure of peptide monolayer. Surface morphology of LB films showed arranged nano-fibers which have differences in interval between fibers and their orientation depending on pH of the subphase. On the other hand, the self-organized nano-fiber was also re-arranged by compressing process. The nano-fibers became elongated and aligned upon compression to higher surface pressures. The exploitation of an electrostatic interaction and compressing process will allow a larger two-dimensionally regulated nano-structure.

Computational Analysis of Adhesion of Primer Ligands to Dentinal Collagen: Effect of Spacer Groups in Ligand and Amino Acid Residue Sequence Differences in Collagen

This study sought to assess by computer modeling the interactions between dentinal collagen and primer monomer ligands used to promote bonding of restorations to tooth. Modeling was carried out both by direct and indirect methods to probe interaction mechanisms. Ligands studied in this investigation conformed chemically to methacrylate phosphates of alkane diol, with changes in the number of methylene spacer groups. Increase in number of methylene groups in the series introduces increasing levels of ligand conformational freedom. An automatic docking program was used to analyze the effect of these changes on primer-collagen interactions in direct (target-based) modeling. The effect of limited modifications of amino acid residue sequences in structural variants of type 1 dentinal collagen was also assessed in this approach. The indirect (ligand-based) modeling used a pharmacaphore search to mimic primer binding to type 1 collagen using a common functional alignment algorithm. Docking energy, and the non-bonded and electrostatic contributions to it, showed statistically highly significant differences (p<0.0001) with ligand conformational freedom. But the effect of collagen composition differences was, although statistically significant (p<0.05), relatively small. Both target-based direct docking and ligand-based indirect modeling visualizations showed that conformations tended to align in a 3-D geometric pattern in bound states, and that the conformational flexibility of the ligands played a critical role in alignment. The results suggest that incorporation of spacer groups in primer monomers may modify dentin bonding to improve overall adhesion under optimum conditions.

The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases

The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.

HTHP: A Novel Class of Hexameric, Tyrosine-coordinated Heme Proteins

We have cloned, expressed, isolated and characterized a hexameric tyrosine-coordinated heme protein (HTHP) from the marine bacterium Silicibacter pomeroyi. HTHP shows peroxidase and catalase activity and has a high thermal stability. As-isolated HTHP has absorption maxima at 407, 495, 504, 532 and 622 nm wavelength. Upon reduction maxima at 430, 564 and 596 nm wavelength are discernible. The crystal structure of HTHP reveals a hexameric, ring-like arrangement of six monomers. Each monomer binds a solvent accessible heme group, which is stabilized by the interaction of three neighboring monomers. The pocket around the heme distal side is positively charged due to three conserved arginine residues in direct vicinity. The heme iron is penta-coordinated with a tyrosine residue as proximal ligand. The coordinating hydroxyl-group of the tyrosine ligand interacts with the guanidinium group of a nearby arginine residue, an arrangement closely resembling the catalytic dyad found in monofunctional heme-containing catalases and coral allene oxide synthases, which are b-type cytochromes with tyrosine coordination trans to an empty coordination site. Despite the similarity in heme coordination HTHP is functionally and structurally unrelated to catalases and other heme-containing proteins. Its hexameric arrangement, solvent accessible heme binding pocket and heme coordination by tyrosine render HTHP a unique protein with unusual properties. A database search against complete and incomplete genomes shows that the 76 amino acid residues sequence of HTHP is unrelated to characterized proteins, but is homologous to orfs found in a phylogenetically diverse set of bacteria with sequence identities of 30–76%. We therefore propose that HTHP is the prototype of a new class of heme proteins.

Improved Whitten−Rabinovitch Approximation for the Rice−Ramsperger−Kassel−Marcus Calculation of Unimolecular Reaction Rate Constants for Proteins

The Whitten-Rabinovitch (WR) approximation used in the semi-classical calculation of the Rice-Ramsperger-Kassel-Marcus (RRKM) unimolecular reaction rate constant was improved for reliable application to protein reactions. The state sum data for the 10-mer of each amino acid calculated by the accurate Beyer-Swinehart (BS) algorithm were used to obtain the residue-specific correction functions (w). The correction functions were obtained down to a much lower internal energy range than reported in the original work, and the cubic, rather than quadratic, polynomial was used for data fitting. For a specified sequence of amino acid residues in a protein, an average was made over these functions to obtain the sequence-specific correction function to be used in the rate constant calculation. Reliability of the improved method was tested for dissociation of various peptides and proteins. Even at low internal energies corresponding to the RRKM rate constant as small as 0.1 s-1, the rate constant calculated by the present method differed from the accurate BS result by 60% only. In contrast, the result from the original WR calculation differed from the accurate result by a factor of 3000. Compared to the BS method, which is difficult to use for proteins, the main advantage of the present method is that the RRKM rate constant can be calculated instantly regardless of the protein mass.

Cysteine-scanning Analysis of the Nucleobase-Ascorbate Transporter Signature Motif in YgfO Permease of Escherichia coli: Gln-324 AND Asn-325 ARE ESSENTIAL, AND Ile-329–Val-339 FORM AN α-HELIX

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.

Modeling of the potential coiled-coil structure of snapin protein and its interaction with SNARE complex.

Autism is a developmental disability causing learning and memory disorder. The heart of the search for a cure for this syndrome is the need to understand dendrite branch patterning, a process crucial for proper synaptic transmission. Due to the association of snapin with the SNARE complex and its role in synaptic transmission it is reported as a potential drug target for autism therapies. We wish to impart the noesis of the 3D structure of the snapin protein, and in this chase we predict the native structure from its sequence of amino acid residues using the classical Comparative protein structure modeling methods. The predicted protein model can be of great assistance in understanding the structural insights, which is necessary to understand the protein function. Understanding the interactions between snapin and SNARE complex is crucial in studying its role in the neurotransmitter release process. We also presented a computational model that shows the interaction between the snapin and SNAP-25 protein, a part of the larger SNARE complex.

Errors in Translation Cause Selective Neurodegeneration

The 3D structure of a protein is determined by the unique sequence of amino acid residues comprising the polypeptide chain. Sequence changes can cause protein misfolding, a potentially toxic phenomenon implicated in various neurodegenerative disorders. In a recent paper, translational misincorporation is proposed to be a new biochemical mechanism for generating mutant proteins that misfold and kill neurons.

Two-Dimensional Ordered β-Sheet Lipopeptide Monolayers

A series of amphiphilic lipopeptides, ALPs, consisting of an alternating hydrophilic and hydrophobic amino acid residue sequence coupled to a phospholipid tail, was designed to form supramolecular assemblies composed of beta-sheet monolayers decorated by lipid tails at the air-water interface. A straightforward synthetic approach based on solid-phase synthesis, followed by an efficient purification protocol was used to prepare the lipid-peptide conjugates. Structural insight into the organization of monolayers was provided by surface pressure versus area isotherms, circular dichroism, Fourier transform infrared spectroscopy, and Brewster angle microscopy. In situ grazing-incidence X-ray diffraction (GIXD) revealed that lipopeptides six to eight amino acids in length form a new type of 2D self-organized monolayers that exhibit beta-sheet ribbons segregated by lipid tails. The conclusions drawn from the experimental findings were supported by a representative model based on molecular dynamics simulations of amphiphilic lipopeptides at the vacuum-water interface.

Human CD4+ T cells stimulated by conserved adenovirus 5 hexon peptides recognize cells infected with different species of human adenovirus

The immune response against human adenovirus (HAdV) has gained interest because of the application of HAdV-based vectors in gene therapy and the high incidence of infections in pediatric recipients of allogeneic stem cell grafts. Because antiviral medication is frequently ineffective, the option of adoptive transfer of HAdV-specific donor-derived T cells in these immunocompromised patients is investigated. To generate good manufacturing practice-compatible reagents, a panel of 63 long, overlapping, peptides of the hexon protein was screened for recognition by T cells. Five conserved peptides of 30 amino acids were identified that were recognized by the majority of adult donors. CD4+ T cells from long-term cultures of PBMC, stimulated with this set of five peptides, recognized cells infected with HAdV serotypes belonging to different species. These data demonstrate that adult human T cells preferentially recognize conserved sequences of amino acid residues from a structural protein of HAdV. In the context of gene therapy, this observation may limit the beneficial effect of switching to HAdV-based vectors derived from less common serotypes of HAdV in an attempt to circumvent pre-existing immunity. However, this cross-reactivity benefits the application of HAdV-specific T cells for adoptive immunotherapy in immunocompromised transplant recipients.

Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information.

We describe an improved artificial neural network (ANN)-based method for predicting peptide retention times in reversed-phase liquid chromatography. In addition to the peptide amino acid composition, this study investigated several other peptide descriptors to improve the predictive capability, such as peptide length, sequence, hydrophobicity and hydrophobic moment, and nearest-neighbor amino acid, as well as peptide predicted structural configurations (i.e., helix, sheet, coil). An ANN architecture that consisted of 1052 input nodes, 24 hidden nodes, and 1 output node was used to fully consider the amino acid residue sequence in each peptide. The network was trained using approximately 345,000 nonredundant peptides identified from a total of 12,059 LC-MS/MS analyses of more than 20 different organisms, and the predictive capability of the model was tested using 1303 confidently identified peptides that were not included in the training set. The model demonstrated an average elution time precision of approximately 1.5% and was able to distinguish among isomeric peptides based upon the inclusion of peptide sequence information. The prediction power represents a significant improvement over our earlier report (Petritis, K.; Kangas, L. J.; Ferguson, P. L.; Anderson, G. A.; Pasa-Tolic, L.; Lipton, M. S.; Auberry, K. J.; Strittmatter, E. F.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2003, 75, 1039-1048) and other previously reported models.

An immunoassay for ochratoxin A without the mycotoxin

Phage display peptide libraries allow the selection of new molecules capable of mimicking the structural and functional features of native proteins or chemicals. This technology can be applied to developing new reagents, which may serve as the surrogate of the original objects. In order to screen peptides capable of mimicking ochratoxin A (OTA) in the interaction with anti-OTA monoclonal antibody (McAb) and establish the immunoassay for OTA, an anti-OTA McAb was used as the target for panning-elution selection from a phage random seven-peptide library. After four rounds of panning, 11 phages were found to be able to mimic OTA in binding with the antibody. Enzyme-linked Immunosorbent Assay (ELISA) for detecting OTA was established with the phages. In the most sensitive assay, the linear range of the inhibition curve was 200–8000 pg/ml; the detection limit was 150 pg/ml. The inserted peptide sequences were deduced by DNA sequencing. The common amino acid residue sequence was IR(V)PMV(L)XX (X is any amino acid residues), which was verified by two synthesized peptides. The results demonstrated that those phage peptides could be used as the surrogate of OTA to establish the immunoassay.

Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.