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This experimental investigation exemplifies the design and testing of diffuser at compressor inlet and nozzle at condenser outlet in vapour compression refrigeration system with the help of R134a refrigerant. The diffuser with divergence angle of 12°,14° and the nozzle with convergent angle 12°,14° are designed for same inlet and outlet diameters. Initially diffusers are tested at compressor inlet diffuser is used with inlet diameter equal to exit tube diameter of evaporator and outlet tube diameter is equal to suction tube diameter of the compressor. Diffuser helps to increases the pressure of the refrigerant before entering the compressor it will be helps to reduces the compression work and achieve higher performance of the vapour compression refrigeration system. Then nozzles are testing at condenser outlet, whereas nozzle inlet diameter equal to discharging tube diameter of condenser and outlet diameter equal to inlet diameter of expansion valve. Additional pressure drop in the nozzle helped to achieve higher performance of the vapour compression refrigeration system. The system is analyzes using the first and second laws of thermodynamics, to determine the refrigerating effect, the compressor work input, coefficient of performance (COP).

Sesquioxides (Al2O3, Y2O3, Sb2O3) doped Oxy fluoro Cobalt borate novel luminescent series of glasses are prepared by melt quenching method. The structural characteristics of these glasses were studied by XRD, SEM and EDS techniques. The change in physical parameters such as density of all the series of glasses prepared gives a pathway for cobalt/Alumina/Antimony/Yttrium ions to enter in to borate glass network with the enhancement in the concentration of corresponding dopants. The depolymerization of the borate glass network is evident by the decline in various elastic parameters with B-O-Co/Y/Sb/Al bonds and by the modifier role of CoO/Y2O3/Sb2O3/Al2O3 transforming a fraction of tetrahedral BO3 units into BO4 units. Both FTIR and IR spectra indicate an increase in octahedral occupancy of cations Co2+, Y3+, Sb3+and Al3+ ions in the glass network with the increase of their concentration. The conversion rate of tetrahedral BO3 units to octahedral BO4 units leading to the formation of NBOs is more in Al3+ and least in Y3+ with the increase of concentration of sesquioxides at 0.05 mol% concentration of CoO. The optical absorption spectra of all series of glasses prepared reveal that octahedral occupancy of cobalt ions is dominant due to the ligand fields created by different sesquioxide ions in the glass network produce NBOs and number of electrons trapped at the donor centers. The PL spectra of all the series of these glasses reveal the octahedral occupancy of Co2+ ions, which is evident from the two emission transitions 2T1g(H) →4T1g(F) (531–556 nm) and 2Eg→5T2g (699–718 nm).The prepared series of glasses show efficient luminescence of Cobalt ions, and Al2O3 series of glasses are better candidates for luminescence as their emission Cross-sections are highest when compared with the remaining series of glasses.

The objective of the study is to compare the Friedewald formula, Modified Friedewald formula, and Anandaraja formula with direct homogeneous assay for low- density lipoprotein cholesterol (LDL-C) levels in CAD patients. Study Design is Cross-sectional study. Healthy subjects of both the gender, aged 18 - 75 years were included in this study. Lipid profile, LDH and Troponin I of both the controls and patients sample were collected from OPD of Central Hospital laboratory of Gayatri Vidhya Parishad Institute of Health Care and Medical Technology, Visakhapatnam. LDL-C estimation was done by direct homogenous assay and also calculated by using the Friedewald’s Formula, Modified Friedewald’s Formula and Anandaraja’s Formula for assessing and validity of the LDL cholesterol. LDH were measured kinetically UV method by Chem Ultra reagent system Pack and Cardiac Troponin I by JusCheck Rapid card test. One hundred samples were analyzed in this study, Out of that 50 are Known CAD patient samples and 50 are controls. The comparison of Lipid profile of Variable versus group. The P value also > 0.05 statistically significant. The Mean value for LDH in Case (Patients) is 486.2, whereas for control, it was 328.3. It shows highly significant for this parameter. The association between Case (Patients) and Control for Troponin I was highly significant in this study. In our study, we conclude that, modified friedewald’s formula was closure to the direct method. The performance of calculated methods was not uniform at different TG levels. Even though, Modified Friedewald’s formula is closure to Direct LDL, For laboratory setup, Novel and Innovative direct homogeneous assays are most reliable and accurate for the analysis of LDL cholesterol.

ZnO-CaF

Clustering is a simple information mining and best tool for analysing big data. There are difficulties in applying grouping procedures to large-scale information, combined with new difficulties that occur with large-scale information. As Big Data refers to TBs and PBs of information and grouping estimates are followed by large computational expenses, the survey is the means by which to adjust to this problem and how to communicate clustering methods to vast information and to achieve results in a reasonable period. In this post, the firefly optimization algorithm was used to find the optimum cluster centres. This algorithm has a global search capability and is commonly used to solve difficult problems.

The human immunodeficiency virus (HIV-1 virus) exploits several host factors for assembly, infection, and replication within the infected cells. In this work, we describe the evidence for an interaction of the N-terminal domain of the HIV-1 capsid protein with human calmodulin. The precise role of this interaction within the life cycle of the HIV-1 virus is yet to be defined. Potential roles for this interaction in the viral capsid uncoating are discussed.

Cellular calmodulin binds to the SH2 domain of Src kinase, and upon Fas activation it recruits Src into the death-inducing signaling complex. This results in Src-ERK activation of cell survival pathway through which pancreatic cancer cells survive and proliferate. We had proposed that the inhibition of the interaction of calmodulin with Src-SH2 domain is an attractive strategy to inhibit the proliferation of pancreatic cancer. Thus we have performed screening of compound libraries by a combination of methods and identified some compounds (initial leads) that target the calmodulin-binding region on the SH2 domain and inhibit the proliferation of pancreatic cancer cells in in vitro assays. Most of these compounds also exhibited varying degrees of cytotoxicity when tested against immortalized breast epithelial cell line (MCF10A). These initial leads are likely candidates for development in targeted delivery of compounds to cancer cells without affecting normal cells.

Background: Tuberculosis a major public health problem in India with highest burden of cases. India accounts for about 24% of global prevalence, 23% of the global incidence cases and 21% of global TB deaths. Irregular and inappropriate treatment of persons with active TB, unawareness about TB symptoms and treatment course, illiteracy, may be the major hurdles for TB eradication.Methods: A cross sectional, community based study was conducted in urban slum dwellers (n=153). Data compilation and analysis: All the data collected was entered and analyzed with MS excel software 2007 and Epi info 3.5.3. All tests were considered significant at p

Background: Globally an estimated 422 million adults are living with Diabetes as per WHO data 2014, and 85 to 90% cases are type 2 diabetes. Maximum number of people with diabetes mellitus are in the age group of 40 to 59 yrs. Among them 50% are undiagnosed. Sedentary lifestyle, diet, dietary fiber, mal-nutrition, some chemical agents, stress, alcohol, viral infections, etc., are some environmental risk factors.Methods: A cross sectional, community based study. Details of the study subjects were recorded using structured predesigned and pretested questionnaire. All the data collected was entered and analyzed with MS Excel software 2007 and Epi info 3.5.3. All tests were considered significant at p

The relationship between the network structure and properties of iron containing novel oxyfluorophosphate glasses with the composition 20ZnO–20CaF 2 –(60 − x)P 2 O 5 :xFe 2 O 3 with 0 ≤ x ≤ 5 mol% has been studied. The density, molar volume, oxygen molar volume and oxygen packing density studies helped to understand the structural changes occurring in these glasses. The dissolution rate and pH of the immersion liquid are measured to explain the chemical durability of the glasses. Differential thermal analysis studies are carried out to analyze the thermal stability of the studied glasses. Deconvoluted infrared and Raman spectra of the glasses are analyzed to determine the relative areas of the vibrational bands corresponding to different structural units. Analysis of the vibrational spectra in conjunction with the other properties reveals that Fe 2 O 3 enters the glass matrix in the form of Fe 2 + and Fe 3 + ions through the formation of more stable P O Fe 2 + and P O Fe 3 + bonds at the expense of easily hydrated P O P bonds.

Design, synthesis and characterization of four novel series of Zafirlukast analogs are described. The modifications are substitution on indole nitrogen, C-5 position of indole moiety and amide nitrogen of zafirlukast.

The mature fullerene cone-shaped capsid of the human immunodeficiency virus 1 is composed of about 1,500 copies of the capsid protein (CA). The CA is 231 residues long, and consists of two distinct structural domains, the N-terminal domain and the C-terminal domain (CTD), joined by a flexible linker. The wild type CA exhibits monomer-dimer equilibrium in solution through the CTD-CTD dimerization. This CTD-CTD interaction, together with other intermolecular interdomain interactions, plays significant roles during the assembly of the mature capsid. In addition, CA-CA interactions also play a role in the assembly of the immature virion. The CA also interacts with some host cell proteins within the viral replication cycle. Thus, the capsid protein has been of significant interest as a target for designing inhibitors of assembly of immature virions and mature capsids and inhibitors of its interactions with host cell proteins. However, the equilibrium exhibited by the wild-type CA protein between the monomeric and dimeric states, along with the inherent flexibility from the interdomain linker, have hindered attempts at structural determination by solution NMR and X-ray crystallography methods. In this study, we have utilized a CA protein with W184A and M185A mutations that abolish the dimerization of CA protein as well as its infectivity, but preserve most of the remaining properties of the wild type CA. We have determined the detailed solution structure of the monomeric W184A/M185A-CA protein using 3D-NMR spectroscopy. Here, we present the detailed sequence-specific NMR assignments for this protein.

The surprising observation that a 10-residue class G⁎ peptide from apolipoprotein J, [113–122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113–122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5 mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (−16.75 ± 0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (−3.67 ± 0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5 mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (−13.43 ± 0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113–122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.

KISS1 was first discovered as a metastasis suppressor, but also plays crucial roles in the onset of puberty. The KISS1 gene encodes a secreted protein of 145 amino acids that exhibits no sequence similarity with any known proteins. KISS1 protein is proteolytically processed to generate a number of so-called kisspeptins (KP), the most well characterized is known as KP-54 or metastin. KP-54 is carboxy-terminally amidated and binds to and activates the KISS1 receptor (KISS1R). The current studies were undertaken in order to determine structure of KP-54 using nuclear magnetic resonance and circular dichroism. KP-54 is mostly disordered both in water and in trifluoroethanol/water mixed solvent, with no structural motifs. In sodium dodecyl sulfate micelles, KP-54 remains mostly disordered except for a small increase in helical propensity (from 3.7% in water to 9.9% in micelles). Despite this apparent lack of structure, KP-54 is biologically active. The intrinsic disorder of KP-54 may confer advantages in its ability to recognize and bind a wide range of target proteins.

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Conjugates of curcumin to two differently sized poly(ethylene glycol) molecules were synthesized in an attempt to overcome the low aqueous solubility of this natural product with cytotoxic activity against some human cancer cell lines. The soluble conjugates exhibited enhanced cytotoxicity as compared to that of the parent drug. Synthesis, analyses of the rate of drug release, and cytotoxicity studies are herein reported. The water-soluble conjugates may provide information useful for the development of injectable curcumin conjugates.

Class A amphipathic helical peptides have been shown to mimic apolipoprotein A-I, the major protein component of high density lipoproteins and have been shown to inhibit atherosclerosis in several dyslipidemic mouse models. Previously we reported the NMR structure of Ac-18A-NH2, the base-line model class A amphipathic helical peptide in a 50% (v/v) trifluoroethanol-d3/water mixture, a membrane-mimic environment (Mishra, V. K., Palgunachari, M. N., Anantharamaiah, G. M., Jones, M. K., Segrest, J. P., and Krishna, N. R. (2001) Peptides 22, 567–573). The peptide Ac-18A-NH2 forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Because subtle structural changes in the peptide·lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of recombinant peptide·1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes. The peptide adopts a well defined amphipathic α helical structure in association with the lipid at a 1:1 peptide:lipid weight ratio. Nuclear Overhauser effect spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. The pattern of observed peptide-lipid nuclear Overhauser effects is consistent with a parallel orientation of the amphipathic α helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). Based on the results of chemical cross-linking and molecular modeling, we propose that peptide helices are arranged in a head to tail fashion to cover the edge of the disc. This arrangement of peptides is also consistent with the pKa values of the Lys residues determined previously. Taken together, these results provide for the first time a high resolution structural view of the peptide·lipid discoidal complexes formed by a class A amphipathic α helical peptide.

Dihydrofolate reductase (DHFR) is a pharmacologically important intracellular target enzyme for folate antagonists, including the antibacterial agent trimethoprim (TMP). The structures of DHFR from various sources with and without the bound ligands have been determined by X-ray crystallography and solution NMR spectroscopy. However, there is no crystal or solution NMR structure for the bovine DHFR/TMP complex. Here we report the solution structure of TMP within the binding pocket of bovine DHFR using a novel method developed in our laboratory, viz., STD-NMR intensity-restrained CORCEMA-ST optimization (SICO) utilizing experimental STD data on this complex, and demonstrate that its solution structure is essentially identical to the one in the crystal structure of the homologous chicken liver DHFR/TMP complex. The excellent agreement we obtain between the experimental and predicted STDs also serves as a validation of the CORCEMA-ST methodology.

Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-d-glucopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention.

A couple of recent applications of intermolecular NOE (INOE) experiments as applied to biomolecular systems involve the (i) saturation transfer difference NMR (STD-NMR) method and (ii) the intermolecular cross-saturation NMR (ICS-NMR) experiment. STD-NMR is a promising tool for rapid screening of a large library of compounds to identify bioactive ligands binding to a target protein. Additionally, it is also useful in mapping the binding epitopes presented by a bioactive ligand to its target protein. In this latter application, the STD-NMR technique is essentially similar to the ICS-NMR experiment, which is used to map protein-protein or protein-nucleic acid contact surfaces in complexes. In this work, we present a complete relaxation and conformational exchange matrix (CORCEMA) theory (H. N. B. Moseley et al., J. Magn. Reson. B 108, 243-261 (1995)) applicable for these two closely related experiments. As in our previous work, we show that when exchange is fast on the relaxation rate scale, a simplified CORCEMA theory can be formulated using a generalized average relaxation rate matrix. Its range of validity is established by comparing its predictions with those of the exact CORCEMA theory which is valid for all exchange rates. Using some ideal model systems we have analyzed the factors that influence the ligand proton intensity changes when the resonances from some protons on the receptor protein are saturated. The results show that the intensity changes in the ligand signals in an intermolecular NOE experiment are very much dependent upon: (1) the saturation time, (2) the location of the saturated receptor protons with respect to the ligand protons, (3) the conformation of the ligand-receptor interface, (4) the rotational correlation times for the molecular species, (5) the kinetics of the reversibly forming complex, and (6) the ligand/receptor ratio. As an example of a typical application of the STD-NMR experiment we have also simulated the STD effects for a hypothetical trisaccharide bound to a protein. The CORCEMA theory for INOE and the associated algorithm are useful in a quantitative interpretation of the intensity changes in the ligand in both the STD-NMR and ICS-NMR, provided the identity of the receptor protons experiencing direct RF saturation is known. The formalism presented here is likely to be useful in the design of bioactive ligands to a specific target protein and in the quantitative mapping of binding epitopes and interfaces between molecules in complexes.

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.

The gene for a β-neurotoxin [ Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [ 3 H]GABA [γ-aminobutyric acid (GABA)] in neuronal cells as effectively as natural β-toxins.

Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family. They function by transiently binding and positioning the coat protein subunits during capsid assembly. In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action. NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core. One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions. Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure.

The preparation and properties of some novel inhibitors of calmodulin function are described. The compounds are cationic derivatives of phenyl-substituted thiazoles which inhibit the calmodulin stimulation of cyclic-AMP phosphodiesterase and are active against animal tumor cells in culture. These derivatives form the basis for the preparation of new, more potent inhibitors of calmodulin function which could take advantage of the reported elevated levels of calcium-bound calmodulin in tumor cells and show preferential anti-tumor activity.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.

Interferon-tau (IFN-tau) is a novel type I IFN that was originally identified as a pregnancy recognition hormone. IFN-tau shares all of the biological properties of other type I IFNs including antiviral activity and antiproliferative activity through induction of the cell cycle inhibitor gene product p21WAF1. It is a promising therapy for cancers, viral infections, and for autoimmune disorders such as multiple sclerosis, without the adverse side effects associated with IFN-alpha and IFN-beta. Here, we describe novel growth and induction conditions for the expression of functionally active and uniformly 15N-labeled IFN-tau from Pichia pastoris in a minimal media for use in initial 2D- and 3D-NMR studies in solution. Purified 15N-IFN-tau was homogenous, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometer (MS), and retained full biological activity. MS analysis confirmed uniform isotopic labeling of IFN-tau with 15N incorporation exceeding 99%. Circular dichroism (CD) as well as 1D-NMR and 15N-1H heteronuclear single quantum coherence (HSQC) spectra confirmed that purified 15N-labeled IFN-tau has a stable secondary structure. Besides providing a route for isotope labeling of IFN-tau, our procedure may be useful for the expression and purification of other proteins that are difficult to obtain in Pichia pastoris grown in minimal media.

The scaffolding protein of bacteriophage P22 directs the assembly of an icosahedral procapsid, a metastable shell that is the precursor for DNA packaging. The full-length protein has been shown previously to exist in a monomer-dimer-tetramer equilibrium of elongated and predominantly α-helical molecules. Two deletion-mutant fragments of the scaffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichia coli , and purified. Removal of residues 1 to 140 yields a protein that is assembly-active both in vitro and in vivo , while the removal of the C-terminal 11 residues (293 to 303) leads to complete loss of scaffolding activity. Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent dissociation constants K d(141–303) = 640 μM and K d(141–292) = 880 μM. Tetramer formation is not observed for either fragment; thus, the tetramerization domain of the scaffolding subunit resides in the N-terminal portion of the polypeptide chain. Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly α-helical fold in each case. Nonetheless, pronounced differences are observed between spectral signatures of the two fragments. Notably, Raman spectra of fragments 141–292 and 141–303 indicate that elimination of residues 293 to 303 results in unfolding of an α-helical coat protein “recognition” domain encompassing about 20 to 30 residues. The thermostability of fragment 141–303, monitored over a wide concentration range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) form, while more cooperative unfolding is observed for the dimeric (high concentration) form. A lesser increase in cooperativity upon dimerization is obtained for fragment 141–292. Additionally, the C-terminal recognition domain constitutes the most stable and cooperative unit in the 141–303 fragment. Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows that the C terminus is the only protected segment of the polypeptide chain. On the basis of the measured hydrodynamic and spectroscopic properties, a domain structure is proposed for the scaffolding subunit. The roles of these domains in P22 procapsid assembly are discussed.