Protein and Peptide Letters - Volume 14, Issue 1, 2007

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.

The transcription factor Brn-1, which belongs to POU-domain family, has been shown to play critical roles in the development of the nervous system. A cDNA clone coding for a quail Brn-1 homologue, qBrn-1, was isolated. To investigate whether this gene plays a role in the development of the quail nervous system, an anti-N-terminal peptide antibody was prepared. The coding region for amino acids 1-79 (the N-terminal domain of qBrn-1) was subcloned into Trx fusion expression vector pET32c and introduced into the Escherichia coli Origami(DE3) cells for efficient expression. After purification, Trx-fused polypeptides, called Trx-qBrn-1N, were used to immunize the rabbits to prepare polyclonal antibodies against qBrn-1. The produced and purified antiserum showed specificity not only to the in vitro expressed qBrn- 1, but also to the natural qBrn-1 in tissues. Immunolabeling on sections by the anti-qBrn-1 serum showed that qBrn-1 was specifically expressed in the developing spinal cord and kidney. This suggests that qBrn-1 may play some roles in the development of avian nervous system and kidney, and the preparation of anti-qBrn-1 polyclonal antibody will facilitate further detection of, and functional study on, qBrn-1 both in vivo and in vitro.

Modification of proteins by nonenzymatic glycation is one of the underlying factors that contribute to the development of the complications of diabetes. Human serum albumin (HSA) is one of the major targets of interaction with glucose through the Maillard reaction. The effects of 1 and 5 mg/ml glucose concentrations, which are consistent with blood glucose levels found in diabetic patients, on human serum albumin were studied by circular dichroism and fluorescence spectroscopy in sodium phosphate buffer, pH 7.4. Partial denaturation and changes in the structural integrity of HSA are caused by glycation at lower (1 mg/ml) and higher (5 mg/ml) concentrations of glucose. To study the relationship between structure and function, we investigated the interaction of L-tryptophan (L-Trp) with glycated and nonglycated HSA. The results showed that L-Trp, as the only free amino acid that substantially binds to HSA, has a lower affinity for the glycated form (especially at low concentrations of glucose) than for non-glycated HSA.

Sites of exendin-4 that that are relatively susceptible to degradation in plasma were identified with the aim of providing information for designing new exendin-4 analogues. The stability of exendin-4 in human plasma was evaluated in vitro. The results showed that the peptide was slowly degraded with a half-life of 9.57 h and the principal cleavage sites are between Thr5 and Phe6, Phe6 and Thr7, and Thr7 and Ser8 of the N-terminus region of exendin-4.

Nineteen mutants of decaprenyl diphosphate synthase from Gluconobacter suboxydans were generated and their production of ubiquinones (UQs) was compared to that of the wild type protein. The accessible surface area and the polarity of amino acid around the catalytic pocket wield remarkable influences on the isoprenoid chain elongation of UQs.

Bacteria use the receptor-adhesion-like interaction between laminin and the laminin receptor in the process of infection. We determined that bacteria do not interact with the receptor-binding site on laminin which could be expected for the bacterial laminin receptor. Rather, binding occurs via the laminin-binding site on the 67-kDa laminin receptor, which has a function similar to the one the bacterial laminin receptor possesses. This finding has implications for the effective use of antimicrobial peptides.

Discriminating outer membrane proteins for globular proteins (GPs) and other types of membrane proteins from genomic sequences is an important and hot topic. In this paper, a measure based on information discrepancy is proposed and applied to the discrimination of outer membrane proteins. It differs from previous methods which are based on amino acid composition. Our approach focuses on the comparison of subsequence distributions and takes into account the effect of residue order in protein primary structures. As a result, the new approach outperforms all previous methods on the same benchmark datasets. In particular, we show that the proposed approach has correctly identified the outer membrane proteins at an accuracy of 99% for the training set of 337 proteins and has correctly excluded the GPs at an accuracy of 86% in a non-redundant dataset of 668 proteins. Furthermore, this method is able to correctly exclude α-helical membrane proteins at an accuracy of 100%.

F0F1-ATPase is a rotary molecular motor. It is well known that the rotary torque is generated by ATP hydrolysis in F1 but little is known about how it produces the proton-motive force (PMF) in F0. Here a cross-linking approach was used to estimate the rotary torque produced by PMF. Three mutant E. coli strains were used in this study: SWM92 (δW28L F0F1, as control), MM10 (αP280C γA285C F0F1) and PP2 (αA334C/ δL262C F0F1). The oxidized inner membranes from mutant MM10 having a disulfide bridge in the top of γ subunit exhibited good ATP synthesis activity, while the oxidized PP2 inner membranes having a disulfide bridge in the middle of δ subunit synthesized ATP very poorly. We conclude that the rotary torque generated by PMF is sufficient to uncoil the α-helix in the top of δ subunit (MM10) and to overcome the Ramachandran activation barriers (25-30kJ/mol, i.e. about 40-50pN·nm), but cannot cleave the disulfide bond in the middle of the δ subunit (200 kJ/mol, i.e. 330pN·nm) (PP2). Consequently a preliminary estimation is that the rotary torque generated by PMF in the fully functional F0F1 motor is greater than 40-50pN·nm but less than 330pN·nm.

Although there have been several reports on the conformational analysis of endomorphin-1 (YPWF-NH2) and related MOR (μ-opioid receptor) agonists, a definitive, convincing model of the biologically active structure is not yet available. We recently reported the synthesis and pharmacological characterization of the atypical endomorphin-analogue agonist c[YpwFG]. In this paper we discuss the conformational analysis of c[YpwFG] in comparison to its epimers, for investigating the topological features responsible for ligand recognition and receptor activation, and the role of the different pharmacophores.

Antimicrobial peptides, or host defense peptides, are universal signaling and effector molecules in host defense and innate immunity. This article highlights various tools developed for cathelicidins and defensins, ranging from peptide identification, production, and structural biology, including the eight databases for antimicrobial peptides. Novel peptides can be identified from natural sources at both gene and protein levels. Solid-phase synthesis and bacterial expression are the two important methods for peptide production. Three-dimensional structures of antimicrobial peptides, primarily determined by solution NMR techniques, are essential for an in-depth understanding of the mode of action. The introduction of octanoyl phosphatidylglycerol as a bacterial membrane-mimetic model provides new insights into peptide-lipid interactions. The incorporation of structure and activity data into the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html) will lead to an integrated understanding of these peptides via structural bioinformatics.

A monocot araceous lectin from tubers of Gonatanthus pumilus (GPL) was earlier purified in our laboratory and reported as T-cell mitogen having homotetrameric structure with subunit molecular mass of 13 kDa. Besides asialofetuin as reported earlier, in the present study it was also inhibited by N-acetyl-D-lactosamine but was non-reactive towards mannose or its derivatives. The lectin is rich in acidic amino acids and cysteine is completely absent. Chemical modification of GPL revealed requirement of tryptophan and tyrosine for lectin sugar interaction. The secondary structure content of GPL, as estimated with CD spectrum in K2D programme, has 37% α-helix, 26% β-sheet and 38% random contributions. Fluorescence spectrum of the lectin solution at 280 nm was typical for tryptophan residues buried inside the protein. Lectin activity decreased when treated with denaturants like guanidine-HCl, urea and thiourea. GPL inhibited the growth of three plant pathogenic fungi namely Colletotrichum lindemuthianum, Fusarium oxysporum and Botrytis cinerea. Out of 11 human cancer cell lines tested, GPL significantly inhibited proliferation of five lines viz. Colo-205, IMR-32, HCT-15, SK-N-SH and HT-29.

We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.

The 42 amino acid Alzheimer's Aβ peptide has been produced in E. coli as a soluble fusion to maltose binding protein (MBP). Affinity purification on amylose columns of MBP-Aβ and MBP led to the recovery of proteins at purities that were suited for physicochemical analyses. MBP-Aβ was able to bind approximately 2 mole equivalents of copper or 4 mole equivalents of zinc, while MBP alone bound negligible amounts of zinc or copper. We conclude that A can bind 2 copper or 4 zinc ions in its fusion format. Because MBP-Aβ is a convenient protein to work with, this system is well suited for further studies on the structure of Aβ and its interactions with metals.

Due to asymmetrical charge distribution of the mammalian high mobility group protein A2 (HMGA2), which makes HMGA2 bind to both cation- and anion-exchange columns, we developed a rapid procedure for purifying HMGA2 in the milligram range. This purification procedure greatly facilitated biophysical studies, which require large amounts of the protein.

Porcine pancreatic elastase (PPE) was crystallized in complex with a novel inhibitor at pH 5 and X-ray diffraction data were collected at a synchrotron source to 1.66 Å. Crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 50.25 Å, b = 57.94 Å and c = 74.69 Å. PPE is often used as model for drug target, due to its structural homology with the important therapeutic target human leukocyte elastase (HLE). Elastase is a serine protease that belongs to the chymotrypsin family, which has the ability to degrade elastin, an important component in connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases.

PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P212121 space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding.