Protein and Peptide Letters - Volume 16, Issue 4, 2009

The role of the internal water molecules in vasopressin and oxytocin receptors has been investigated via molecular dynamics simulations in hydrated membrane model. Several water molecules have been identified within the binding pockets of receptors, where they interact with the conserved residues. In all unliganded receptors, the water molecules bound to the highly conserved D2.50 cluster have been observed. It has been proposed which water molecules may significantly contribute to the stability of overall receptor structure. In receptor-ligand complexes the water molecules are involved in the receptor-ligand interactions by forming water-mediated hydrogen bonds at their contact surface.

Discriminating cell wall lytic enzymes from non lytic enzymes is a very important task for curing bacterial infections. In this paper, based on Chou's amphiphilic pseudo amino acid composition, we develop fisher-discriminant based classifier to predict cell wall lytic enzymes. Experiments show that 66.7% sensitivity with 88.6% specificity is obtained. The method is further able to predict endolysin and autolysin with an overall accuracy of 92.9%. Results demonstrated that our method can provide highly useful information for further bacterial control research.

The xylanase (Xyn11A) gene (883 bp) was amplified using C. thermophilum DNA as template and cloned into pET-32a (+) and expressed in E. coli BL21 under T7 promoter. The recombinant xylanase on SDS-PAGE had a molecular mass of 30 kDa. Productivity profiles of xylanase in E. coli recombinant are more than 4-fold of that produced from T. reesei RUTC-30, 5-fold of that produced by the donor and significantly higher than the values reported on other E. coli, and Saccharomyces cerevisiae recombinants. Temperature stability, pH stability, and other kinetic parameters confirmed that the gene product was thermo-stable in alkaline buffer favoring its suitability to bio-bleaching of kraft pulp.

Novel affinity biosensors for detecting cellular prions, PrPC, based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrPC with the limits of detection of 20 to 50 pM.

We synthesized a 15-amino acid bi-functional synthetic peptide, RPC2, with the sequence Ac-CGKRKWSQ PKKKRKV-Cysteamide, which consists of a 7-amino acid nuclear localization signal (NLS) domain at the carboxyl terminus that electrostatically binds DNA and a 5-amino-acid tumor-homing domain at the amino terminus. This peptide efficiently delivered GFP and Renilla luciferase reporter genes into rat primary osteoblastic cells while exhibiting low cytotoxicity. The optimal delivery was achieved when the ratio of DNA: RPC2 reached 1:10 (w/w). Transfection efficiency can be further enhanced by the addition of Lipofectamine 2000 and modification of RPC2. These results indicated that RPC2 can deliver exogenous DNA into primary osteoblastic cells with low cytotoxicity and be potentially utilized in experimental and clinical applications in the field of bone tissue engineering.

Hepatitis C virus (HCV) infection is a major cause of liver disease and a dangerous threat to public health. Hence, the problem of finding interactions between HCV and human proteins has received much attention. In this paper, we present an approach to predicting binding residues in HCV proteins using a support vector machine (SVM) classifier. Based on six biochemical properties of amino acids (sequence profile, accessible surface area, residue binding propensity, sequence entropy, hydrophobicity and conservation weight), the SVM classifier achieved an average accuracy of 93%. Contiguous residues in the sequence act together to determine a binding site, and a window of 11 residues (the target residue and 5 adjacent residues on each side) gave the best result in our study. Our approach has been implemented in a program called BSFinder (Binding Site Finder), which is available at http://wilab.inha.ac.kr/bsfinder. BSFinder will be of considerable help in predicting binding residues and potential interacting partners of a protein.

Prohormone or proprotein convertases (PC2) are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues within the general motif (K/R)-(X)n- (K/R)2, where n is 0, 2, 4 or 6 and X is usually not Cys. Among the members of this prohormone convertase family, Neuroendocrine Convertase-2 (NEC-2) is regarded as one of the important proteins involved in the maturation of many precursor proteins. Being widely distributed in the neuroendocrine cells, these proteins play a vital role in causing malignant gliomas. They can serve as important drug targets in the treatment of cancers. In the present study, a 3D model of NEC-2 was generated using homology modeling. The model was optimized by a brief energy minimization in CHARMM and dynamics simulation of 250ps in MOE. The validation results of PROCHECK and Profile 3D show that the stereochemical quality of the model is good. The Cα backbone of the template and the target (NEC-2) when superimposed showed RMSD of 0.39Å. The model showed Asp51, His92 and Ser268 in the active site as seen in most of the PC2 members. The NEC-2 structure differs from that of furin at the catalytic pocket region with relevance to the amino acid composition which can be exploited for the design of specific inhibitors towards NEC-2.

2'/3'-O-[Bz(NO2)-Orn(Boc)]-5'-O-Piv-Ado (1) and its deoxy analog: 3'-O-[Bz(NO2)-Orn(Boc)]-5'-O-Piv-2'- dAdo (2) were designed and synthesized as substrates for the model ribosome reaction we used to demonstrate the crucial role of A76 2'-OH of peptidyl-tRNA in the rate acceleration of peptide bond formation during protein biosynthesis.

The role of the TPR2B domain of Hop is as yet unknown. We have shown here by site directed mutagenesis and size exclusion chromatography for the first time that the TPR1 and TPR2B domains of Hop independently dimerized, and that the dimerization of TPR2B was not dependent on its predicted two-carboxylate clamp residues. Furthermore, our data indicated that the dimerization of Hop and its domains was not disrupted in the presence of Hsp70 and Hsp90 peptides.

A series of aldehyde inhibitors with the general formula Ac-Phe-Val-Thr-X-CHO, where X = Lys, Arg, Phe, Tyr, p-nitro-L-phenylalanine (Nif), p-amino-L-phenylalanine (Amf), p-guanidine-L-phenylalanine (Gnf), pyridyl-L-alanine (Pal), was synthesized. The starting structure of this series based on our previous work on cathepsin G chromogenic substrates. The synthesis of all compounds was performed in solid phase applying Fmoc chemistry. We investigated the inhibitory potency of the obtained compounds against cathepsin G and bovine α-chymotrypsin and evaluated their dissociation constants (Ki). The studied peptides displayed different inhibition profiles and potency. As a result, a potent and selective inhibitor of cathepsin G with the sequence Ac-Phe-Val-Thr-Gnf-CHO, displaying Ki = 22 nM was obtained.

The hemolytic lectin CEL-III and its site-directed mutants were expressed in Escherichia coli cells. Replacement of the valine clusters in domain 3 with alanine residues led to increased self-oligomerization in solution and higher hemolytic activity. The results suggest the involvement of these valine clusters in CEL-III oligomerization and hemolytic activity.

The role of interaction between Asn259 (catalytic domain) with Gln821 (C-terminal domain) in PeptidaseN was investigated. The kcat of PeptidaseN containing Asn259Asp or Gln821Glu is enhanced whereas it is suppressed in Asn259AspGln821Glu. Structural analysis shows this interaction to change the relative disposition of active site residues, which modulates catalytic activity.

The equilibrium denaturation pathway of Succinyl Con A exhibited three-state mechanism with the transition midpoints at 1 and 3 M GdnHCl and at 2.6 and 5 M urea. Unfolding resulted in stabilization of molten-globule (MG) like intermediate states at 2 M GdnHCl and 3 M urea. It was particularly interesting that state obtained at 3 M urea was functionally active.

Comparing available Poaceae defensins with sugarcane ESTs, a putative defensin gene was identified in sugarcane and cloned from genomic sugarcane DNA. The deduced encoded peptide shows the structure and amino acid composition typical of other plant defensins. Using RTPCR, defensin expression in sugarcane and differences between “normal” and infected sugarcane were evidenced.

The Drosophila proteases Gastrulation Defective and Snake function in embryonic polarity establishment and bind heparin, a surrogate for anionic species present in the extracellular matrix. Here we demonstrate binding of GD, but not Snake, to anionic species that appear to be tightly associated with a highly purified eggshell matrix.

Hemoglobin is the versatile oxygen carrier in the blood of vertebrates and a key factor for adaptation to live in high altitudes. Several structural changes are known to account for increased oxygen affinity in hemoglobin of altitude adapted animals such as llama and barheaded goose. Guinea pigs are adapted to live in high altitudes in the Andes and consequently their hemoglobin has an increased oxygen affinity. However, the structural changes responsible for the adaptation of guinea pig hemoglobin are unknown. Here we report the crystallization of guinea pig hemoglobin in the presence of 2.6 M ammonium sulfate and a preliminary analysis of the crystals. Crystals diffract up to a resolution of 2.0 Å. They are orthorhombic with space group C 2 2 21 and cell dimensions a = 84.08 Å, b = 90.21 Å and c = 83.44 Å.

Human leukocyte antigen-G (HLA-G) is a nonclassical MHC class I (MHCI) molecule that is expressed mainly on placenta trophoblast cells. Leukocyte Ig-like receptor B2 (LILRB2) is a human inhibitory immune receptor that recognizes HLA-G with a higher affinity than any other MHCI although this interaction is only in the μM range. The interaction between HLA-G and LILRB2 seems to play a dominant role in the escape of the fetus from the maternal immune response. Here we report the crystallization and x-ray analysis of the LILRB2/HLA-G complex. The extracellular domains of HLA-G and LILRB2 were expressed in Escherichia coli, refolded and purified. The initial crystallization trials using novel PEG-based screening sets provided crystals of the LILRB2/HLA-G complex with 40-50% PEG400 as the precipitant. These crystals belong to space group P3121 (a=b=81.4 Å, c=186.7 Å, γ=120°). Dehydration of the crystals by soaking them in a solution containing a higher concentration of PEG400 dramatically improved the resolution and also the mosaicity.

Saccharomyces cerevisae ARO9 protein, an aromatic aminotransferase II, catalyzes the transamination step of the catabolism of aromatic amino acids, mainly tryptophan. ARO9 also belongs to a novel subfamily of enzymes within the aminotransferase subgroup I. Crystals of ARO9 protein have been grown using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 75.6 Å, b = 117.5 Å, c = 134.9 Å. Diffraction data were collected to a resolution of 2.6 Å using a rotating-anode X-ray source. Analysis indicates the presence of two molecules in an asymmetric unit.

Hemoglobin is a vital protein present in almost all higher species. It is a transport protein involved in carrying oxygen from lungs to tissues and carbon dioxide back to lungs by an intrinsically coordinated manner. Even though a good amount of work has been carried out in this direction there exists scarcity of structural insight on low oxygen affinity species. Attempts are being made to unravel the structural insight of this low oxygen affinity species. Goat blood plasma was collected, treated with EDTA to avoid blood clotting and purification was accomplished using DEAE-anion chromatographic column. The goat hemoglobin was crystallized using 50mM of phosphate buffer at pH 6.7 with 1M NaCl and PEG 3350 as precipitant by hanging drop vapor diffusion method. Crystals obtained are screened and suitable crystals are taken for data collection using mar345dtb as image plate detector system. Goat hemoglobin crystal diffracted up to 2.61 Å resolution. Goat hemoglobin crystallizes in orthorhombic space group P212121 as a whole biological molecule in the asymmetric unit with cell dimensions a=53.568Å, b=67.365Å, c=154.183Å.