Biomolecular Activity Research Articles

Zebrafish as an in Vivo Screen for Early Black Cranberry Proanthocyanidin Biomolecular Activity

Antioxidants have been widely studied in various naturally occurring substances as a bioavailable cancer prevention treatment. Proanthocyanidins (PACs), which are abundant polyphenols in Early Black (EB) Cranberry (Vaccinium macrocarpon), are readily available and we have shown their anticancer activity in several cancer cell lines. This work focused on the activity of these compounds when incorporated into the zebrafish (Danio rerio) system. We began investigating the in vivo effect of these phytochemicals, the protective role of several other cranberry compounds, and the metabolic activity of the vertebrate model organism. Proanthocyanidin fractions were separated from fresh EB Cranberry fruit by chromatography on Sephadex LH-20 in order to acquire a workable stock solution in DMSO. Various concentrations of proanthocyanidins in solution were tested against fish ranging in age from 1-cell stage to adult level of growth. Acridine orange apoptosis indicator dye was incorporated into the treatment protocol, and it was observed that irregular epithelial cell death was occurring in treated embryos but not in the control group. Further apoptosis assays were carried out utilizing Dihydroethidium (DHE) superoxide sensitive dye in the treatment protocol. Fluorescing red nuclei were visible along the outer surface of the epithelium cell layer; an indication of superoxide release within cells leading to the nicking of DNA within the nucleus. It was also possible to screen for superoxide release in PACs treated CCD-CO18 and HT-29 cells using confocal microscopy and cell apoptosis was investigated by trypan blue cytotoxicity assay; cell apoptosis results were statistically significant as confirmed by ANOVA analysis. Results indicate that the phytochemicals may induce apoptosis in rapidly dividing cells.

Molecular docking studies for the identification of novel melatoninergic inhibitors for acetylserotonin-O-methyltransferase using different docking routines

BackgroundN-Acetylserotonin O-methyltransferase (ASMT) is an enzyme which by converting nor-melatonin to melatonin catalyzes the final reaction in melatonin biosynthesis in tryptophan metabolism pathway. High Expression of ASMT gene is evident in PPTs. The presence of abnormally high levels of ASMT in pineal gland could serve as an indication of the existence of pineal parenchymal tumors (PPTs) in the brain (J Neuropathol Exp Neurol 65: 675–684, 2006). Different levels of melatonin are used as a trait marker for prescribing the mood disorders e.g. Seasonal affective disorder, bipolar disorder, or major depressive disorder. In addition, melatonin levels can also be used to calculate the severity of a patient’s illness at a given point in time.MethodsSeventy three melatoninergic inhibitors were docked with acetylserotonin-O-methyltransferase in order to identify the potent inhibitor against the enzyme. The chemical nature of the protein and ligands greatly influence the performance of docking routines. Keeping this fact in view, critical evaluation of the performance of four different commonly used docking routines: AutoDock/Vina, GOLD, FlexX and FRED were performed. An evaluation criterion was based on the binding affinities/docking scores and experimental bioactivities.Results and conclusionResults indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for its ligand binding and the compound selected as potent inhibitor is having minimum binding affinity, maximum GoldScore and minimum FlexX energy. The correlation value of r2 = 0. 66 may be useful in the selection of correct docked complexes based on the energy without having prior knowledge of the active site. This may lead to further understanding of structures, their reliability and Biomolecular activity especially in connection with bipolar disorders.

LASSO-ing Potential Nuclear Receptor Agonists and Antagonists: A New Computational Method for Database Screening

Nuclear receptors (NRs) are important biological macromolecular transcription factors that are implicated in multiple biological pathways and may interact with other xenobiotics that are endocrine disruptors present in the environment. Examples of important NRs include the androgen receptor (AR), estrogen receptors (ER), and the pregnane X receptor (PXR). In this study we have utilized the Ligand Activity by Surface Similarity Order (LASSO) method, a ligand-based virtual screening strategy to derive structural (surface/shape) molecular features used to generate predictive models of biomolecular activity for AR, ER, and PXR. For PXR, twenty-five models were built using between 8 to 128 agonists and tested using 3000, 8000, and 24,000 drug-like decoys including PXR inactive compounds(N=228). Preliminary studies with AR and ER using LASSO suggested the utility of this approach with 2-fold enrichment factors at 20%. We found that models with 64–128 PXR actives provided enrichment factors of 10-fold (10% actives in the top 1% of compounds screened). The LASSO models for AR and ER have been deployed and are freely available online, and they represent a ligand-based prediction method for putative NR activity of compounds in this database.

Method Development for Metaproteomic Analyses of Marine Biofilms

The large-scale identification and quantitation of proteins via nanoliquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate (SDS) protein extraction + 1D gel LC; phenol protein extraction + 1D gel LC] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85-15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (≥1 peptide) or 54.7% (≥2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.

Neural Regenerative Strategies Incorporating Biomolecular Axon Guidance Signals

There are currently no acceptable cures for central nervous system injuries, and damage induced large gaps in the peripheral nervous system have been challenging to bridge to restore neural functionality. Innervation by neurons is made possible by the growth cone. This dynamic structure is unique to neurons, and can directly sense physical and chemical activity in its environment, utilizing these cues to propel axons to precisely reach their targets. Guidance can occur through chemoattractive factors such as neurotrophins and netrins, chemorepulsive agents like semaphorins and slits, or contact-mediated molecules such as ephrins and those located in the extracellular matrix. The understanding of biomolecular activity during nervous system development and injury has generated new techniques and tactics for improving and restoring function to the nervous system after injury. This review will focus on the major neuronal guidance molecules and their utility in current tissue engineering and neural regenerative strategies.

Prebiotic Significance of Extraterrestrial Ice Photochemistry: Detection of Hydantoin in Organic Residues

The delivery of extraterrestrial organic materials to primitive Earth from meteorites or micrometeorites has long been postulated to be one of the origins of the prebiotic molecules involved in the subsequent apparition of life. Here, we report on experiments in which vacuum UV photo-irradiation of interstellar/circumstellar ice analogues containing H(2)O, CH(3)OH, and NH(3) led to the production of several molecules of prebiotic interest. These were recovered at room temperature in the semi-refractory, water-soluble residues after evaporation of the ice. In particular, we detected small quantities of hydantoin (2,4-imidazolidinedione), a species suspected to play an important role in the formation of poly- and oligopeptides. In addition, hydantoin is known to form under extraterrestrial, abiotic conditions, since it has been detected, along with various other derivatives, in the soluble part of organic matter of primitive carbonaceous meteorites. This result, together with other related experiments reported recently, points to the potential importance of the photochemistry of interstellar "dirty" ices in the formation of organics in Solar System materials. Such molecules could then have been delivered to the surface of primitive Earth, as well as other telluric (exo-) planets, to help trigger first prebiotic reactions with the capacity to lead to some form of primitive biomolecular activity.

Polyethylene Glycol Grafted Polyethylene: A Versatile Platform for Nonmigratory Active Packaging Applications

Nonmigratory active packaging, in which bioactive components are tethered to the package, offers the potential to reduce the need for additives in food products while maintaining safety and quality. A challenge in developing nonmigratory active packaging materials is the loss of biomolecular activity that can occur when biomolecules are immobilized. In this work, we describe a method in which a biocompatible polymer (polyethylene glycol, PEG) is grafted from the surface of ozone-treated low-density polyethylene (LDPE) resulting in a surface functionalized polyethylene to which a range of amine-terminated bioactive molecules can be immobilized. Free radical graft polymerization is used to graft PEG onto the LDPE surface, followed by immobilization of ethylenediamine onto the PEG tether. Ethylenediamine was used to demonstrate that amine-terminated molecules could be covalently attached to the PEG-grafted film. Changes in surface chemistry and topography were measured by attenuated total reflectance Fourier transform infrared spectroscopy, contact angle, atomic force microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. We demonstrate the ability to graft PEG onto the surface of polymer packaging films by free radical graft polymerization, and to covalently link an amine-terminated molecule to the PEG tether, demonstrating that amine-terminated bioactive compounds (such as peptides, enzymes, and some antimicrobials) can be immobilized onto PEG-grafted LDPE in the development of nonmigratory active packaging. Nonmigratory active packaging offers the potential for improving food safety and quality while minimizing the migration of the active agent into food. In this paper, we describe a technique to modify polyethylene packaging films such that active agents can be covalently immobilized by a biocompatible tether. Such a technique can be adapted to a number of applications such as antimicrobial, antioxidant, or immobilized enzyme active packaging.

ChemInform Abstract: Capturing Protein Structural Kinetics by Mass Spectrometry

AbstractReview: 63 refs.

The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

Nuclear Magnetic Resonance Provides a Quantitative Description of Protein Conformational Flexibility on Physiologically Important Time Scales

A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years, novel nuclear magnetic resonance-based techniques that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important time scales have emerged. Residual dipolar couplings (RDCs) provide precise information about time- and ensemble-averaged structural and dynamic processes with correlation times up to the millisecond and thereby encode key information for understanding biological activity. In this review, we present the application of two very different approaches to the quantitative description of protein motion using RDCs. The first is purely analytical, describing backbone dynamics in terms of diffusive motions of each peptide plane, using extensive statistical analysis to validate the proposed dynamic modes. The second is based on restraint-free accelerated molecular dynamics simulation, providing statistically sampled free energy-weighted ensembles that describe conformational fluctuations occurring on time scales from pico- to milliseconds, at atomic resolution. Remarkably, the results from these two approaches converge closely in terms of distribution and absolute amplitude of motions, suggesting that this kind of combination of analytical and numerical models is now capable of providing a unified description of protein conformational dynamics in solution.

Coupled folding and specific binding: fishing for amphiphilicity.

Proteins are uniquely capable of identifying targets with unparalleled selectivity, but, in addition to the precision of the binding phenomenon, nature has the ability to find its targets exceptionally quickly. Transcription factors for instance can bind to a specific sequence of nucleic acids from a soup of similar, but not identical DNA strands, on a timescale of seconds. This is only possible with the enhanced kinetics provided for by a natively disordered structure, where protein folding and binding are cooperative processes. The secondary structures of many proteins are disordered under physiological conditions. Subsequently, the disordered structures fold into ordered structures only when they bind to their specific targets. Induced folding of the protein has two key biological advantages. First, flexible unstructured domains can result in an intrinsic plasticity that allows them to accommodate targets of various size and shape. And, second, the dynamics of this folding process can result in enhanced binding kinetics. Several groups have hypothesized the acceleration of binding kinetics is due to induced folding where a “fly-casting” effect has been shown to break the diffusion-limited rate of binding. This review describes experimental results in rationally designed peptide systems where the folding is coupled to amphiphilicity and biomolecular activity.

Effect of oxidized silicon (SiOx) surfaces functionalization on real‐time PCR by Lab‐on‐a‐chip microdevices

There is a great need to improve the biocompatibility of silicon‐based lab‐on‐chip substrate materials for reliable quantitative analysis of biological solutions. These advanced microdevice surfaces need not only be biocompatible but also have surfaces of defined wettability characteristics. The inhibition of biomolecular activity due to microdevice surface interaction is common and can result in inaccurate results or decreased reaction yields. In this work we investigate different techniques for the chemical functionalization of oxidized silicon (SiOx) surfaces in order to: (i) obtain defined hydrophobic/hydrophilic surfaces; and (ii) increase the efficiency of performing Real‐Time Polymerase Chain Reaction (PCR) on a silicon‐based lab‐on‐chip. Silicon oxide surfaces are functionalized by grafting alkylic chain silanes and poly(ethylene glycol) (PEG) chains to the surfaces, rendering them hydrophobic or hydrophilic. Functionalized surfaces are characterized through contact angle and atomic force microscopy (AFM) measurements, showing stable hydrophobic surfaces with contact angles of 69–78° and layer thicknesses of 11–15 Å and hydrophilic surfaces displaying contact angles of 5–6° and thicknesses of 22–52 Å. PCR experiments carried out directly on bare silicon oxide lab‐on‐chip surfaces show low yields of DNA amplification. Hydrophobic surfaces decrease the inhibition of PCR. Hydrophilic surfaces are a major improvement on the bare silicon oxide exhibiting the same maximum reaction yield as obtained with a standard thermocycler. We have found that the best results are associated with PEG modified surfaces, which prove very suitable for the fabrication of reliable PCR silicon lab‐on‐chips. Copyright © 2011 John Wiley & Sons, Ltd.

Capturing protein structural kinetics by mass spectrometry

Precise knowledge of the three-dimensional structure of a protein is critical, if we are to understand its biological role and mode of action. However, today it is becoming increasingly clear that dissecting the protein's structural architecture is not enough: a complete description of biomolecular activity must also include the dimension of time. Protein motion and dynamics are crucial for protein stability and reactivity. A range of techniques have been developed for probing dynamic processes. In this tutorial review, we focus on one of these approaches--structural mass spectrometry (MS). MS has the ability to capture functional conformational transitions in the slow time regime, from a few milliseconds to hours. The power of this approach lies not only in its sensitivity and speed of analysis, but also in the fact that it is a non-ensemble technique. Thus, within a single spectrum, the entire distribution of co-existing states can be resolved. In discussing the challenges, advantages and limitations of the field, as well as future directions, we highlight the applicability of MS for quantitative monitoring of structural kinetics. In particular, we describe the array of MS-based strategies that are available for capturing protein folding, enzymatic reactions, ligand interactions, subunit exchange and biogenesis pathways.

A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

We present a microfluidic approach for single-molecule studies of the temperature-dependent behavior of biomolecules, using a platform that combines microfluidic sample handling, on-chip temperature control, and total internal reflection fluorescence (TIRF) microscopy of surface-immobilized biomolecules. With efficient, rapid, and uniform heating by microheaters and in situ temperature measurements within a microfluidic flowcell by micro temperature sensors, closed-loop, accurate temperature control is achieved. To demonstrate its utility, the temperature-controlled microfluidic flowcell is coupled to a prism-based TIRF microscope and is used to investigate the temperature-dependence of ribosome and transfer RNA (tRNA) structural dynamics that are required for the rapid and precise translocation of tRNAs through the ribosome during protein synthesis. Our studies reveal that the previously characterized, thermally activated transitions between two global conformational states of the pre-translocation (PRE) ribosomal complex persist at physiological temperature. In addition, the temperature-dependence of the rates of transition between these two global conformational states of the PRE complex reveal well-defined, measurable, and disproportionate effects, providing a robust experimental framework for investigating the thermodynamic activation parameters that underlie transitions across these barriers.

Engineered Neuronal Circuits: A New Platform for Studying the Role of Modular Topology

Neuron–glia cultures serve as a valuable model system for exploring the bio-molecular activity of single cells. Since neurons in culture can be conveniently recorded with great fidelity from many sites simultaneously, it has long been suggested that uniform cultured neurons may also be used to investigate network-level mechanisms pertinent to information processing, activity propagation, memory, and learning. But how much of the functionality of neural circuits can be retained in vitro remains an open question. Recent studies utilizing patterned networks suggest that they provide a most useful platform to address fundamental questions in neuroscience. Here we review recent efforts in the realm of patterned networks’ activity investigations. We give a brief overview of the patterning methods and experimental approaches commonly employed in the field, and summarize the main results reported in the literature. The general picture that emerges from these reports indicates that patterned networks with uniform connectivity do not exhibit unique activity patterns. Rather, their activity is very similar to that of unpatterned uniform networks. However, by breaking the connectivity homogeneity, using a modular architecture, it is possible to introduce pronounced topology-related gating and delay effects. These findings suggest that patterned cultured networks may serve as a new platform for studying the role of modularity in neuronal circuits.

Reversible dioxygen binding in solvent-free liquid myoglobin

The ensemble of forces that stabilize protein structure and facilitate biological function are intimately linked with the ubiquitous aqueous environment of living systems. As a consequence, biomolecular activity is highly sensitive to the interplay of solvent-protein interactions, and deviation from the native conditions, for example by exposure to increased thermal energy or severe dehydration, results in denaturation and subsequent loss of function. Although certain enzymes can be extracted into non-aqueous solvents without significant loss of activity, there are no known examples of solvent-less (molten) liquids of functional metalloproteins. Here we describe the synthesis and properties of room-temperature solvent-free myoglobin liquids with near-native structure and reversible dioxygen binding ability equivalent to the haem protein under physiological conditions. The realization of room-temperature solvent-free myoglobin liquids with retained function presents novel challenges to existing theories on the role of solvent molecules in structural biology, and should offer new opportunities in protein-based nanoscience and bionanotechnology.

Orientation difference of chemically immobilized and physically adsorbed biological molecules on polymers detected at the solid/liquid interfaces in situ.

A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.

Single-quantum dot imaging with a photon counting camera.

The expanding spectrum of applications of single-molecule fluorescence imaging ranges from fundamental in vitro studies of biomolecular activity to tracking of receptors in live cells. The success of these assays has relied on progress in organic and non-organic fluorescent probe developments as well as improvements in the sensitivity of light detectors. We describe a new type of detector developed with the specific goal of ultra-sensitive single-molecule imaging. It is a wide-field, photon-counting detector providing high temporal and high spatial resolution information for each incoming photon. It can be used as a standard low-light level camera, but also allows access to a lot more information, such as fluorescence lifetime and spatio-temporal correlations. We illustrate the single-molecule imaging performance of our current prototype using quantum dots and discuss on-going and future developments of this detector.

Dynamic control of biomolecular activity using electrical interfaces.

The development of novel interfaces between electronic devices and biological systems is a rapidly evolving research area that may lead to new insights into biological behavior, clinical diagnostics and therapeutic treatments. Full electrical integration into biological networks will require bioactuators which can translate an electrical pulse into a specific biochemical signal the system can understand. One approach has been the use of electrostatic fields near the surface of an electrode to locally alter the ionic and electrostatic environment within an ionic double layer. In this scheme, normally active biological macromolecules are suspended in a 'low-salt buffer' that is depleted of necessary ions, such as Mg2+, rendering them inactive. Upon application of an electrical potential these ions are concentrated at the electrode surface, locally activating biomolecular function. An initial demonstration of this method is presented for the dynamic polymerization of actin filaments from electrode surfaces. In principle, electrodes functionalized with different proteins could be individually activated to translate an electrical potential into a specific biochemical signal or behavior.

Polymerization of Nanocrystal Quantum Dot–Tubulin Bioconjugates

Nanocrystal quantum dot (NQD)-tubulin bioconjugates were prepared using a two-step crosslinking procedure. NQD-decorated microtubules were successfully polymerized directly from the NQD-tubulin conjugates to form nonaggregated, full-length (several micrometers) biopolymers. However, polymerization kinetics were slowed in comparison with unmodified tubulin and tubulin modified with small-molecule dyes or biotin. Association with the relatively large nanocrystals, therefore, interferes to some extent with tubulin's ability to polymerize. These results suggest that before NQDs are used extensively as fluorescent labels in studies of biomolecular activity, the impact of NQD bioconjugation must be well understood.