Circular Dichroism Spectropolarimetry Research Articles

Dynamic Peptide Folding and Assembly for DNA Separations

The phenomenon of counterion-mediated DNA-condensation is fundamental to most DNA related activity in the cell, from chromosome packaging to control over translational mechanisms. Developing synthetic systems to manipulate DNA-condensation is essential for the development of biotechnologies for gene encapsulation and DNA-separation. We investigate the dynamics of the DNA-condensation by using our model peptide where the interaction between DNA and peptide is non-specific. We have designed a peptide that shows switchable surface activity, where the folded form of the peptide is amphiphilic and the unfolded form is not amphiphilic. The peptide is α-helical, containing 23 amino acids with variation in the number and distribution of hydrophobic and charged amino acids. The designs incorporate hydrophobic residues on one side (leucines and alanines) and hydrophilic residues on the opposite side so that helix is surface active. The secondary structure has been characterized by using circular dichroism spectropolarimetry, and we show that the peptide has a transient secondary structure as a function of monovalent salt concentration. The behavior of the peptide at air-water interface is characterized by pendant drop/bubble method and modeled accordingly. Our hypothesis is that the unfolded peptide is in equilibrium with the folded peptide in the bulk solution but in presence of DNA, the unfolded peptide folds and then binds to DNA. Critical Aggregate concentration of the peptide for DNA condensation is determined by using multi angle light scattering, which is also used to calculate the radius of gyration and molecular weight of these condensates. We investigate the kinetics of the condensation process by using Circular dichroism in Stop-flow mode and also by isothermal titration calorimetry.

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS–EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS–EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS–EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN alpha-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN alpha-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN alpha-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN alpha-2b remains mostly unchanged at a variety of BSA to IFN alpha-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN alpha-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN alpha-2b secondary structure when BSA is in molar excess.

The Adherence of platelets to adsorbed albumin by receptor-mediated recognition of binding sites exposed by adsorption-induced unfolding

Although albumin (Alb) is the most abundant plasma protein, it is considered to be non-adhesive to platelets, as it lacks any known amino acid sequences for binding platelet receptors. Recent studies have suggested that platelets adhere to adsorbed Alb by mechanisms linked to its conformational state. To definitively address this issue we used circular dichroism (CD) spectropolarimetry to characterize the conformation of Alb adsorbed on a broad range of surface chemistries from a wide range of Alb solution concentrations, with platelet adhesion examined using a lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results prove that platelets bind to adsorbed Alb through receptor-mediated processes, with binding sites in Alb exposed and/or formed by adsorption-induced protein unfolding. Most importantly, beyond a critical degree of unfolding, the platelet adhesion levels correlated strongly with the adsorption-induced unfolding in Alb. The blockage of Arg-Gly-Asp (RGD) specific platelet receptors using an Arg-Gly-Asp-Ser (RGDS) peptide led to significant inhibition of platelet adhesion to adsorbed Alb, with the extent of inhibition and morphology of adherent platelets being similar for both Alb and Fg. Chemical neutralization of arginine (Arg) residues in the adsorbed Alb layer inhibited platelet–Alb interactions significantly, indicating that Arg residues play a prominent role in mediating platelet adhesion to Alb. These results provide deeper insight into the molecular mechanisms that mediate the interactions of platelets with adsorbed proteins, and how to control these interactions to improve the blood compatibility of biomaterials for cardiovascular applications.

The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen

While platelet adhesion to biomaterial surfaces is widely recognized to be related to adsorbed fibrinogen (Fg), it has remained controversial whether platelet adhesion is in response to the adsorbed amount or the adsorbed conformation of this protein. To address this issue, we designed a series of platelet adhesion studies to clearly separate these two factors, thus enabling us to definitively determine whether it is the amount or the conformation of adsorbed Fg that mediates platelet response. Fg was adsorbed to a broad range of surface chemistries from a wide range of solution concentrations, with the amount and conformation of adsorbed Fg determined by absorbance and circular dichroism (CD) spectropolarimetry, respectively. Platelet adhesion response was determined by lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results show that platelet adhesion is strongly correlated with the degree of adsorption-induced unfolding of Fg ( r 2 = 0.96) with essentially no correlation with the amount of Fg adsorbed ( r 2 = 0.04). Platelet receptor inhibitor studies using an RGDS peptide reduced platelet adhesion by only about 50%, and SEM results show that adherent platelets after RGDS blocking were much more rounded with minimal extended filopodia compared with the unblocked platelets. These results provide definitive proof that the conformation of adsorbed Fg is the critical determinant of platelet adhesion, not the amount of Fg adsorbed, with adsorption-induced unfolding potentially exposing two distinctly different types of platelet binding sites in Fg; one that induces platelet adhesion alone and one that induces both platelet adhesion and activation.

Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing Small Molecules Based on the 1,3-Phenylene-Bis(piperazinyl benzimidazole) System

Achieving stabilization of telomeric DNA in G-quadruplex conformation by various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li(+), Na(+), or K(+)), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (i) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further suggest that these compounds are able to abet the conversion of the intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.

The 3D structures of G-Quadruplexes of HIV-1 integrase inhibitors: molecular dynamics simulations in aqueous solution and in the gas phase

The unimolecular G-quadruplex structures of d(GGGTGGGTGGGTGGGT) (G1) and d(GTGGTGGGTGGGTGGGT) (G2) are known as the potent nanomolar HIV-1 integrase inhibitors, thus investigating the 3D structures of the two sequences is significant for structure-based rational anti-HIV drug design. In this research, based on the experimental data of circular dichroism (CD) spectropolarimetry and electrospray ionization mass spectrometry (ESI-MS), the initial models of G1 and G2 were constructed by molecular modeling method. The modeling structures of G1 and G2 are intramolecular parallel-stranded quadruplex conformation with three guanine tetrads. Particularly, the structure of G2 possesses a T loop residue between the first and the second G residues that are the component of two adjacent same-stranded G-tetrad planes. This structure proposed by us has a very novel geometry and is different from all reported G-quadruplexes. The extended (35 ns) molecular dynamic (MD) simulations for the models indicate that the G-quadruplexes maintain their structures very well in aqueous solution whether the existence of K(+) or NH (4) (+) in the central channel. Furthermore, we perform 500 ns MD simulations for the models in the gas phase. The results show that all the ion-G-quadruplex complexes are maintained during the whole simulations, despite the large magnitude of phosphate-phosphate repulsions. The gas phase MD simulations provide a good explanation to ESI-MS experiments. Our 3D structures for G1 and G2 will assist in understanding geometric formalism of G-quadruplex folding and may be helpful as a platform for rational anti-HIV drug design.

Headgroup-dependent membrane catalysis of apelin-receptor interactions is likely.

Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin−APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6−K12 is highly structured, with R6−L9 directly interacting with headgroups of the micelle. Type I β-turns are initiated between R6 and L9, and a well-defined type IV β-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide−receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced.

Heterochromatinization as a Potential Mechanism of Nickel-Induced Carcinogenesis

Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation.

Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine–isoleucine or glycine–leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K m similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.

Self Assembly of Coiled-Coil Peptide−Porphyrin Complexes

We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

Investigation of the Effects of Surface Chemistry and Solution Concentration on the Conformation of Adsorbed Proteins Using an Improved Circular Dichroism Method

In this paper we present the development of methods using circular dichroism spectropolarimetry with a custom-designed cuvette to increase the signal-to-noise ratio for the measurement of the secondary structure of adsorbed proteins, thus providing enhanced sensitivity and reproducibility. These methods were then applied to investigate how surface chemistry and solution concentration influence both the amount of adsorbed proteins and their secondary structure. Human fibrinogen and albumin were adsorbed onto alkanethiol self-assembled monolayers (SAMs) on gold with CH3, OCH2-CF3, NH2, COOH, and OH terminal groups from both dilute (0.1 mg/mL) and moderately concentrated (1.0 mg/mL) solutions. An increase in surface hydrophobicity was found to cause an increase in both the amount of the protein adsorbed and the degree of structural change that was caused by the adsorption process, while an increase in solution concentration caused an increase in the amount of protein adsorbed but a decrease in the degree of conformational change, with these effects being more pronounced on the more hydrophobic surfaces. The combined use of these two parameters (i.e., surface chemistry and solution concentration) thus provides ameans of independently varying the degree of structural change following adsorption from the amount of adsorbed protein. Further studies are underway to examine which of these factors most strongly influences platelet response, with the overall goal of developing a better understanding of the fundamental factors governing the hemocompatibility of biomaterial surfaces.

The influence of sterols on the conformation of recombinant mitochondrial porin in detergent

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent-sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent-sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin.

The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus‐1 gp41 ectodomain by electrophoresis

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.

Alpha‐helix formation studied with pressure perturbation calorimetry

Pressure perturbation calorimetry (PPC) is a relatively unexplored technique that quantifies the temperature dependence of a solute's thermal expansion coefficient. PPC data provides information about solute‐solvent interactions. To investigate the formation of an alpha‐helix in solution, PPC was used to characterize helix‐solvent and random coil‐solvent interactions. Helix and random coil models were obtained by using two peptides, Ac‐DKDGYISAAE‐NH2 (P12) and Ac‐DKDGDGYISAAEAAAQ‐NH2 (P16) which contain the same sequence for the first twelve amino acids. Circular dichroism spectropolarimetry was used to determine the helix content. In the presence of La3+ the last four residues of P16 form an alpha‐helix. When La3+ is absent, both peptides are random coils, with a small amount of residual helix, which decreases with increasing temperature. The temperature dependence of the corrected thermal expansion coefficient suggests that the helix‐solvent interactions are more hydrophobic, while random coil‐solvent interactions are more hydrophilic. Therefore, alpha‐helix formation is marked by a change from hydrophilic to hydrophobic solvent interaction. This result is consistent with the accepted view that helix formation results in a net loss of hydrophilic surface area.

Two-Step Folding of Recombinant Mitochondrial Porin in Detergent

Precise information regarding the transmembrane topology of mitochondrial porin is essential for understanding the mechanisms by which this protein functions. Porin acts as a channel in the outer membrane and interacts with small solutes and proteins to regulate mitochondrial function. The acquisition of high-resolution structural data requires a method of maintaining high concentrations of unaggregated, properly folded porin. In the current studies, several mixed detergent systems were analyzed for their ability to fold Neurospora mitochondrial porin expressed in and isolated from Escherichia coli. A mixture of sodium dodecyl sulfate and dodecyl- β-D-maltopyranoside in a 1:6 molar ratio supports a β-strand-rich conformation. In this state, the two tryptophan residues in the protein reside in hydrophobic environments, and about half of the nine tyrosines are solvent exposed. Most importantly, heat-labile tertiary contacts, as detected by near-UV circular dichroism spectropolarimetry, in the sodium dodecyl sulfate/dodecyl- β-D-maltopyranoside-solubilized porin are very similar to those of the protein following functional reconstitution into liposomes. Similarly, both forms are protease resistant. Thus, a method has been identified with the potential to solubilize high concentrations of mitochondrial porin in a state virtually indistinguishable from the membrane-embedded form.

Design of a Selective Metal Ion Switch for Self-Assembly of Peptide-Based Fibrils

The self-assembling peptide TZ1H, a structural variant of the trimeric isoleucine zipper GCN4-pII, contains histidine residues at core d-positions of alternate heptads that define three trigonal coordination sites within the coiled-coil trimer. Circular dichroism spectropolarimetry indicated that peptide TZ1H undergoes a random coil to alpha-helical conformational change upon binding of 1 equiv of silver(I) ion, but not zinc(II), copper(II), or nickel(II) ions. Isothermal titration calorimetry provided evidence for a single binding-site model in which each peptide contributes one net silver(I) coordination site, in agreement with the proposed structural model. Transmission electron microscopy revealed that TZ1H self-assembles into long aspect ratio helical fibers in the presence of silver(I) ion. These results demonstrate that the rational design of selective metal ion binding sites within de novo designed peptides represents a promising approach to the controlled fabrication of nanoscale, self-assembled materials.

Studies on the DNA binding and cleavage activity of a synthesized polyamide containing dipeptide Ser-His

The DNA binding and cleavage activity of a synthetic polyamide containing dipeptide Ser-His has been investigated by spectroscopic techniques, such as electronic absorbance and circular dichroism (CD) spectropolarimetry, as well as gel electrophoresis. The results show that the molecule has a strong interaction with DNA and can improve DNA-cleavage ability about 100 fold as compared with single dipeptide Ser-His.

In vitro utilization of ferromagnetic nanoparticles in hemodialysis therapy

The in vitro utilization of biocompatible ferromagnetic nanoparticles (BFNs) inhemodialysis (HD), routinely used today for the treatment of end stage renal disease(ESRD), is introduced in this work. The proposed strategy is termed magnetically assistedhemodialysis (MAHD) and it aims to become a more efficient development ofconventional HD. The method is based on the production of biocompatible ferromagneticnanoparticles–targeted binding substances conjugates (BFNs–TBSs Cs) constructed ofBFNs and specifically designed TBSs that should have high affinity and binding capacityfor target toxic substances (TTSs) which must be removed from the ESRD patientsubjected to HD. Antibodies or even specific proteins could serve as the TBS ofthe desired BFNs–TBSs Cs. The BFNs–TBSs Cs should be administered to thepatient timely prior to the MAHD session so as to bind with the desired TTSsduring their free circulation in the vascular network. Eventually, the completeBFNs–TBSs–TTSs structure can be selectively removed during the MAHD session bymeans of an external inhomogeneous magnetic field that is applied either at thedialyzer or at other collection point(s) along the blood circulation line of the dialysismachine. The advantages of MAHD over conventional HD regarding the patient’scomfort and overall health status are discussed in detail among practical issues.To examine this proposition we employedFe3O4 and bovine serum albumin (BSA) as the BFN and the TBS constituents respectively, sincethey are both highly biocompatible. By means of x-ray diffraction, atomic forcemicroscopy, circular dichroism spectropolarimetry, UV–vis spectrophotometry,SQUID magnetometry, and nuclear magnetic resonance we evaluated (i) thestructural/morphological characteristics, (ii) the magnetic retraction efficiency,and most importantly (iii) the toxin binding affinity and capacity of both bareFe3O4 BFNsand Fe3O4–BSA Cs by performing in vitro experiments on specific TTSs. Homocysteine and p-cresolwere chosen as representative TTSs and were investigated in great detail. Theresults obtained prove the in vitro applicability of the proposed MAHD method.Corrections were made to this article on 6 November 2007 (see figure 10 caption and lines 10 and 11 of page 11). The corrected electronic version is identical to the print version.

A Template‐assembled Model of the N‐peptide Helix Bundle from HIV‐1 Gp‐41 with High Affinity for C‐peptide

The infection of target cells by HIV-1 is initiated by fusion of the viral and cell membranes, which is mediated by the viral glycoproteins, gp120 and gp41. After initial cell binding by gp120, the folding of gp41 to form a stable six-helix bundle structure is directly associated with membrane fusion. This helix bundle is composed of an alpha-helical trimer of gp41 N-peptide, with three copies of the alpha-helical gp41 C-peptide folded onto it in an antiparallel orientation. This report describes the synthesis and study of an N-peptide three-helix bundle structure, KTA-3N29b, that is assembled on a threefold symmetric template derived from Kemp's triacid. At neutral pH and in the presence of physiologic salt, KTA-3N29b, exists as a monomer with high helix content. Binding isotherms measured by circular dichroism spectropolarimetry indicate that KTA-3N29b binds three copies of the C-peptide native sequence, with a KD of about 260 nM. These features of KTA-3N29b demonstrate that this templated three-helix bundle serves as a functional model for the native N-peptide structure that will allow detailed studies of the folding and thermodynamic stability of the gp41 six-helix bundle, and may aid the future development of potent HIV-1 fusion inhibitors and immunogens.