Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water*

A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H₂O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.     

The interaction of proteins and cells with affinity ligands covalently coupled to silicon surfaces as monitored by ellipsometry

Two methods for the chemical binding of biomolecules to silicon surfaces are described. The first method utilizes an alkyl silane and a nucleophilic reagent to join the biomolecule to the silicon surface; the second method involves crosslinking with glutaraldehyde in order to couple the biomolecule and albumin molecules, which have first been physically adsorbed. The course of binding to the silicon surface has been followed with the aid of ellipsometry. This optical measuring technique estimates the thicknesses of, e.g., organic layers, by measuring the polarization properties of a light beam before and after reflection at surfaces. The method by which the binding of a biomolecule to its corresponding affinity ligand on silicon wafers can be followed with this technique is reported. The systems studied are concanavalin A-Saccharomyces cerevisiae cells, immunoglobulin G-Staphylococcus aureus cells, and an NAD-analog-lactate dehydrogenase. With ellipsometry it was possible to assess how the incubation time and the concentration of the cells and the biomolecules added influenced the results. It was found that an increasing time of incubation and higher concentration resulted in a more complete coverage of the silicon wafer surfaces.     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

Electrochemical biomemory device consisting of recombinant protein molecules

The new concept micro devices consisting of various biomolecules have been developed in clinical, pharmaceutical, and environmental fields. Particularly, various diagnostics using biomolecule related device have been investigated and commercialized to detect specific molecules in complex matrix. In recent days, biomolecules have been employed to electronic device to generate new alternatives of silicon based nano electronics by applying natural behaviors of biomolecules. We reviewed here the bioelectronic device consisting of proteins developed by mimicking natural phenomena. We surveyed the working principle, fabrication technologies, and memory function validation of metalloprotein based biomemory device.     

Preservation of key biomolecules in the fossil record: current knowledge and future challenges

We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record.     

A hybrid approach to theoretical analysis of bovine pancreatic trypsin inhibitor.

A static analysis of bovine pancreatic trypsin inhibitor (BPTI) is presented based on a new discrete/continuum approach to modeling the dynamics of biomolecules. This hybrid method utilizes knowledge of the intramolecular potential and molecular configuration to generate a field of elastic modulus tensors. These tensors, which relate the local stress and strain for each atom in the biomolecule, can be used to judge the local rigidity as well as indicate regions of high stress. Comparing the tensor fields for an unrelaxed and a relaxed configuration, the microscopic structure of BPTI is found to be anisotropic and to have regions of stress even when it is relaxed in the potential field. However, when these fields are averaged over the whole protein or over individual residues the structure becomes more isotropic and the stressed regions vanish. Using these averaged tensors, we calculated bulk properties such as Young's modulus and the Lamé constants and they agreed with previously reported values.     

Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls

The labeling of targeting peptides with (99m)Tc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac-[(99m)Tc(CO)(3)](+) core for introducing (99m)Tc in biomolecules in general and in peptides in particular. The three coordination sites available in [(99m)Tc(OH(2))(3)(CO)(3)](+) can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed-ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.     

Analysis of heterogeneity in nonspecific PEGylation reactions of biomolecules

The compositional heterogeneity associated with polymer conjugation reactions of biomolecules is analyzed for the particular case of nonspecific PEGylation reactions. It is shown that the distribution of the number of PEG moieties grafted to biomolecules such as proteins is a binomial‐type function of two parameters—the reaction efficiency as well as the number of binding sites per biomolecule. The nature of this distribution implies that uniform compositions are favored for increasing number of coupling sites per biomolecule as well as for increasing efficiency of the modification process. Therefore, the binomial distribution provides a rationale for the pronounced heterogeneity that is observed for PEGylated small enzyme systems even at high coupling efficiencies. For the particular case of PEGylated trypsin it is shown that the heterogeneity results in a broad distribution of deactivation times that is captured by a stretched exponential decay model. The presented analysis is expected to apply to general modification processes of compounds in which partial functionalization of a fixed number of reactive sites is achieved by means of a nonspecific coupling reaction. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 427–435, 2013.     

Clathrate hydrates are poor models of biomolecule hydration

Clathrate hydrates form the basis of a general model of biomolecule hydration. In clathrate hydrate crystal structures, the size of hydrogen-bonded water rings is highly constrained to five members. The clathrate hydrate model predicts that the size of water rings near biomolecule surfaces is similarly constrained to five members. This report describes a test of this model of biomolecule hydration. We have demonstrated that five-membered water rings are not a general feature of protein or nucleic acid hydration. The clathrate hydrate model appears to be inappropriate for biomolecules. © 1996 John Wiley & Sons, Inc.     

Quantifying Biomolecule Diffusivity Using an Optimal Bayesian Method

We propose a Bayesian method to extract the diffusivity of biomolecules evolving freely or inside membrane microdomains. This approach assumes a model of motion for the particle considered, namely free Brownian motion or confined diffusion. In each framework, a systematic Bayesian scheme is provided for estimating the diffusivity. We show that this method reaches the best performances theoretically achievable. Its efficiency overcomes that of widely used methods based on the analysis of the mean-square displacement. The approach presented here also gives direct access to the uncertainty on the estimation of the diffusivity and predicts the number of steps of the trajectory necessary to achieve any desired precision. Its robustness with respect to noise on the position of the biomolecule is also investigated.     

Biomolecule–nanoparticle interactions: Elucidation of the thermodynamics by isothermal titration calorimetry

BackgroundNanomaterials (NMs) are often exposed to a broad range of biomolecules of different abundances. Biomolecule sorption driven by various interfacial forces determines the surface structure and composition of NMs, subsequently governs their functionality and the reactivity of the adsorbed biomolecules. Isothermal titration calorimetry (ITC) is a nondestructive technique that quantifies thermodynamic parameters through in-situ measurement of the heat absorption or release associated with an interaction.Scope of reviewThis review highlights the recent applications of ITC in understanding the thermodynamics of interactions between various nanoparticles (NPs) and biomolecules. Different aspects of a typical ITC experiment that are crucial for obtaining accurate and meaningful data, as well as the strengths, weaknesses, and challenges of ITC applications to NP research were discussed.Major conclusionsITC reveals the driving forces behind biomolecule–NP interactions and the effects of the physicochemical properties of both NPs and biomolecules by quantifying the crucial thermodynamics parameters (e.g., binding stoichiometry, ΔH, ΔS, and ΔG). Complimentary techniques would strengthen the interpretation of ITC results for a more holistic understanding of biomolecule–NP interactions.General significanceThe thermodynamic information revealed by ITC and its complimentary characterizations is important for understanding biomolecule–NP interactions that are fundamental to the biomedical and environmental applications of NMs and their toxicological effects. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Biocompatible photolithographic process for the patterning of biomolecules

A new approach for the patterning of biomolecule layers is introduced based on the design of a new photoresist material with biocompatible lithographic processing requirements. The photoresist is based on poly(t-butyl acrylate), which allows positive imaging with very dilute basic solutions, tolerable by selected biomolecules used in immunoanalysis. Sensitivity at lambda>300 nm is obtained using a suitable sulfonium salt photoacid generator. Thermal steps also take place under conditions tolerable by biomolecules. Lithographic results on Si wafer substrates show resolution capabilities for equal lines/spaces, down to the range of 5-10 microm under biocompatible conditions. The process is also used on substrates of different geometries, including inner capillary surfaces. The patterning of the inner surface of a polystyrene capillary with mouse IgG is reported to demonstrate the principles of the above approach.     

Brownian dynamics simulations of ion atmospheres around polyalanine and B-DNA: effects of biomolecular dielectric

We have extended an earlier Brownian dynamics simulation algorithm for simulating the structural dynamics of ions around biomolecules to accommodate dielectric inhomogeneity. The electrostatic environment of a biomolecule immersed in water was obtained by numerically solving the Poisson equation with the biomolecule treated as a low dielectric region and the solvent treated as a high dielectric region. Instead of using the mean-field type approximations of ion interactions as in the Poisson-Boltzmann model, the ions were treated explicitly by allowing them to evolve dynamically under the electrostatic field of the biomolecule. This model thus accounts for ion-ion correlations and the finite-size effects of the ions. For a 13-residue alpha-helical polyalanine and a 12-base-pair bp B-form DNA, we found that the choice of the dielectric constant of the biomolecule has much larger effects on the mean ionic structure around the biomolecule than on the fluctuational and dynamical properties of the ions surrounding the biomolecule.     

Two-photon uncaging: New prospects in neuroscience and cellular biology

An uncaging process refers to a fast and efficient release of a biomolecule after photochemical excitation from a photoactivatable precursor. Two-photon excitation produces excited states identical to standard UV excitation while overcoming major limitations when dealing with biological materials, like spatial resolution, tissue penetration and toxicity and has therefore been applied to the uncaging of different biological effectors. A literature survey of two-photon uncaging of biomolecules is described in this article, including applications in cellular- and neurobiology.     

Effect of artificial periodicity in simulations of biomolecules under Ewald boundary conditions: a continuum electrostatics study.

Ewald and related methods are nowadays routinely used in explicit-solvent simulations of biomolecules, although they impose an artificial periodicity in systems which are inherently non-periodic. The consequences of this approximation should be assessed, since they may crucially affect the reliability of computer simulations under Ewald boundary conditions. In the present study we use a method based on continuum electrostatics to investigate the nature and magnitude of possible periodicity-induced artifacts on the potentials of mean force for conformational equilibria in biomolecules. Three model systems and pathways are considered: polyalanine oligopeptides (unfolding), a DNA tetranucleotide (separation of the strands), and the protein Sac7d (conformations from a molecular dynamics simulation). Artificial periodicity may significantly affect these conformational equilibria, in each case stabilizing the most compact conformation of the biomolecule. Three factors enhance periodicity-induced artifacts: (i) a solvent of low dielectric permittivity; (ii) a solute size which is non-negligible compared to the size of the unit cell; and (iii) a non-neutral solute. Neither the neutrality of the solute nor the absence of charge pairs at distances exceeding half the edge of the unit cell do guarantee the absence of artifacts.     

Displacement effects in large-scale chromatography?

Displacement effects in large-scale (total column volume v(t) = 150 L) and preparative ion-exchange chromatography purifying human erythrocyte superoxide dismutase are described in the present article. The biomolecules are eluted in a very small peak elution volume (<0.2 v(o)) behind the salt wave using a step gradient. The theoretical peak width and retention behavior are calculated according to the model of Yamamoto. The theoretical values are then compared with the experimental data. There was a difference observed between the elution type I (also called fronting) and the experimentally obtained elution. Some instructions are given on how to achieve these phenomenona because a beneficial effect in respect to resolution and recovery of a biomolecule is observed.     

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme–electrode contact junction electrode

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5–40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.