Entrapment of a Water Wire in a Hydrophobic Peptide Channel with an Aromatic Lining

A one-dimensional water wire has been characterized by X-ray diffraction in single crystals of the tripeptide Ac-Phe-Pro-Trp-OMe. Crystals in the hexagonal space group P6(5) reveal a central hydrophobic channel lined by aromatic residues which entraps an approximately linear array of hydrogen bonded water molecules. The absence of any significant van der Waals contact with the channel walls suggests that the dominant interaction between the ``water wire'' and ``peptide nanotube'' is electrostatic in origin. An energy difference of 16 KJmol(-1) is estimated for the distinct orientations of the water wire dipole with respect to the macrodipole of the peptide nanotube. The structural model suggests that Grotthuss type proton conduction may, through constricted hydrophobic channels, be facilitated by concerted, rotational reorientation of water molecules.

Synthesis and characterization of novel cationic lipid and cholesterol-coated gold nanoparticles and their interactions with dipalmitoylphosphatidylcholine membranes

Novel gold nanoparticles bearing cationic single-chain, double-chain, and cholesterol based amphiphilic units have been synthesized. These nanoparticles represent size-stable entities in which various cationic lipids have been immobilized through their thiol group onto the gold nanoparticle core. The resulting colloids have been characterized by UV-vis, (1)H NMR, FT-IR spectroscopy, and transmission electron microscopy. The average size of the resultant nanoparticles could be controlled by the relative bulkiness of the capping agent. Thus, the average diameters of the nanoparticles formed from the cationic single-chain, double-chain, and cholesterol based thiolate-coated materials were 5.9,2.9, and 2.04 nm, respectively. We also examined the interaction of these cationic gold nanoparticles with vesicular membranes generated from dipalmitoylphosphatidylcholine (DPPC) lipid suspensions. Nanoparticle doped DPPC vesicular suspensions displayed a characteristic surface plasmon band in their UV-vis spectra. Inclusion of nanoparticles in vesicular suspensions led to increases in the aggregate diameters, as evidenced from dynamic light scattering. Differential scanning calorimetric examination indicated that incorporation of single-chain, double-chain, and cholesteryl-linked cationic nanoparticles exert variable effects on the DPPC melting transitions. While increased doping of single-chain nanoparticles in DPPC resulted in the phases that melt at higher temperatures, inclusion of an incremental amount of double-chain nanoparticles caused the lowering of the melting temperature of DPPC. On the other hand, the cationic cholesteryl nanoparticle interacted with DPPC in membranes in a manner somewhat analogous to that of cholesterol itself and caused broadening of the DPPC melting transition.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the beta-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

Co-liposomes comprising a lipidated multivalent RGD-peptide and a cationic gemini cholesterol induce selective gene transfection in alpha v beta 3 and alpha v beta 5 integrin receptor-rich cancer cells

The alpha v beta 3 and alpha v beta 5 integrins, transmembrane glycoprotein receptors, are over-expressed in numerous tumors and in endothelial cells that constitute tumor blood vessels. As this protein selectively binds to the Arg-Gly-Asp (RGD) sequence containing peptides, it is an attractive way to target tumors. Herein we have developed novel formulations for integrin mediated selective gene delivery. These formulations are composed of a novel palmitoylated tetrameric RGD containing scaffold (named RAFT-RGD), cationic gemini cholesterol (GL5) and a natural helper lipid 1,2-dioleoyl-L-alpha-glycero-3-phosphatidylethanolamine (DOPE). We have optimized a co-liposomal formulation to introduce the multivalent RGD-containing macromolecule in GL5: DOPE (GL5D) mixture to produce GL5D-RGD. We have unambiguously shown the selectivity of these formulations towards cancer cells that over express alpha v beta 3 and alpha v beta 5 integrins. Two reporter plasmids, pEGFP-C3 and PGL-3, were employed for the transfection experiments and it was shown that GL5D-RGD Liposomes increased exclusively the transfection in alpha v beta 3 and alpha v beta 5 overexpressing HeLa cells.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA, The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a. property similar to the eukaryotic type I topoisomerases, The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

Helix-sheet interconversion in a synthetic pore lining peptide derived from a designed ion channel

We have designed a four-helix protein that is expected to tetramerize in the membrane to form an ion channel with a structurally well defined pore. A synthetic peptide corresponding to the channel lining helix facilitates ion transport across liposomal membranes and largely helical in membranes. Detailed circular dichroism studies of the peptide in methanol, water and methanal-water mixtures reveal that it is helical in methanol, beta-structured in 97.5% water and a combination of these two structures at intermediate compositions of methanol and water. A fluorescence resonance energy transfer study of the peptide shows that the peptide is monomeric in methanol but undergoes extensive anti-parallel aggregation in aqueous solution.

Studies on interaction of Paenibacillus polymyxa with iron ore minerals in relation to beneficiation

Interaction between Paenibacillus polymyxa with minerals such as hematite, corundum, quartz and kaolinite brought about significant surface chemical changes on all the minerals. Quartz and kaolinite were rendered more hydrophobic, while hematite and corundum, became more hydrophilic after biotreatment. The predominance of bacterial polysaccharides on interacted hematite and corundum and of proteins on quartz and kaolinite was responsible for the above surface-chemical changes. Bio-pretreatment of the above iron ore mineral mixtures resulted in the selective separation of silica and alumina from iron oxide, through bioflotation and bioflocculation. The utility of bioprocessing in the beneficiation of iron ores is demonstrated.

Hydrogen bonding in peptide helices - Analysis of two independent helices in the crystal structure of a peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe

The crystal structure determination of the heptapeptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe reveals two peptide helices in the asymmetric unit, Crystal parameters are: space group P2(1), a = 10.356(2) Angstrom, b = 19.488(5) Angstrom, c = 23.756(6) Angstrom, beta = 102.25(2)degrees), V = 4685.4 Angstrom(3), Z = 4 and R = 5.7% for 7615 reflections [I>3 sigma(I)]. Both molecules adopt largely alpha-helical conformations with variations at the C-terminus, Helix type Is determined by analysing both 4-->1 and 5-->1 hydrogen-bond interactions and comparison with the results of analysis of protein structures. The presence of two 4-->1 hydrogen-bond interactions, besides four 5-->1 interact ions in both the conformations provides an opportunity to characterize bifurcated hydrogen bonds at high resolution, Comparison of the two helical conformations with related peptide structures suggests that distortions at the C-terminus are more facile than at the N-terminus.

Difference spectroscopic studies on binding of Cibacron blue F3GA to ribosome inactivating proteins: effect of beta-mercaptoethanol on the interaction with ricin

The interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of beta-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of beta-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by beta-mercaptoethanol showed that reduction pet se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond.

First donor-acceptor interaction promoted gelation of organic fluids

In recent years there has been considerable interest in developing new types of gelators of organic solvents.1 Despite the recent advances, a priori design of a gelator for gelling a given solvent has remained a challenging task. Various noncovalent interactions like hydrogen-bonding,2 metal coordination3 etc. have been used as the driving force for the gelation process. A special class of cholesterol-based gelators were reported by Weiss,4 and by Shinkai.5 Gels derived from these molecules have been used for chiral recognition/sensing,6 for studying photo- and metal-responsive functions,7 and as templates to make hollow fiber silica.8 Other types of organogels have been used for designing polymerized 9 and reverse aerogels,10 and in molecular imprinting.11 Hanabusa’s group has recently reported organogels with a bile acid derivative.12 This has prompted us to disclose our results on a novel electron donor–acceptor (EDA) interaction mediated two-component13 gelator system based on the bile acid14 backbone.