Showing papers in "Journal of Molecular Recognition in 2005"

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling.

Abstract: Regulation, recognition and cell signaling involve the coordinated actions of many players. To achieve this coordination, each participant must have a valid identification (ID) that is easily recognized by the others. For proteins, these IDs are often within intrinsically disordered (also ID) regions. The functions of a set of well-characterized ID regions from a diversity of proteins are presented herein to support this view. These examples include both more recently described signaling proteins, such as p53, alpha-synuclein, HMGA, the Rieske protein, estrogen receptor alpha, chaperones, GCN4, Arf, Hdm2, FlgM, measles virus nucleoprotein, RNase E, glycogen synthase kinase 3beta, p21(Waf1/Cip1/Sdi1), caldesmon, calmodulin, BRCA1 and several other intriguing proteins, as well as historical prototypes for signaling, regulation, control and molecular recognition, such as the lac repressor, the voltage gated potassium channel, RNA polymerase and the S15 peptide associating with the RNA polymerase S-protein. The frequent occurrence and the common use of ID regions in important protein functions raise the possibility that the relationship between amino acid sequence, disordered ensemble and function might be the dominant paradigm for the molecular recognition that serves as the basis for signaling and regulation by protein molecules.

Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production

Abstract: The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and muts phenotypes, intracellular expression and folding pathways is examined. The roles of pre–pro signal sequences such as the alpha mating factor (α-MF) and the Glu–Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed. Copyright © 2004 John Wiley & Sons, Ltd.

Survey of the year 2003 commercial optical biosensor literature

Abstract: In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

Theoretical aspects of virus capsid assembly.

Abstract: A virus capsid is constructed from many copies of the same protein(s). Molecular recognition is central to capsid assembly. The capsid protein must polymerize in order to create a three-dimensional protein polymer. More than structure is required to understand this self-assembly reaction: one must understand how the pieces come together in solution.

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Abstract: Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.

A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions.

Abstract: Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.

Kinetic determinations of molecular interactions using Biacore—minimum data requirements for efficient experimental design

Abstract: Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M(-1) s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between approximately 10 microM and approximately 1 nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided.

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture†

Abstract: The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.

Casein-hydrolyzing activity of sIgA antibodies from human milk.

Abstract: During pregnancy and immediately after delivery (i.e. at the beginning of lactation), the female organism is frequently characterized by an immune status similar to that of patients with autoimmune diseases. In addition, lactation is associated with an appearance of catalytically active antibodies or abzymes (Abzs) with DNAse, RNase, ATPase, amylolitic, protein kinase and lipid kinase activities in breast milk. However, until now there were no examples of human milk Abzs with a proteolytic activity. We present the first evidence that electrophoretically and immunologically homogeneous human milk sIgAs possess a beta-casein-hydrolyzing activity different from known proteases. Abzs specifically hydrolyze both human and bovine beta-caseins but not many other proteins tested. Using different methods including in situ analysis of proteolytic activity in a gel after SDS-PAGE it was shown that the observed proteolytic activity is an intrinsic property of human milk polyclonal sIgAs. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of Abzs, while a specific inhibitor of serine proteases (AEBSF) significantly inhibited the proteolytic activity of the abzymes. The K(M) value for human casein as a substrate was estimated (7.3 microM). Our findings suggest that the immune system of clinically healthy mothers can generate IgAs with a beta-casein-specific serine protease-like activity.

Location and nature of the residues important for ligand recognition in G-protein coupled receptors.

Abstract: The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.

Synthesis and screening of a rationally designed combinatorial library of affinity ligands mimicking protein L from Peptostreptococcus magnus.

Abstract: Rational design and combinatorial chemistry were utilized to search for lead protein L (PpL) mimetics for application as affinity ligands for the purification of antibodies and small fragments, such as Fab and scFv, and as potential diagnostic or therapeutic agents. Inspection of the key structural features of the complex between PpL and human Fab prompted the de novo design and combinatorial synthesis of a 169-membered solid-phase ligand library, which was assessed for binding to human IgG and subsequent selectivity for the Fab fragment. Eight ligands were selected, chemically characterized and compared with a commercial PpL-adsorbent for binding pure immunoglobulin fractions. The most promising lead, ligand 8/7, when immobilized on an agarose support, behaved in a similar fashion to PpL in isolating Fab fragments from papain digests of human IgG to a final purity of 97%.

Selective recognition of neodymium (III) using ion imprinted polymer particles.

Abstract: Neodymium (III) ion-imprinted polymer (IIP) materials were prepared by the copolymerization of neodymium (III)–5,7-dichloroquinoline-8-ol–4-vinylpyridine ternary complex with styrene(monomer), divinyl benzene (crosslinking monomer) in the presence of 2,2′-azobisisobutyronitrile (initiator). The synthesis was carried out in 2-methoxy ethanol medium (porogen) and the resultant material was filtered, washed, dried and powdered to form unleached IIP particles. The imprint ion was removed by stirring the above particles with 50% (v/v) HCl for 6 h to obtain leached IIP particles with cavities in the polymer particles. Control polymer (CP) particles were similarly prepared without imprint ion, i.e. neodymium (III). CP, unleached and leached IIP particles were characterized by TLC, IR, microanalysis, XRD and UV–visible spectrophotometric studies. The preconcentration of 5–150 μg of neodymium (III) ions present in 500 ml of solution was possible with as little as 40 mg of neodymium (III) IIP particles in the pH range 7.5–8.0 with a detection limit of 50 ng/l. Five replicate determinations of 25 μg of neodymium (III) present in 500 ml of solution gave a mean absorbance of 0.120 with a relative standard deviation of 2.65%. The imprinting effect of IIP particles was noticed in all preconcentration and selectivity studies when compared with CP particles. Furthermore, the selectivity coefficients of neodymium (III) IIP particles were much higher compared with the reported separation factors for the best liquid–liquid extractants, viz. di-2-ethylhexyl phosphoric acid and 2-ethylhexyl-ethylhexyl phosphonate. Kinetic and isotherm studies during rebinding of neodymium (III) onto IIP particles were also carried out. Copyright © 2004 John Wiley & Sons, Ltd.

Discovery of high-affinity peptide binders to BLyS by phage display.

Abstract: B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.

The structural basis of recognition and removal of cellular mRNA 7-methyl G 'caps' by a viral capsid protein: a unique viral response to host defense.

Abstract: The single segment, double-stranded RNA genome of the L-A virus (L-A) of yeast encodes two proteins: the major coat protein Gag (76 kDa) and the Gag–Pol fusion protein (180 kDa). The icosahedral L-A capsid is formed by 120 copies of Gag and has architecture similar to that seen in the reovirus, blue tongue virus and rice dwarf virus inner protein shells. Gag chemically removes the m7GMP caps from host cellular mRNAs. Previously we identified a trench on the outer surface of Gag that included His154, to which caps are covalently attached. Here we report the refined L-A coordinates at 3.4 A resolution with additional structural features and the structure of L-A with bound m7GDP at 6.5 A resolution, which shows the conformational change of the virus upon ligand binding. Based on site-directed mutations, residues in or adjacent to the trench that are essential (or dispensable) for the decapping reaction are described here. Along with His154, the reaction requires a cluster of positive charge adjoining the trench and residues Tyr 452, Tyr150 and either Tyr or Phe at position 538. A tentative mechanism for decapping is proposed. Copyright © 2004 John Wiley & Sons, Ltd.

Peptide self-aggregation and peptide complementarity as bases for the evolution of peptide receptors: a review.

Abstract: This paper reviews the three major theories of peptide receptor evolution: (1) Dwyer's theory that peptide receptors evolved from self-aggregating peptides; (2) Root-Bernstein's theory that peptide receptors evolved from functionally and structurally complementary peptides; and (3) Blalock's theory that receptors evolved from hydropathically complementary sequences encoded in the antisense strand of the DNA encoding each peptide. The evidence to date suggests that the co-yevolution of peptides and their receptors is strongly constrained by one or more of these physicochemically based mechanisms, which argues against a random or frozen accident' model. The data also suggest that structure and function are integrally related from the earliest steps of receptor–ligand evolution so that peptide functionality is non-random and highly conserved in its origin. The result is a molecular paleontology' that reveals the evolutionary constraints that shaped the interaction of structure and function. Copyright © 2004 John Wiley & Sons, Ltd.

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides.

Abstract: In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.

Discovery of human antibodies against the C5aR target using phage display technology.

Abstract: Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.

Nanoparticle energy transfer on the cell surface.

Abstract: Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2–10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal–fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle–fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range esentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore–nanoparticle distance. The intensity of 90° scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90° light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging microscope. Modulations of 90° light scattering intensity and photobleaching time constant indicate the role of the local field in the fluorescence enhancement. A mathematical simulation based on the electrodynamic theory of fluorescence enhancement showed a consistency between the measured enhancement values, the inter-epitope distances and the quantum yields. The feasibility of realizing proximity sensors operating at distance ranges larger than that of the conventional Forster transfer is demonstrated on the surface of living cells. Copyright © 2004 John Wiley & Sons, Ltd.

Selective recognition of enzymatically active prostate‐specific antigen (PSA) by anti‐PSA monoclonal antibodies

Abstract: Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines.

Abstract: 'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.

High-resolution crystallography and drug design

Abstract: Ultra-high-resolution X-ray crystallography of macromolecules (i.e. resolution better than 0.8 A) is a rising field that promises to provide new insight into the structure–function relationships of biomacromolecules. The picture emerging from macromolecular structures at this resolution is far more complex than previously understood, requiring for its study improved tools for structure refinement, analysis and annotation. Some of these problems were highlighted during the recent High Resolution Drug Design Meeting (Bischenberg-Strasbourg, France, 13–16 May 2004). We will review here some of the results and discussions that took place during that meeting and elaborate on the trends and challenges ahead in this emerging new field of research. Copyright © 2005 John Wiley & Sons, Ltd.

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

Abstract: Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.

Structural basis for preferential binding of non‐ortho‐substituted polychlorinated biphenyls by the monoclonal antibody S2B1

Abstract: Polychlorinated biphenyls (PCBs) are a family of 209 isomers (congeners) with a wide range of toxic effects. In structural terms, they are of two types: those with and those without chlorines at the ortho positions (2, 2′, 6 and 6′). Only 20 congeners have no ortho chlorines. Three of these are bound by the aryl hydrocarbon receptor and are one to four orders of magnitude more toxic than all others. A monoclonal antibody, S2B1, and its recombinant Fab have high selectivity and nanomolar binding affinities for two of the most toxic non-ortho-chlorinated PCBs, 3,4,3′,4′-tetrachlorobiphenyl and 3,4,3′,4′,5′-pentachlorobiphenyl. To investigate the basis for these properties, we built a three-dimensional structure model of the S2B1 variable fragment (Fv) based on the high-resolution crystallographic structures of antibodies 48G7 and N1G9. Two plausible conformations for the complementarity-determining region (CDR) H3 loop led to two putative PCB-binding pockets with very different shapes (models A and B). Docking studies using molecular mechanics and potentials of mean force (PMF) indicated that model B was most consistent with the selectivity observed for S2B1 in competition ELISAs. The binding site in model B had a deep, narrow pocket between VL and VH, with a slight constriction at the top that opened into a wider pocket between CDRs H1 and H3 on the antibody surface. This binding site resembles those of esterolytic antibodies that bind haptens with phenyl rings. One phenyl ring of the PCB fits into the deep pocket, and the other ring is bound in the shallower one. The bound PCB is surrounded by the side chains of TyrL91, TyrL96 and TrpH98, and it has a π-cation interaction with ArgL46. The tight fit of the binding pocket around the ortho positions of the bound PCBs indicates that steric hindrance of ortho chlorines in the binding site, rather than induced conformational change of the PCBs, is responsible for the selectivity of S2B1. Copyright © 2005 John Wiley & Sons, Ltd.

A molecular recognition paradigm: promiscuity associated with the ligand-receptor interactions of the activin members of the TGF-beta superfamily.

Abstract: The structure-function properties of the pleiotropic activins and their relationship to other members of the transforming growth factor-beta superfamily of proteins are described. In order to highlight the molecular promiscuity of these growth factors, emphasis has been placed on molecular features associated with the recognition by activin A and the bone morphogenic proteins of the corresponding extracellular domains of the ActRI and ActRII receptors. The available evidence suggests that the homodimeric activin A in its various functional roles has the propensity to fulfill key tasks in the regulation of mammalian cell behaviour, through coordination of numerous transcriptional and translational processes. Because of these profound effects, under physiologically normal conditions, activin A levels are closely controlled by a variety of binding partners, such as follistatin-288 and follistatin-315, alpha(2)-macroglobulin and other proteins. Moreover, the subunits of other members of the activin subfamily, such as activin B or activin C, are able to form heterodimers with the activin A subunit, thus providing a further avenue to positively or negatively control the physiological concentrations of activin A that are available for interaction with specific receptors and induction of cell signaling events. Based on data from X-ray crystallographic studies and homology modeling experiments, the molecular architecture of the ternary receptor-activin ligand complexes has been dissected, permitting rationalization in structural terms of the pattern of interactions that are the hallmark of this protein family.

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Abstract: Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.

Molecularly imprinted polymer with salicylaldehyde-Cu(OAc)2 as template.

Abstract: In the report molecularly imprinted polymer (MIP) with salicylaldehyde–Cu(OAc)2 as the template was synthesized and characterized by SEM, porosity and elemental analysis. Copper acetate was introduced since salicylaldehyde alone cannot display imprinting effect for its intramolecular hydrogen bond. The strong coordination interaction between salicylaldehyde and copper acetate made the complex have high retention on the HPLC column based on the SAD–Cu(OAc)2 imprinted polymer. Several structural analogues such as salicylaldoxime, sulfosalicylic acid, p-hydroxybenzaldehyde and their complexes with copper acetate were chosen to study the selectivity of the MIPs. The influence of acetic acid and H2O in methanol mobile phase was studied. The experimental results showed that small amount of either acetic acid or H2O in mobile phase would weaken the interaction between the complex and the polymer, therefore, the retention of the complex was lowered to a large extent, but that of salicylaldehyde remained almost unchanged. The polymer imprinted with the complex showed high selectivity to both the acetate and copper (II). In addition, the MIP showed an enhanced selectivity to its template compared with the polymer prepared without copper acetate. Copyright © 2004 John Wiley & Sons, Ltd.

Efficient recognition of protein fold at low sequence identity by conservative application of Psi‐BLAST: application

Abstract: Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins.

RNase S complex bearing arginine-rich peptide and anti-HIV activity

Abstract: Basic peptide-mediated protein delivery into living cells is becoming recognized as a potent approach for the understanding of cellular mechanisms and drug delivery. We have prepared the conjugates of the S-peptide (1-15) derived from RNase S with membrane-permeable basic peptides, octaarginine and the human immunodeficient virus (HIV)-1 Rev (34-50). The RNase S complexes, formed among these S-peptide (1-15)-basic peptide conjugates and the S-protein and having a dissociation constant in the range of 10(-5) M, efficiently penetrated into the HeLa cells. These RNase S complexes exerted an anti-HIV replication activity. The time-of-drug-addition assay suggested that the site of action for these complexes would reside in the stages between the viral entry into the cells and reverse transcription. The present study exemplified the applicability of the arginine-rich peptides to the intracellular targeting of non-covalent protein complexes and supramolecular assemblies for the research in chemical and cellular biology.

Stereoselective antibodies to free α-hydroxy acids

Abstract: This work describes antibodies exhibiting high stereoselectivity and class-specificity towards the enantiomers of free alpha-hydroxy acids. Since the antibodies interact primarily with the carboxyl-hydroxyl-hydrogen triad about the stereogenic center, they are useful for enantiomer analysis of a variety of structurally different alpha-hydroxy acids including aromatic and aliphatic compounds, e.g. lactic acid. The utility of such antibodies for enantiomer separation in chromatography was demonstrated. Comparative studies of these and previously described anti-alpha-amino acid antibodies revealed that both types of antibodies bind only to analytes that possess both the corresponding target structure and the correct configuration. Thus, substitution of an amino group for the alpha-hydroxyl group results in a complete loss of binding activity with the anti-alpha-hydroxy acid antibodies, while an alpha-amino group is essential for the interaction between analytes and anti-alpha-amino acid antibodies.

Investigation of potential RNA bulge stabilizing elements.

Abstract: As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2′-modified nucleoside moieties (2′-O-methyl, 2′-deoxy and 2′-deoxy-2′-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G–U wobble base pair to a G–C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (ΔTm + 4.6 to + 5.4°C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2′-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (ΔTm + 3.6 and + 2.9°C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson–Crick recognition of RNA. Copyright © 2005 John Wiley & Sons, Ltd.