Protein and Peptide Letters - Volume 17, Issue 2, 2010

36 mutants of the Sulfolobus solfataricus amidase were analyzed by comparing biochemical data to structural data obtained by a learning machine. The analysis shows that beside well known catalytic residues, amino acid residues Arg197, Lys209 and Asp228 are important for the catalytic activity of the signature thermophilic amidase.

The peptidyl prolyl isomerase (Pin1) that catalyzes the isomerization of peptide bonds involving proline and phosphorylated serine/threonine/tyrosine and alters the conformation and differential folding has been implicated in the regulation and function of phosphorylated proteins including mitotic and cell cycle proteins viz. Cdc25c, Bcl2, p53 etc. DNA topoisomerase IIα is one of the nuclear enzymes that maintain the DNA topology and regulates nuclear transactions like chromatin segregation and mitosis. In the present studies, we have carried out in-silico investigations on the possibilities of pin1 interaction with topo IIα and its functional regulation. We found ten potential pin1 interacting sites within topo IIα, which were part of loop and/or low complexity regions except helix at S802 within the catalytic domain. Proline directed phosphorylation was found to be possible at 1354, 1361, 1393 positions by cdk. Change in dihedral angle (ω) to 0 degree at all potential pin1 interacting sites at 575, 602, 802 and 950 for cis conformation of peptide bond introduced significant structural change with higher potential energy. All-cis-topo IIα structure reveals that potential pin1 sites come closer to each other, perhaps forming a motif, thereby suggesting cooperative phenomenon to maintain higher potential energy conformation. The bio-informatic analysis of topo IIα showed that multisite interaction of pin1 is possible at all the predicted sites. However, a strong possibility of pin1 interaction exist within c-terminal at 1213, 1247, 1354, 1361, 1393 sites, which may lead to either alterations in localization or modification in the activity and perhaps stability of the enzyme.

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)3-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.

Tert- butylation of tryptophan (2', 5', 7'- tri tertiary butyl tryptophan), formed during acidolytic cleavage of synthetic peptides Ac-KLVYWAE-CONH2 (A-YW) and Ac-KLVWWAE-CONH2 (A-WW), that are analogs of the fragment of Alzheimer's β-amyloid peptide Ac-KLVFFAE-CONH2, during solid-phase peptide synthesis, was characterized by matrix- assisted laser desorption/ionization time of flight/time of flight (MALDI TOF/TOF) mass spectrometry. Crude peptide was fractionated by high performance liquid chromatography. Peptide fractions were sequenced and modified tryptophan was determined with the help of MALDI TOF/TOF mass spectra. Thus, it is possible to pinpoint the particular tryptophan residue that undergoes modification during synthesis of peptides containing multiple tryptophan residues.

Tapes japonica lysozyme (TJL), which belongs to the invertebrate-type lysozyme family, has a unique dimer formation. The residues, which include catalytic residues (glutamate 18 and aspartate 30), at the dimer interface form electrostatic interactions. Our previous study suggested that increasing the NaCl concentration switched TJL from a dimer to monomer structure, which increased TJL activity. Therefore, conversion from the dimeric to the monomeric structure is crucial for the TJL activity. In the present study, to further understand the effect of NaCl on TJL dimer formation, we examined the protein concentration and pH dependence of TJL activity in the presence or absence of 500 mM NaCl. TJL activity was suppressed at the high protein concentration. And the optimum pH of TJL activity was decreased in the absence of NaCl. These dependencies confirm the presence of electrostatic interactions between molecules of TJL in the dimeric form in an aqueous solution.

Amyloid fibril formation of amyloid beta peptide 1-40 (Aβ 1-40) was reported to be retarded in the presence of 150mM phosphate buffer at pH 7 [Monji, Ustumi, Ueda, Imoto, Yoshida, Hashioka, Tashiro and Tashiro, J. Neurochemistry, 77, 1425-1432 (2007)]. In order to elucidate the reason why phosphate ion retards the amyloid fibril formation, we examined the preferential binding sites of phosphate ion to Aβ 1-40 using chemical shift perturbation analysis of heteronuclear NMR. In titration analysis of 15N-labeled Aβ1-40 in the presence of 150 mM phosphate ion or 150 mM chloride ion, we identified the residues affected by these ions in Aβ 1-40. As a result, we found the tendency that phosphate ion preferentially bound to some residues located on the C-terminus region where the region was reported to be the potential β-strand region in Aβ1-40. Therefore, we suggested that phosphate ions interacted with the potential β-strand region in Aβ1-40 to be hard to form β-sheet in Aβ 1-40, resulting in retardation of the amyloid fibril formation.

Aquaporin Z (AqpZ) is a typical orthodox aquaporin with 6 transmembrane domains and five connecting loops. In order to express this complex membrane protein efficiently, E. coli cell-free expression system was employed as an alternative to produce aquaporin Z. Using different fusion vectors containing AqpZ gene, the expression level of fusion proteins in cell-free system varied from 7.97 to 578.35 μg/ml, while 7.34 to 340.81 μg/ml for target protein (AqpZ). The free energy of mRNA secondary structure at translation initiation region (TIR) was predicted and demonstrated a positive relationship with the expression level of AqpZ in cell-free system. This is the first report of expressing water channel protein in E. coli cell-free system, which has become a highly promising tool for fast and efficient production of integral membrane proteins.

Tumor necrosis factor-α (TNF-α) is a glycoprotein hormone with important functions in inflammation and apoptosis. It plays a significant role as a pro-inflammatory cytokine in the defense against viral, bacterial and parasitic infections and autoimmune disorders. Furthermore, it influences energy homeostasis and has an anorexigenic effect on the hypothalamus. TNF-α has also been shown to be involved in the pathogenesis of psychiatric disorders such as depression or narcolepsy. Ghrelin is a peptide hormone which primarily regulates eating behavior through modulation of expression of orexigenic peptides in the hypothalamus. Ghrelin administration increases food intake and body weight, while weight loss in turn increases ghrelin levels. Secondly, it posesses anti-inflammatory properties. It also seems to have an impact on mental health as it is has been suggested to have antidepressant and anxiolytic properties. Therefore, TNF-α and ghrelin seem to have opposite effects regarding the hypothalamic regulation of eating behavior, modulation of the immune response and the state of mental health.

Oxazepam and lorazepam inhibit the adenosine deaminase (ADA) differently. In the case of lorazepam temperature increment causes an increase in the inhibition potency whereas higher temperature reduces the inhibitory effect of oxazepam; which proposes the overall profounder structural changes in the case of lorazepam relative to those caused by oxazepam.

Comparison between the BAD complexes indicated that BAD all docks a hydrophobic surface of PKAc regardless of its phosphorylation. PKAc may prevent Bcl-xL from rebinding to BAD by phosphorylating human BAD at Ser118; whereas human BAD is phosphorylated on Ser75 in a BAD-Bcl-xL complex, resulting in the dissociation of BAD.

A lectin specific for glucuronic acid and galacturonic acid has been isolated from seeds of the French bean Phaseolus vulgaris using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 32-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-13 and the temperature range of 10-60 C. The lectin neither exhibited any antiproliferative activity against tumor cells nor stimulated nitric oxide production by murine peritoneal macrophages at doses as high as 1mM , The lectin failed to evoke any mitogenic response from murine splenocytes as measured by [3H-methyl]-thymidine incorporation and did not inhibit the activity of HIV-1 reverse transcriptase. The lectin had no antifungal activity.

The three new Au (III), Pt (II) and Pd (II) complexes with pentapeptide glycyl-glycyl-L-methyonyl-glycylglycine have been synthesized, isolated, and spectroscopically and structurally elucidated in solution and in the solid-state. Solid-state linear-dichroic infrared (IR-LD) spectroscopy of oriented colloids in a nematic liquid crystal host, 1H- and 13CNMR, TGA and DSC, UV-VIS spectroscopy, EPR, ESI- and FAB- mass spectrometry and HPLC tandem mass spectrometry (HPLC-MS/MS) have been used. Quantum chemical calculations and molecular modelling were carried out in order to determine the structures and spectroscopic properties of the ligand, the newly synthesised metal complexes and their interactions with calf thymus DNA. The pentapeptide coordinates in a tetradentate manner with the metal ions via an S-atom on the methyonyl-side chain, two N-amide nitrogens, (after a deprotonation of gly1 and gly2 residues) and the primary NH2 nitrogen of Ntermini. The MN3S chromophores are distorted to near square planar geometry. Their interaction with calf thymus DNA shows the competitive N7 (G) coordination position, where the pentapeptide residues is coordinated with the metal centers in a tridentate manner through the S- atom and both N-amide centers. This interaction leads to a transfer from a distorted square planar geometry (D4h symmetry) to a pseudo tetrahedral (Td symmetry) of the metal ions with the obtained dihedral angle values of the MN3N7(G) chromophors within 114.67 to 110.92 . The isolated Au (III) complex is stable for about 1.5 months, while the stability of the Au (III) complex/DNA adduct is decreased to 33 days.

Cathepsin L (ctsl), a lysosomal cyteine protease over expressed and secreted by cancer cells, has been implicated in a number of physiological and pathological processes including tumor cell proliferation and metastasis. In the present study we demonstrate that an unknown mRNA of human origin (Gene Bank accession number AF 217997) is a splice variant of human cathepsin L mRNA (hCATL A IV) and encodes a truncated form of cathepsin L (Δctsl) containing only 151 C-terminal amino acids. This isoform is cytotoxic to the mammalian cells. Transient transfection studies revealed that unlike ctsl, upon over expression in eukaryotic cells Δctsl is not secreted in to the media. Immunogold electron microscopy revealed its localization to nuclear, perinuclear and cytosolic regions. In view of its cytotoxic property, targeted expression of Δctsl in tumor cells may prove useful in the management of cancer.

In this study, we used two categories of molecular descriptors as CODESSA and DPPS (divided physicochemical property scores of amino acids) to parameterize structural characteristics of 2015 human amphiphysin SH3 domainbinding decapeptides at atom and residue levels. Based upon that, several robust quantitative structure-affinity relationship (QSAR) models were then constructed using partial least squares regression (PLS) and least squares-support vector machine (LSSVM) coupled with genetic algorithm (GA)-variable selection. Results show that (1) GA is a powerful tool for variable selection by which the most informative variable combinations can be efficiently determined for PLS and LSSVM modeling, (2) regression models constructed using nonlinear LSSVM approach are more robust and predictable than those by linear PLS method, (3) the residue level descriptor (DPPS) performs better in capturing peptide structural characteristics, more amenable than those from the atom level descriptor (CODESSA). By investigating the optimal DPPS-based GA-LSSVM model, it is indicated that the core motif of SH3 domain-binding peptides contributes significantly to the binding affinity, whereas the two end residues, especially the N-terminal residue, have a little effect on the binding process.

Lipases share an overall α/β hydrolase fold structure characteristic of serine hydrolases. Nevertheless, each lipases group possesses its characteristic 3-D structure and catalytic properties. The purified N-terminal truncated forms of a pancreatic (from ostrich) and a fungal (from Rhizopus oryzae, ROL32) (sayari et al., 2005) lipases displayed much lower activities as compared to the native proteins. The aim of this study is to explain this common functional feature on a structural basis. The molecular modelling showed that the N-terminal peptide of the fungal lipase displays an extended “V” shaped structure motif (sayari et al., 2005). We observed that the N-terminal peptide of a pancreatic lipase shares the same extended structure with that of the ROL32, despite the low sequence homology between the two peptides. Upon superimposition of the 3-D structure of the N-terminal catalytic domain of the pancreatic lipase with the model of the ROL32, we have shown that the N-terminal peptide and the open lid domain, of each lipase, are located distally within the putative interfacial binding surface. In particular, two hydrophobic residues, Leu and Ile belonging to the N-terminal peptide of each lipase are well placed to interact with the lipidic substrate. Furthermore, the N-terminal peptide of each lipase seems to be well placed to interact with the loop bearing the catalytic aspartic acid. All these observations might explain the fact that the loss of the N-terminal peptide affects the lipase activity. This work shows that the two lipases share striking structural and functional features with respect to their N-terminal peptide despite the fact that they belong to very distant kingdoms such as fungal and higher animals' ones.

The superoxide anion radical is a highly reactive toxic species produced during metabolic processes. Several copper (II) complexes with peptides are known to show superoxide dismutase (SOD) activity but those having a peptide with a non-natural amino acid are limited. The synthesis of HisAibGly peptide and its complexation with copper (II) ions has been reported. The interaction of the synthetic peptide with Cu(II) was studied by electron spray ionization-mass (ESIMS), circular dichroism (CD), absorption (UV-Vis) and electron paramagnetic resonance (EPR) spectroscopic methods. The solution studies and species distribution were performed by both spectrophotometric and potentiometric methods. The studies were performed at 25 ± 0.1 °C with constant ionic strength (μ = 0.1 M NaNO3) in aqueous solution using Bjerrum- Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The species distribution stidies indicated that the complexation occurred from 3-11 pH and a three nitrogen coordinated species predominates at 8-9 whereas a four nitrogen coordinated species was formed between pH 9-11. The copper-peptide complex was tested for SOD activity using xanthine-xanthine oxidase ‐ nitroblue tetrazolium (NBT) methods.

DNA ligase is an important enzyme and it plays vital role in the replication and repair; also catalyzes nick joining between adjacent bases of DNA. The NAD+ dependent DNA ligase is selectively present in eubacteria and few viruses; but missing in humans. Homology modeling was used to generate 3-D structure of NAD+ dependent DNA ligase (LigA) of Mycobacterium tuberculosis using the known template (PDB: 2OWO). Furthermore, the stereochemical quality and torsion angle of 3-D structure was validated. Numerous effective drugs were selected and the active amino acid residue in LigA was targeted and virtual screening through molecular docking was done. In this analysis, four drugs Chloroquine, Hydroxychloroquine, Putrienscine and Adriamycin were found more potent in inhibition of M. tuberculosis through the robust binding affinity between protein-drug interactions in comparison with the other studied drugs. A phylogenetic tree was constructed and it was observed that homology of LigA in M. tuberculosis resembled with other Mycobacterium species. The conserved active amino acids of LigA may be useful to target these drugs. These findings could be used as the starting point of a rational design of novel antibacterial drugs and its analogs.