Showing papers in "Journal of Molecular Recognition in 2007"

A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions.

Abstract: The widespread exploitation of biosensors in the analysis of molecular recognition has its origins in the mid-1990s following the release of commercial systems based on surface plasmon resonance (SPR). More recently, platforms based on piezoelectric acoustic sensors (principally 'bulk acoustic wave' (BAW), 'thickness shear mode' (TSM) sensors or 'quartz crystal microbalances' (QCM)), have been released that are driving the publication of a large number of papers analysing binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights salient theoretical and practical aspects of the technologies that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and lipidic and polymeric interfaces. Key differentiators between optical and acoustic sensing modalities are also reviewed.

Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools

Abstract: A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools.

Past, present and future of atomic force microscopy in life sciences and medicine

Abstract: To introduce this special issue of the Journal of Molecular Recognition dedicated to the applications of atomic force microscopy (AFM) in life sciences, this paper presents a short summary of the history of AFM in biology. Based on contributions from the first international conference of AFM in biological sciences and medicine (AFM BioMed Barcelona, 19-21 April 2007), we present and discuss recent progress made using AFM for studying cells and cellular interactions, probing single molecules, imaging biosurfaces at high resolution and investigating model membranes and their interactions. Future prospects in these different fields are also highlighted. Copyright (c) 2007 John Wiley & Sons, Ltd.

Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology†

Abstract: This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.

Energy landscape roughness of the streptavidin-biotin interaction.

Abstract: Molecular interactions between receptors and ligands can be characterized by their free energy landscape. In its simplest representation, the energy landscape is described by a barrier of certain height and width that determines the dissociation rate of the complex, as well as its dynamic strength. Some interactions, however, require a more complex landscape with additional barriers and roughness along the reaction coordinate. This roughness slows down the dissociation kinetics of the interaction and contributes to its dynamic strength. The streptavidin-biotin complex has been extensively studied due to its remarkably low dissociation kinetics. However, single molecule measurements from independent experiments showed scattered and disparate results. In this work, the energy landscape roughness of the streptavidin-biotin interaction was estimated to be in the range of 5-8kBT using dynamic force spectroscopy (DFS) measurements at three different temperatures. These results can be used to explain both its slow dissociation kinetics and the discrepancies in the reported force measurements.

High‐speed atomic force microscopy for observing dynamic biomolecular processes

Abstract: The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability will become more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging, because 'direct and real-time visualization' is a straightforward and powerful means to understand biomolecular processes. With conventional AFMs, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale or less. In order to fill this large gap, various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of high-speed AFM, and discuss possibilities that may break the limitations and lead to the development of a truly useful high-speed AFM for biological sciences.

Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity.

Abstract: The capability of atomic force microscopes (AFM) to generate atomic or nanoscale resolution images of surfaces has deeply transformed the study of materials. However, high resolution imaging of biological systems has proved more difficult than obtaining atomic resolution images of crystalline surfaces. In many cases, the forces exerted by the tip on the molecules (1-10 nN) either displace them laterally or break the noncovalent bonds that hold the biomolecules together. Here, we apply a force microscope concept based on the simultaneous excitation of the first two flexural modes of the cantilever. The coupling of the modes generated by the tip-molecule forces enables imaging under the application of forces ( approximately 35 pN) which are smaller than those needed to break noncovalent bonds. With this instrument we have resolved the intramolecular structure of antibodies in monomer and pentameric forms. Furthermore, the instrument has a force sensitivity of 0.2 pN which enables the identification of compositional changes along the protein fragments.

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies.

Abstract: There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies.

A benzimidazole-based chromogenic anion receptor.

Abstract: A benzimidazole-based anion receptor 1 has been designed and synthesized. The structure of 1 is confirmed through single crystal X-ray diffraction analysis. Anion-binding studies carried out using 1H NMR and UV–visible revealed that this compound exhibits selective recognition toward F− over other halide anions. The highest selectivity for F− among the halides is attributed mainly to the strongest hydrogen-bond interaction of the receptor with F−; in addition, the higher match in geometry between the receptor and F− also plays a role in the selective recognition of the receptor for F−. Receptor 1 (1 × 10−5 M) shows dramatic color change from light-yellow to red in the presence of tetrabutylammonium fluoride (TBAF) (1 × 10−5 M). Moreover, F−-induced color changes remain the same even in the presence of a large excess of Cl−, Br−, I−. Copyright © 2006 John Wiley & Sons, Ltd.

Docking study and free energy simulation of the complex between p53 DNA-binding domain and azurin.

Abstract: Molecular interaction between p53 tumor suppressor and the copper protein azurin (AZ) has been demonstrated to enhance p53 stability and hence antitumoral function, opening new perspectives in cancer treatment. While some experimental work has provided evidence for AZ binding to p53, no crystal structure for the p53-AZ complex was solved thus far. In this work the association between AZ and the p53 DNA-binding domain (DBD) was investigated by computational methods. Using a combination of rigid-body protein docking, experimental mutagenesis information, and cluster analysis 10 main p53 DBD-AZ binding modes were generated. The resulting structures were further characterized by molecular dynamics (MD) simulations and free energy calculations. We found that the highest scored docking conformation for the p53 DBD-AZ complex also yielded the most favorable free energy value. This best three-dimensional model for the complex was validated by using a computational mutagenesis strategy. In this structure AZ binds to the flexible L(1) and s(7)-s(8) loops of the p53 DBD and stabilizes them through protein-protein tight packing interactions, resulting in high degree of both surface matching and electrostatic complementarity.

What is the biological relevance of the specific bond properties revealed by single-molecule studies?

Abstract: During the last decade, many authors took advantage of new methodologies based on atomic force microscopy (AFM), biomembrane force probes (BFPs), laminar flow chambers or optical traps to study at the single-molecule level the formation and dissociation of bonds between receptors and ligands attached to surfaces. Experiments provided a wealth of data revealing the complexity of bond response to mechanical forces and the dependence of bond rupture on bond history. These results supported the existence of multiple binding states and/or reaction pathways. Also, single bond studies allowed us to monitor attachments mediated by a few bonds. The aim of this review is to discuss the impact of this new information on our understanding of biological molecules and phenomena. The following points are discussed: (i) which parameters do we need to know in order to predict the behaviour of an encounter between receptors and ligands, (ii) which information is actually yielded by single-molecule studies and (iii) is it possible to relate this information to molecular structure?

Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips.

Abstract: Cell adhesion is required for essential biological functions such as migration, tissue formation and wound healing, and it is mediated by individual molecules that bind specifically to ligands on other cells or on the extracellular matrix. Atomic force microscopy (AFM) has been successfully used to measure cell adhesion at both single molecule and whole cell levels. However, the measurement of inherent cell adhesion properties requires a constant cell–probe contact area during indentation, a requirement which is not fulfilled in common pyramidal or spherical AFM tips. We developed a procedure using focused ion beam (FIB) technology by which we modified silicon pyramidal AFM cantilever tips to obtain flat-ended cylindrical tips with a constant and known area of contact. The tips were validated on elastic gels and living cells. Cylindrical tips showed a fairly linear force–indentation behaviour on both gels and cells for indentations >200 nm. Cylindrical tips coated with ligands were used to quantify inherent dynamic cell adhesion and elastic properties. Force, work of adhesion and elasticity showed a marked dynamic response. In contrast, the deformation applied to the cells before rupture was fairly constant within the probed dynamic range. Taken together, these results suggest that the dynamic adhesion strength is counterbalanced by the dynamic elastic response to keep a constant cell deformation regardless of the applied pulling rate. Copyright © 2007 John Wiley & Sons, Ltd.

Mapping correlated membrane pulsations and fluctuations in human cells.

Abstract: The cell membrane and cytoskeleton are dynamic structures that are strongly influenced by the thermo-mechanical background in addition to biologically driven mechanical processes. We used atomic force microscopy (AFM) to measure the local membrane motion of human foreskin fibroblasts (HFFs) which were found to be governed by random and non-random correlated mechanical processes. Interphase cells displayed distinct membrane pulsations in which the membrane was observed to slowly contract upwards followed by a recovery to its initial position. These pulsations occurred one to three times per minute with variable amplitudes (20-100 pN) separated by periods of random baseline fluctuations with amplitudes of <20 pN. Cells were exposed to actin and microtubule (MT) destabilizing drugs and induced into early apoptosis. Mechanical pulsations (20-80 pN) were not prevented by actin or MT depolymerization but were prevented in early apoptotic cells which only displayed small amplitude baseline fluctuations (<20 pN). Correlation analysis revealed that the cell membrane motion is largely random; however several non-random processes, with time constants varying between approximately 2 and 35 s are present. Results were compared to measured cardiomyocyte motion which was well defined and highly correlated. Employing automated positioning of the AFM tip, interphase HFF correlation time constants were also mapped over a 10 microm2 area above the nucleus providing some insights into the spatial variability of membrane correlations. Here, we are able to show that membrane pulsations and fluctuations can be linked to physiological state and cytoskeletal dynamics through distinct sets of correlation time constants in human cells.

Detection of antigen‐specific T cells on p/MHC microarrays

Abstract: The development of high-throughput protein microarrays for rapidly determining antigen-specific T-cell receptor repertoires of diverse T-cell populations can enable comprehensive, broad-based analyses of T-cell responses. Promising applications include medical diagnostics, vaccine development, treatment of autoimmune diseases and detection of potential agents of bioterrorism. In this study, we examined the feasibility of using peptide/major histocompatibility complex (p/MHC) microarrays to selectively capture and enumerate antigen-specific T cells. Results are presented for p/MHC microarrays consisting of a dimeric MHC-immunoglobulin complex, K(b)-Ig, loaded with either a cognate or non-cognate peptide for binding CD8(+) T cells. We quantified the sensitivity of these K(b)-Ig microarrays by measuring a lower detection limit of 0.05% antigen-specific CD8(+) T cells mixed with splenocytes from C57BL/6J mouse. A fivefold increase in this lower detection limit (0.01%) was achieved using a secondary capture anti-Ig antibody to coat the microarray surface. This higher sensitivity is comparable to that obtained using standard state-of-the-art fluorescence activated cell sorting (FACS) instruments. We also found that contacting the T-cell suspension with the K(b)-Ig microarrays under mild shear flow conditions produced more uniform distributions of captured T cells on the individual spots and better spot-to-spot reproducibility across the entire microarray.

Identification of discontinuous antigenic determinants on proteins based on shape complementarities.

Abstract: Diverse procedures for identifying antigenic determinants on proteins have been developed, including experimental as well as computational approaches. However, most of these techniques focus on continuous epitopes, whereas fast and reliable identification and verification of discontinuous epitopes remains barely amenable. In this paper, we describe a computational workflow for the detection of discontinuous epitopes on proteins. The workflow uses a given protein 3D structure as input, and combines a per residue solvent accessibility constraint with epitope to paratope shape complementarity measures and binding energies for assigning antigenic determinants in the conformational context. We have developed the procedure on a given set of 26 antigen-antibody complexes with a known structure, and have further expanded the available paratope shapes by generating a virtual paratope library in order to improve the screening for candidate residues constituting discontinuous epitopes. Applying the workflow on the 26 given antigens with known discontinuous epitopes resulted in the correct identification of the spatial proximity of 12 antigen-antibody interaction sites. Combining solvent accessibility, shape complementarity and binding energies towards the identification of discontinuous epitopes clearly outperforms approaches solely considering accessibility and residue distance constraints.

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin.

Abstract: The thyrotropin receptor (TSHR) has a unique 50 residue (317–366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334–349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation. Copyright © 2007 John Wiley & Sons, Ltd.

Biosensor‐based characterization of serum antibodies during development of an anti‐IgE immunotherapeutic against allergy and asthma

Abstract: Antibody responses, induced in Cynomolgus monkey by recombinant IgE-derived immunotherapeutic protein against atopic allergies and asthma, were characterized using label-free, real-time protein interaction analysis. The effects of two different immunotherapeutic proteins were compared. Active concentrations of specific anti-IgE antibodies formed were determined in sera sampled at multiple time points, using conditions of total mass transport limitation that were proved to exist on the sensor surface. These concentrations varied from about 0.4 to 35 microg/ml among the monkeys and throughout the immunization period. Based on these concentrations, the rate and affinity constants for the binding of antibody populations to the antigen could be determined. The apparent equilibrium dissociation constant decreased during the immunization period, for all the monkeys, by a factor between 6 and 50, ending at values from approximately 2 x 10(-9) to approximately 2 x 10(-11) M among the animals. This affinity maturation was attributable to the changes in both rate constants, although the magnitude of the contribution of each constant depended partly on specimen, but primarily on the immunotherapeutic used. The immunotherapeutic proteins examined showed excellent immunogenic properties, providing the basis for a new and effective treatment for allergy and asthma.

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

Abstract: Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.

Self-organization versus Watchmaker: ambiguity of molecular recognition and design charts of cellular circuitry.

Abstract: A large body of experimental evidence indicates that the specific molecular interactions and/or chemical conversions depicted as links in the conventional diagrams of cellular signal transduction and metabolic pathways are inherently probabilistic, ambiguous and context-dependent Being the inevitable consequence of the dynamic nature of protein structure in solution, the ambiguity of protein-mediated interactions and conversions challenges the conceptual adequacy and practical usefulness of the mechanistic assumptions and inferences embodied in the design charts of cellular circuitry It is argued that the reconceptualization of molecular recognition and cellular organization within the emerging interpretational framework of self-organization, which is expanded here to include such concepts as bounded stochasticity, evolutionary memory, and adaptive plasticity offers a significantly more adequate representation of experimental reality than conventional mechanistic conceptions do Importantly, the expanded framework of self-organization appears to be universal and scale-invariant, providing conceptual continuity across multiple scales of biological organization, from molecules to societies This new conceptualization of biological phenomena suggests that such attributes of intelligence as adaptive plasticity, decision-making, and memory are enforced by evolution at different scales of biological organization and may represent inherent properties of living matter Copyright © 2007 John Wiley & Sons, Ltd

Complement C1q-target proteins recognition is inhibited by electric moment effectors.

Abstract: Classical complement pathway is an important innate immune mechanism, which is usually triggered by binding of C1q to immunoglobulins, pentraxins and other target molecules. Although the activation of the classical pathway is crucial in the host defence, its undesirable and uncontrolled activation can lead to tissue damage. Thus, understanding the molecular basis of complement activation and its inhibition are of great biomedical importance. Recently, we proposed a mechanism for target recognition and classical pathway activation by C1q, which is likely governed by calcium-controlled reorientation of macromolecular electric moment vectors. Here we sought to define the mechanism of C1q inhibition by low molecular weight disulphate compounds that bind to the globular (gC1q) domain, using experimental, computational docking and theoretical modelling approaches. Our experimental results demonstrate that betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of C1q and its recombinant globular modules with target molecules IgG1, C-reactive protein (CRP) and long pentraxin 3 (PTX3). In most C1q-inhibitor docked complexes, there is a reduction of electric moment scalar values and similarly altered direction of electric/dipole moment vectors. This could explain the inhibitory effect by impaired electrostatic steering, lacking optimal target recognition and formation of functional complex. In the presence of the inhibitor, the tilt of gC1q domains is likely to be blocked by the altered direction of the electric moment vector. Thus, the transition from the inactive (closed) towards the active (open) conformation of C1q (i.e. the complement activation signal transmission) will be impaired and the cascade initiation disrupted. These results could serve as a starting point for the exploration of a new form of ‘electric moment inhibitors/effectors’. Copyright © 2007 John Wiley & Sons, Ltd.

A novel chiral separation material: polymerized organogel formed by chiral gelators for the separation of D- and L-phenylalanine.

Abstract: N-Stearine-N'-stearyl-L-phenylalanine, a chiral compound, was synthesized and used as a gelator for the gelation of polymerizable solvents, such as ss-hydroxyethyl methacrylate (HEMA), styrene, etc. The scanning electron microscope (SEM) images of the gelator aggregates show fibril-like helices, typical chiral aggregates with diameters of 100-200 nm. The solvent molecules were immobilized by capillary forces in the three-dimensional network structures of the organogels. The HEMA organogels containing crosslinker polyethylene glycol dimethacrylates (PEG200DMA) were subsequently polymerized by in situ UV irradiation. A porous polymerized organogels were obtained after removal of gelator aggregates through ethanol extraction. The chiral separation of D- and L-phenylalanine was carried out by the adsorption of the polymerized organogels. The adsorption efficiency of L-phenylalanine on the polymerized organogels was found to be dependent on the concentration of the gelator and crosslinker.

Atomic force microscopy characterization of supported planar bilayers that mimic the mitochondrial inner membrane.

Abstract: In this study we examined the properties of supported planar bilayers (SPBs) formed from phospholipid components that comprise the mitochondrial inner membrane. We used 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL). Liposomes of binary POPE:POPC (1:1, mol:mol) and ternary (POPE:POPC:CL (0.5:0.3:0.2, mol:mol:mol) composition were used in the formation of SPBs on mica. The characterization of the SPBs was carried out below (4 degrees C) and above (24 and 37 degrees C) the phase transition temperature (Tm) of the mixtures in solution. We observed: (i) that the thickness of the bilayers, calculated from a cross-sectional analysis, decreased as the visualization temperature increased; (ii) the existence of laterally segregated domains that respond to temperature in SPBs of POPE:POPC:CL; (iii) a decrease in height and an increase in roughness (Ra) of SPBs after cytochrome c (cyt c) injection at room temperature. To obtain further insight into the nature of the interaction between cyt c and the bilayers, the competition between 8-anilino-1-naphthalene sulfonate (ANS) and the protein for the same binding sites in liposomes was monitored by fluorescence. The results confirm the existence of preferential interaction of cyt c with CL containing liposomes. Taking these results and those of previous papers published by the group, we discuss the preferential adsorption of cyt c in CL domains. This provides support for the relevance of these phospholipids as a proton trap in the oxidative phosphorylation process that occurs in the energy transducing membranes.

A tale in molecular recognition: the hammerhead ribozyme.

Abstract: A new crystal structure of the hammerhead ribozyme demonstrates the influence of peripheral tertiary contacts on the local conformations around the active site. This structure resolves many conflicting results obtained on reduced systems.

Ligand-receptor interactions and membrane structure investigated by AFM and time-resolved fluorescence microscopy.

Abstract: The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery.

Statistical aspects of van't Hoff analysis: a simulation study.

Abstract: The thermodynamics of biological interactions is frequently studied by the van't Hoff analysis whereby data on variation of the binding constant K(D) with temperature are used to obtain estimates of standard enthalpy (Delta H degrees ), entropy (Delta S degrees ), and heat capacity (Delta C degrees P) of complex formation. A Monte Carlo simulation demonstrates that the absolute error of the above parameters is proportional to the relative error of KD and independent of the actual values of KD and of the way they vary with temperature. The error of Delta H degrees is approximately the same as that of T Delta S degrees (within 14% in the temperature range 5-45 degrees C). The error depends both on the number of temperature points within the experimental temperature range and on the size of the range, but it is more sensitive to the latter. Using the linear form of the van't Hoff equation to fit data with non-zero Delta C degrees P gives erroneous Delta H degrees and DeltaS degrees estimates at standard temperature except for the case when the T points are placed symmetrically with respect to the standard temperature. With the range of Delta C degrees P values usual for protein-protein interactions, the KD error must be very low to confidently infer that Delta C degrees P is non-zero or to claim that two interactions have different Delta C degrees P.

Docking and molecular dynamics simulation of the Azurin–Cytochrome c551 electron transfer complex

Abstract: We coupled protein-protein docking procedure with molecular dynamics (MD) simulation to investigate the electron transfer (ET) complex Azurin-Cytochrome c551 whose transient character makes difficult a direct experimental investigation. The ensemble of complexes generated by the docking algorithm are filtered according to both the distance between the metal ions in the redox centres of the two proteins and to the involvement of suitable residues at the interface. The resulting best complex (BC) is characterized by a distance of 1.59 nm and involves Val23 and Ile59 of Cytochrome c551. The ET properties have been evaluated in the framework of the Pathways model and compared with experimental data. A 60 ns long MD simulation, carried on at full hydration, evidenced that the two protein molecules retain their mutual spatial positions upon forming the complex. An analysis of the ET properties of the complex, monitored at regular time intervals, has revealed that several different ET paths are possible, with the occasional intervening of water molecules. Furthermore, the temporal evolution of the geometric distance between the two redox centres is characterized by very fast fluctuations around an average value of 1.6 nm, with periodic jumps at 2 nm with a frequency of about 70 MHz. Such a behaviour is discussed in connection with a nonlinear dynamics of protein systems and its possible implications in the ET process are explored.

The influence of rigid or flexible linkage between two ligands on the effective affinity and avidity for reversible interactions with bivalent receptors.

Abstract: Bivalent or polyvalent cooperative binding between ligand and receptor is much tighter and more efficient than monovalent binding of the same counterparts. Because of this, many biological processes involve polyvalent binding for realization of regulatory mechanisms. For this reason it is necessary to develop a general formalism for prediction of the relationship between the binding affinities of each ligand subunits, the length of the flexible linker between them and avidity of the interaction. Here, we consider an approach that is based on the description of the state of equilibrium for the reaction of mono- and multivalent ligand-receptor binding of algebraic equation systems. This approach allows the evaluation of the avidity of bivalent binding and to determine the concentrations of ligand–receptor complexes, which will be obtained at the equilibrium state. The analysis presented here may be useful in analysing the binding behaviour of a bivalent receptor and a ligand consisting of two subunits covalently connected with a rigid or flexible linker. Copyright © 2007 John Wiley & Sons, Ltd.

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

Abstract: A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating ‘SF-link’. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC50 of around 30 nM, a Kd of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed. Copyright © 2006 John Wiley & Sons, Ltd.

Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain

Abstract: Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.

Probing the interaction forces between hydrophobic peptides and supported lipid bilayers using AFM.

Abstract: Despite the vast body of literature that has accumulated on tilted peptides in the past decade, direct information on the forces that drive their interaction with lipid membranes is lacking. Here, we attempted to use atomic force microscopy (AFM) to explore the interaction forces between the Simian immunodeficiency virus peptide and phase-separated supported bilayers composed of various lipids, i.e. dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidic acid and dipalmitoylphosphatidylethanolamine. Histidine-tagged peptides were attached onto AFM tips terminated with nitrilotriacetate and tri(ethylene glycol) groups, an approach expected to ensure optimal exposure of the C-terminal hydrophobic domain. Force-distance curves recorded between peptide-tips and the different bilayer domains always showed a long-range repulsion upon approach and a lack of adhesion upon retraction, in marked contrast with the hydrophobic nature of the peptide. To explain this unexpected behaviour, we suggest a mechanism in which lipids are pulled out from the bilayer due to strong interactions with the peptide-tip, in agreement with the very low force needed to extract lipids from supported bilayers.