Tunable Affinity Research Articles

Characterizing Steric Limitations of the Heme Pocket in the Gas-Binding Tt H-Nox Protein using Site-Specific Incorporation of Unnatural Amino Acids

Heme Nitric Oxide and/or Oxygen (H-NOX) binding proteins are bacterial O2 and/or NO gas-sensing proteins involved in signaling a variety of functions to the cell. Much work has been done to characterize the heme-binding pocket in Thermoanaerobacter tencongensis H-NOX (Tt H-NOX) using site-directed mutagenesis with the 20 naturally occurring amino acids. We aim to further characterize the heme-binding pocket of Tt H-NOX by incorporating unnatural amino acids (UAAs) into the H-NOX scaffold, shedding light on both ligand discrimination and the tuning of ligand affinity. Currently, we are working to understand the steric limitations in this pocket by incorporating halogenated phenylalanine residues and characterizing the spectroscopic, gas-binding, and structural properties of these proteins.

Central metal ion exchange in a coordination polymer based on lanthanide ions and di(2-ethylhexyl)phosphoric acid: Exchange rate and tunable affinity

In this paper the exchange of lanthanide(III) ions (Ln3+) between a solution and a coordination polymer (CP) of di(2-ethylhexyl)phosphoric acid (Hdehp), [Ln(dehp)3], is studied. Kinetic and selectivity studies suggest that a polymeric network of [Ln(dehp)3] has different characteristics than the corresponding monomeric complex. The reaction rate is remarkably slow and requires over 600h to reach in nearly equilibrium, and this can be explained by the polymeric crystalline structure and high valency of Ln3+. The affinity of the exchange reaction reaches a maximum with the Ln3+ possessing an ionic radius 7% smaller than that of the central Ln3+, therefore, the affinity of the [Ln(dehp)3] is tunable based on the choice of the central metal ion. Such unique affinity, which differs from the monomeric complex, can be explained by two factors: the coordination preference and steric strain caused by the polymeric structure. The latter likely becomes predominant for Ln3+ exchange when the ionic radius of the ion in solution is smaller than the original Ln3+ by more than 7%. Structural studies suggest that the incoming Ln3+ forms a new phase though an exchange reaction, and this could plausibly cause the structural strain.

Evaluating performance of MARTe as a real-time framework for feed-back control system at tokamak device

The Korea Super conducting Tokamak Advanced Research (KSTAR) is performing the task of “Demonstration and Evaluation of ITER CODAC Technologies at KSTAR” whose objective is the evaluation of real-time technologies for decision making on real-time operating systems (RTOS), real-time frameworks and 10GbE networks. In this task, the Multi-threaded Application Real-Time executor (MARTe) has been evaluated as a real-time framework for real-time feedback control system.The performance of MARTe has been verified by measuring response time and jitter in a path of feedback control from an analog input of a monitoring system to an analog output of an actuator system. In addition, the evaluation has been performed in terms of applicability of MARTe and its performance depending on types of operating system and tuning of CPU affinity and priority.This paper describes the overview of MARTe as a real-time framework, the results of evaluation performance and its implementation.

Multivalent Protein Binding Kinetics of Variable Lymphocyte Receptors Probed using Dynamic Force Spectroscopy

The binding of an antibody to a target antigen underpins an array of biological warfare agent detection schemes and pharmaceutical therapies. However, current antibody-based detection and treatments suffer from multiple drawbacks, including the lack of an antibody that offers a range of affinities for a target antigen. Here, we measure the binding kinetics of a recently discovered class of antibody derived from jawless vertebrates called Variable Lymphocyte Receptors (VLRs), which can achieve IgG-like binding specificity. We first tether low densities of mono-, di-, and pentavalent VLRs to an atomic force microscope cantilever via polyethylene glycol linkers, and then measure the forces required to rupture their bonds with a substrate tethered anthrax antigen. We find that in all three cases the interactions are load-rate dependent - probed far from equilibrium - over the entire range of load rates for which our apparatus is capable. As expected, binding affinity is enhanced in a noncooperative fashion with increasing VLR valency. Furthermore, for the di- and pentavalent molecules, the at-surface dwell time is varied, with longer dwell times increasing the probability of states in which multiple VLR binding domains of a single VLR protein are complexed with surface bound BclA. Thus, through single molecule force spectroscopy, VLRs with greater valency show increased binding stability and lower dissociation rates, and therefore tunable affinity, while the nature of how the binding state probabilities vary with time provides insight into the intermediate binding state kinetics of this system.

Modulation of Intersubunit Interactions during tRNA Translocation through the Ribosome

During protein synthesis, the ribosome undergoes large-scale conformational changes. In particular, the process of tRNA translocation is accompanied by concerted rotations of the 30S ribosomal subunit of more than 20 degrees relative to the 50S. At each step of the translocation process, affinity between the two independent subunits must be finely tuned in order to allow such high conformational flexibility while still maintaining integrity of the ribosomal complex. How the ribosome achieves this is still not fully understood. We address this question by extensive all-atom, explicit solvent molecular dynamics simulations (total simulation time > 1.5 μs) of the 70S ribosome starting from 13 distinct translocation intermediates. The resulting structural ensemble provides a detailed picture of translocation dynamics. Analysis of the trajectories at the residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and located close to the axis of rotation. ii) state specific contacts, seen mostly on the periphery. The baseline of the interaction energy needed to maintain the ribosomal assembly is provided by class i) interactions, while the rupture and formation of class ii) contacts contributes low changes to the overall interaction energy and may serve as an affinity tuning mechanism in switching between different translocation states. Our simulations reveal decreased amplitude of the intersubunit rotations and weakening of stabilizing peripheral contacts upon removal of the tRNAs. This finding is confirmed by cryo-EM analysis of tRNA depleted ribosomes.

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues.

Fine Tuning of Calmodulin's Affinity for Ca2+ by Target Proteins

Calmodulin (CaM) is a prototypical and versatile Ca2+ sensor. CaM is present in all eukaryotic cells, mediating Ca2+-dependent signaling. Upon binding Ca2+, CaM changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on CaM, little is known on how target proteins regulate CaM's ability to bind Ca2+. Recently we identified a novel splice variant (KCNN2-b) of KCNN channels which shows different Ca2+ sensitivity compared to the original KCNN2-a. KCNN2-b has three additional amino acid residues in the CaM binding domain (CaMBD). We take advantage of the CaMBDs from these splice variants to investigate their impact on CaM. Crystal structures show that interactions with these KCNN2 splice variants force CaM to adopt different conformations and thus change its affinity for Ca2+ at its C-lobe. All four EF-hands of CaM in the CaM-CaMBD2-b complex are occupied by Ca2+ ions, whereas in the CaM-CaMBD2-a complex only EF-hands at CaM N-lobe are loaded. Experiments with mutant CaMs support this difference in Ca2+ binding stoichiometry. Mutations of the EF hands at CaM N-lobe (CaM12) almost abolished the interaction with both CaMBDs. The mutant CaM unable to bind Ca2+ at C-lobe (CaM34) interacts with CaMBD2-a substantially, with a similar amplitude compared to wild type CaM. The thermodynamic profile of CaM34-CaMBD2-b, however, is distinct from that of the wild type CaM-CaMBD2-b. This difference in metal ion binding stoichiometry is also proven in solution by Tb3+ Fluorescence measurements. We further manipulated the three residues insertion by shortening it to two residues (CaMBD2-AlaAla) or one residue (CaMBD2-Ala). Different metal ion binding stoichiometry and Ca2+ sensitivity was observed between CaM complexes with these two mutant CaMBDs, which furthers our understanding on the fine-tuning of CaM's affinity for Ca2+ by target proteins.

Modular Hydrogels for Drug Delivery

The development of novel drug delivery systems is an essential step toward controlled site-specific administration of therapeutics within the body. It is desirable for delivery vehicles to be introduced into the body through minimally invasive means and, these vehicles should be capable of releasing drug to their intended location at a controlled rate. Furthermore, it is desirable to develop drug delivery systems that are capable of in vivo to suffer degradation and to deliver the drug completely, avoiding the need to surgically remove the vehicle at the end of its useful lifetime. Hydrogels are of particular interest for drug delivery applications due to their ability to address these needs in addition to their good biocompatibility, tunable network structure to control the diffusion of drugs and, tunable affinity for drugs. However, hydrogels are also limited for drug delivery applications due to the often quick elution of drug from their highly swollen polymer matrices as well as the difficulty inherent in the injection of macroscopic hydrogels into the body. This paper presents an overview to the advances in hydrogels based drug delivery. Different types of hydrogels can be used for drug delivery to specific sites in the gastrointestinal tract ranging from the oral cavity to the colon. These novel systems exhibit a range of several peculiar properties which make them attractive as controlled drug release formulations. Moreover, such materials are biocompatible and can be formulated to give controlled, pulsed, and triggered drug release profiles in a variety of tissues.

Ionic and Polyampholyte N-Isopropylacrylamide-Based Hydrogels Prepared in the Presence of Imprinting Ligands: Stimuli-Responsiveness and Adsorption/Release Properties

The conformation of the imprinted pockets in stimulus-responsive networks can be notably altered when the stimulus causes a volume phase transition. Such a tunable affinity for the template molecule finds interesting applications in the biomedical and drug delivery fields. Nevertheless, the effect that the binding of the template causes on the stimuli-responsiveness of the network has barely been evaluated. In this work, the effect of two ionic drugs used as templates, namely propranolol hydrochloride and ibuprofen sodium, on the responsiveness of N-isopropylacrylamide-based hydrogels copolymerized with acrylic acid (AAc) and N-(3-aminopropyl) methacrylamide (APMA) and on their ability to rebind and to control the release of the template was evaluated. The degree of swelling and, in some cases, energetics (HS-DSC) of the transitions were monitored as a function of temperature, pH, and concentration of drug. Marked decrease in the transition temperature of the hydrogels, accompanied by notable changes in the transition width, was observed in physiological NaCl solutions and after the binding of the drug molecules, which reveals relevant changes in the domain structure of the hydrogels as the charged groups are shielded. The ability of the hydrogels to rebind propranolol or ibuprofen was quantified at both 4 and 37 °C and at two different drug concentrations, in the range of those that cause major changes in the network structure. Noticeable differences between hydrogels bearing AAc or APMA and between imprinted and non-imprinted networks were also observed during the release tests in NaCl solutions of various concentrations. Overall, the results obtained evidence the remarkable effect of the template molecules on the responsiveness of intelligent imprinted hydrogels.

Fabrication of a CO 2-selective membrane by stepwise liquid-phase deposition of an alkylether functionalized pillared-layered metal-organic framework [Cu 2L 2P] n on a macroporous support

Metal-organic framework (MOF) membranes were prepared by stepwise deposition of reactants. Two pillared layered MOFs with the general formula [Cu 2L 2P] n (L = dicarboxylate linker, P = pillaring ligand) were selected. Within this family, fine tuning of adsorption affinity and pore size is possible by variation or functionalization of the L and P linkers. Compound 1 was chosen to be non-polar (L = 1,4-naphtalenedicarboxylate = ndc, P = 1,4-diazabicyclo(2.2.2)octane = dabco); in contrast, compound 2 included a polar linker L with two conformationally flexible ether side chains (L = 2,5-bis(2-methoxyethoxy)-1,4-benzene-dicarboxylate = BME-bdc, P = dabco). The polar functionalization is expected to increase the framework affinity for CO 2 compared to CH 4. The step-by-step, liquid phase deposition of 1 and 2 resulted in pore-plugging of macroporous ceramic supports. The performances of the two MOF membranes were evaluated in gas separation experiments of equimolar CO 2/CH 4 mixtures using a modified Wicke–Kallenbach technique. Anti-Knudsen CO 2/CH 4 separation factors in the range of ∼4–4.5 were obtained for the membrane consisting of the polar 2, whereas the separation of the membrane formed from the non-polar 1 was found to be Knudsen-like.

Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ∼0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.

Microporous organic polymers for carbon dioxide capture

Anthropogenic carbon dioxide emissions are thought to be one cause of global warming. Current methods for CO2 capture result in large energy penalties. Solid adsorbents are a potential method to capture CO2, but the sorbent-sorbate affinity is critical in determining the energetic viability of such processes. In this study, the adsorption of CO2 in a range of microporous organic polymers was tested. These materials adsorb up to 2.20 mmol/g CO2 at 298 K and 1 bar, and thus performance is compared with related porous solids in the literature. The relationship between CO2 uptake and apparent surface area and pore size is described, and this showed that heats of adsorption were important in the low pressure regime. The chemical tuning of gas-sorbent affinity provides a blueprint for the development of improved materials in this area.

Quantitative analysis of multivalent ligand presentation on gold glyconanoparticles and the impact on lectin binding.

Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide an excellent platform for sensitive protein recognition. Using nanomaterials as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, the quantitative analysis of the binding affinity of these glyconanomaterials has been lacking. In this Article, we report a new method to measure the binding affinity of glyconanoparticle (GNP)-protein interactions based on a fluorescent competition binding assay, which yielded the apparent dissociation constant (K(d)) of GNPs with the interacting protein. Au nanoparticles conjugated with underivatized mono-, oligo-, and polysaccharides were synthesized using our recently developed photocoupling chemistry. The affinities of these GNPs with lectins were measured and were several orders of magnitude higher than the corresponding free ligands with lectins. The effect of ligand display on the binding affinity of GNPs was, furthermore, studied where GNPs of varying linker type, spacer length, ligand density, and nanoparticle size were prepared and K(d) values determined. The long spacer linker containing hydrocarbon and ethylene oxide units gave the highest binding affinity as well as assay sensitivity. The binding affinity increased with ligand density in general, showing a drastic increase in affinity at low ligand density. In addition, the affinity enhancement was more pronounced on smaller NPs than the larger ones. These results not only demonstrate that the binding affinity of GNPs is highly influenced by how the ligands are presented on the nanoparticles but also pave the way for tailor-made glyconanomaterials with tunable affinity by way of ligand display.

Clickable peptide nucleic acids (cPNA) with tunable affinity

Peptide nucleic acids (PNAs) are functional analogues of natural oligonucleotides. Herein, we report the synthesis of PNAs bearing a triazole in lieu of the amide bond assembled using a "click" cycloaddition, their hybridization properties as well as the DNA-templated coupling of the azide and alkyne PNA fragments.

Hydrodynamically Tunable Affinities for Fluidic Assembly

Most current micro- and nanoscale self-assembly methods rely on static, preprogrammed assembly affinities between the assembling components such as capillarity, DNA base pair matching, and geometric interactions. While these techniques have proven successful at creating relatively simple and regular structures, it is difficult to adapt these methods to enable dynamic reconfiguration of the structure or on-the-fly error correction. Here we demonstrate a technique to hydrodynamically tune affinities between assembling components by direct thermal modulation of the local viscosity field surrounding them. This approach is shown here for two-dimensional silicon elements of 500 microm length using a thermorheological fluid that undergoes reversible sol-gel transition on heating. Using this system, we demonstrate the ability to dynamically change the assembly point in a fluidic self-assembly process and selectively attract and reject elements from a larger structure. Although this technique is demonstrated here for a small number of passive mobile components around a fixed structure, it has the potential to overcome some of the limitations of current static affinity based self-assembly.

Exploring the Water-Soluble Phosphine Ligand as the Environmentally Friendly Stabilizer for Electroless Nickel Plating

Phosphine (R3P) compounds being a typical type of coordination ligand used in homogeneous catalysis are known to possess a tunable affinity with the nickel atom. As such, it deserves to be studied ...

The Role of Ca2+ Signaling in Cell Function with Special Reference to Exocrine Secretion

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that induces the release of calcium from the endoplasmic reticulum (ER). The IP3 receptor was discovered as a developmentally regulated glycophosphoprotein, P400, that is absent in strains of mutant mice. The crystal structures of the IP3-binding core and N-terminal suppressor sequence of the IP3 receptor have been identified. The IP3-binding core's affinity to IP3 is similar among the three isoforms of IP3 receptors; however, the N-terminal IP3-binding suppressor region is responsible for isoform-specific IP3-binding affinity tuning. Various pathways for the trafficking of IP3 receptors have been identified; for example, the ER forms a meshwork on which IP3 receptors move by lateral diffusion, and vesicular ER subcompartments containing IP3 receptors move rapidly along microtubules using a kinesin motor. Furthermore, IP3 receptor messenger RNA within messenger RNA granules also moves along microtubules. Recently, we discovered that IP3 receptors play a crucial role in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP3 type 1 receptor activity. IP3 receptor also releases IP3 receptor-binding protein released with IP3 (IRBIT). IRBIT is a pseudoligand for IP3 that regulates the frequency and amplitude of calcium oscillations through the IP3 receptor. IRBIT binds to pancreas-type sodium bicarbonate cotransporter 1, which is important for acid-base balance. Type 2 and 3 double-deficient mice show a deficit in saliva and lacrimal and pancreatic juice secretion. Type 1 IP3 receptor influences brain-derived neurotrophic factor production.

Tuning the Electron Affinity and Secondary Electron Emission of Diamond (100) Surfaces by Diels−Alder Reaction

The tuning of electron affinity and secondary electron emission on diamond (100) surfaces due to cycloaddition with 1,3-butadiene is investigated by photoemission experiments and density functional theory (DFT) calculations. A significant reduction in electron affinity up to 0.7 eV and enhancement of secondary electron emission were observed after 1,3-butadiene adsorption. The lowering of vacuum level via 1,3-butadiene adsorption is supported by DFT calculations. The C-H bonds in the covalently bonded organics on diamond contribute to the enhanced secondary electron emission and reduced electron affinity in a mechanism similar to that of C-H bonds on hydrogenated diamond surfaces. This combination of strong secondary emission and low electron affinity by the organic functionalization of diamond has potential applications in diamond-based molecular electronic devices.

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.

Variation of Linker Length in Ratiometric Fluorescent Sensor Proteins Allows Rational Tuning of Zn(II) Affinity in the Picomolar to Femtomolar Range

Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP−Atox1 and WD4−YFP, using a series of flexible peptide linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quantitative understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity.