Biomolecular Receptors Research Articles

Principles for the Rational Design of Allosterically Cooperative Biomolecular Receptors

Allosteric cooperativity is used ubiquitously throughout nature to generate steeper, more responsive input-output behaviors. The focus of my work has been to rationally introduce this valuable property into several classes of normally non-cooperative biomolecular receptors. My approach to this end employs the classical population-shift (between and states) mechanism underlying the cooperative oxygen binding of hemoglobin. Specifically, I have designed receptors that equilibrate between two conformations, the more stable of which binds the target ligand only weakly and the less stable of which exposes two high-affinity ligand binding sites. The binding of the first copy of the ligand shifts this conformational equilibrium towards the later, higher-affinity state, improving the affinity of the second binding event and thus producing a steeper, highly cooperative response. One approach to this end involves receptors that equilibrate between an intrinsically disordered conformation and a folded conformation containing two target-binding sites. The folding of the entire receptor upon binding the first copy of target molecule improves the affinity of the second, leading to cooperative binding. The observed cooperativity of my redesigned receptors is quantitatively dependent on the equilibrium constant KS, describing the switch between the tense and relaxed (e.g., folded and unfolded) states. Effectively maximal sensitivity (i.e., a Hill coefficient within error of the theoretically expected 2 and a 9-fold increase in sensitivity) is achieved as KSfalls below 0.0025. The enhanced responsiveness of these cooperative receptors should improve their utility in applications, such as biosensors, biomolecular logic gates, and smart, responsive biomaterials, in which steeper, more sensitive input−output behavior is of value.

ChemInform Abstract: Biosensors Based on Nanomechanical Systems

AbstractReview: 147 refs.

Biosensors based on nanomechanical systems

The advances in micro- and nanofabrication technologies enable the preparation of increasingly smaller mechanical transducers capable of detecting the forces, motion, mechanical properties and masses that emerge in biomolecular interactions and fundamental biological processes. Thus, biosensors based on nanomechanical systems have gained considerable relevance in the last decade. This review provides insight into the mechanical phenomena that occur in suspended mechanical structures when either biological adsorption or interactions take place on their surface. This review guides the reader through the parameters that change as a consequence of biomolecular adsorption: mass, surface stress, effective Young's modulus and viscoelasticity. The mathematical background needed to correctly interpret the output signals from nanomechanical biosensors is also outlined here. Other practical issues reviewed are the immobilization of biomolecular receptors on the surface of nanomechanical systems and methods to attain that in large arrays of sensors. We then describe some relevant realizations of biosensor devices based on nanomechanical systems that harness some of the mechanical effects cited above. We finally discuss the intrinsic detection limits of the devices and the limitation that arises from non-specific adsorption.

ChemInform Abstract: Application of NMR Methods to the Study of the Interaction of Natural Products with Biomolecular Receptors

AbstractReview: 74 refs.

Application of NMR methods to the study of the interaction of natural products with biomolecular receptors

In this review, we present applications of NMR spectroscopy as a potent tool for the study of molecular interactions. It is clear that a variety of NMR methods may be employed to deduce key features of ligand–receptor molecular recognition processes, looking at the process from the perspective of the receptor or the ligand. We have not provided an exhaustive review, but we have tried to focus on describing the different aspects within this research topic. We have therefore selected examples accordingly, depending on the particular problem under study or the application/development of protocols to circumvent the technical problems that may be found when working in this field.

Molecular Recognition of Melamine by Vesicles Spontaneously Formed from Orotic Acid Derived Bolaamphiphiles

Molecular recognition by means of multiple hydrogen bonds is of great importance in biological functions. In this paper, an orotic acid derived bolaamphiphile 1,12-diaminododecane diorotate (DDO) with molecular recognition function moieties was designed. Both self-aggregation behavior and molecular recognition with melamine were extensively examined. This bolaamphiphile itself can form vesicles easily in aqueous solutions at 25 °C. Steady-state fluorescence was used to characterize the detailed molecular recognition process. The fluorescence of melamine was quenched more effectively by the spontaneously formed vesicles than by the monomers of the surfactant. Two mechanisms were involved in the fluorescence quench process. At lower concentration, the fluorescence of melamine was found to be quenched by static complex formation. While at higher concentration, both static and dynamic quenching mechanisms coexisted in interaction process. Thermodynamic parameters measured by isothermal titration calorimetry showed that the free energy (ΔG) is negative, indicating that binding of DDO molecules with melamine is favorable energetically. Hydrogen-bonded interactions contribute comparatively a lot for the DDO monomer binding with melamine; at the higher concentration above its critical aggregation concentration, the dissociation of the aggregates take place and lead to an entropically driven molecular recognition process. As complicated binding sites can be constructed through self-assembly at the vesicle interface rather than simple molecular modules, this bolaamphiphile with the molecular recognition functional group may make it possible to generate well-defined recognition sites to mimic biomolecular receptors. Moreover, the present research will give a guide to design chemosensors for melamine detection based on molecular recognition.

Examining the Lateral Displacement of HL60 Cells Rolling on Asymmetric P-Selectin Patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.

Dockomatic - automated ligand creation and docking

BackgroundThe application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI) application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions.ResultsDockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations.ConclusionsDockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

ChemInform Abstract: Bioresponsive Peptide—Inorganic Hybrid Nanomaterials

AbstractReview: 62 refs.

Bioresponsive peptide–inorganic hybrid nanomaterials

Bioanalytical techniques that enable simple, fast and reliable high sensitivity monitoring of biomolecular interactions are of immense importance for diagnostics and drug development. This tutorial review provides an overview of recent progress in the development of peptide-based hybrid nanomaterials that transduce molecular interactions by exploiting the optical and magnetic properties of nanoparticles. Peptides have emerged as an interesting alternative to conventional biomolecular receptors, such as antibodies, and are facilitating the design of responsive hybrid nanomaterials that are both robust and sensitive for biodiagnostic applications.

How Do Halogen Substituents Contribute to Protein‐Binding Interactions? A Thermodynamic Study of Peptide Ligands with Diverse Aryl Halides

Hallowed halogens: Ligands with fluorine, chlorine, bromine and iodine can often display enhanced binding affinity for their biomolecular receptors. The underlying thermodynamic driving forces, however, have rarely been studied. Using calorimetry and a series of aryl halide-containing peptides that bind a PDZ domain protein, we examined how binding free energy, enthalpy and entropy are perturbed when the position and identity of halogen substituents are varied.

Automated analytical microarrays: a critical review.

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. FigureMCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays

Label-free optical biosensor on a single chip

The healthcare and pharmaceutical sectors continuously demand more powerful analytical and diagnostic tools for the identification of disease, the development of new medicines, and better diagnostic tests. High-capability optical biosensing systems are emerging as a way to achieve these goals. Based on the combination of optical materials and photonic components, optical sensing offers many advantages. For instance, the technology is non-invasive, immune to electromagnetic interference, and the risk of electrical shocks or explosions is nonexistent. A key enabler for these systems is the development of costeffective miniaturization technology, which permits the efficient integration of mechanical, fluidic, and photonic components. In typical biosensors, detection of specific pathogens or proteins begins by immobilizing appropriate bioreceptors on the sensing areas of a chip. When analytes are introduced into those areas, only the target biomolecules will be bound to their corresponding biomolecular receptors. This binding process is usually monitored with commercial analytical techniques that require transduction labeling elements, such as fluorescent dyes or radioactive isotopes, to generate a physically readable signal from a recognition event. However, labeling chemistry is expensive and time-consuming. In contrast, recent progress in biosensing systems, in which biomolecules are unlabeled or unmodified (label-free biosensing), is showing promising results. The sensing transduction signals in optical label-free biosensing platforms are based on small changes in refractive index caused by the immobilization or binding reaction of biomolecules. Planar photonic biosensors have measured biomolecular binding interactions using well-known photonic structures, including surface plasmon resonance, 4 interferometers, resonators, 10 gratings, 12 and photonic crystals. 14 These photonic sensing biochips are attractive because they integrate multiple compact devices on a single chip. But, although they use low cost mature semiconductor technology, planar photonic biochips normally need complex optical coupling systems, such as inverted taper and grating couplers, which make the devices less cost effective. Our vision is to create a highly sensitive, label-free optical biosensing system that is fully compatible with semiconductor technology and can be fabricated and interrogated at wafer or disposable chip level. Our holistic approach combines biophotonic resonant or interferometric sensing cells and advanced micron and sub-micron spot size high-sensitivity optical interrogation technologies. This approach leads to a new generation of high-value biochips that are scalable at the wafer level without the need for complex coupling systems, thus offering an inexpensive solution for integration and packaging. Our optical sensing system uses a tightly focused beam (see Figure 1), which allows in situ measurement of micron and submicron size geometries. In our current work, we have already demonstrated the capability to measure extremely small volumes of analytes (on the order of 0.1fL per hole). 16 This system makes routine screening more cost effective and, because each wafer can integrate hundreds of sensing structures, it is also suitable for performing hundreds of measurements on one or multiple samples with high throughput and on-wafer monitoring. To determine the interactions with receptor biomolecules with more reliability and sensitivity, the system will be tremendously sensitive to refractive index variations: on the order of 10 or even 10 refractive index units (RIUs). This means that direct detection of picomolar or even lower concentrations is feasible. Both observation of the reflectivity profile and magnification of the biophotonic resonant or interferometric sensing cells will facilitate this sensitivity. Label-free optical biosensors are expected to grow most in importance in the healthcare, biomedical, and biopharmaceutical sectors, since they can provide new analytical tools with reduced size as well as facilitate large-scale screening of a very wide range of samples for many different parameters. Our optical biosensing system, which uses mixed technologies, such as op-

Design and performances of immunoassay based on SPR biosensor with magnetic microbeads

A surface plasmon resonance (SPR) biosensor system was developed for immunoassay, based on the conjugates of magnetic microbeads coupling with antibody which could be trapped on the Au film firmly due to the magnetic force. The magnetic microbeads were used as the solid support for the heat shock protein 70 (Hsp 70) antibody and antibody immobilized magnetic microbeads were utilized instead of the single antibody for the determination of Hsp 70. Since the magnetic bead is coated with dextran, the antibodies and some specific biomolecular receptors can be immobilized using a variety of chemical reactions. Compared to traditional antibody immobilization on the sensing film, there is not a covalent link between the Au film and the antibody. There is a great advantage in that sensor can be stripped and reused, and the same chemistry used to derivative dextran-coated SPR sensors can be used for the magnetic bead-coated sensors. The sensing layer was formed well. Different dilution ratios (v/v) of the conjugates result in different detectable ranges. When the dilution ratios of the conjugate are 1:10 and 1:5, the lowest concentrations of Hsp 70 that can be detected are 1.50 and 0.30 μg ml −1, respectively.

Nucleic acid based sensors.

Nucleic acids may be analyte or molecular recognition elements in biosensors. Both aspects merge in the genosensor approach, where detection of special sequences is facilitated by hybridization of a target nucleic acid to a complementary immobilized template. All three roles of nucleic acids in biosensors are discussed and the state of sensor development reviewed. With the invention of evolutionary synthesis strategies applied to nucleic acids new types of biomolecular receptors are accessible. The impact of aptamers and ribozymes on biosensor development is discussed.