Biophysical Instrumentation Research Articles

Fragment-based screening by protein-detected NMR spectroscopy.

Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spacesthat bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.

Genesis of Open States Zones in a DNA Molecule Depends on the Localization and Value of the Torque.

The formation and dynamics of the open states in a double-stranded DNA molecule are largely determined by its mechanical parameters. The main one is the torque. However, the experimental study of DNA dynamics and the occurrence of open states is limited by the spatial resolution of available biophysical instruments. Therefore, in this work, on the basis of a mechanical mathematical model of DNA, calculations of the torque effect on the process of occurrence and dynamics of open states were carried out for the interferon alpha 17 gene. It was shown that torsion action leads to the occurrence of rotational movements of nitrogenous bases. This influence is nonlinear, and an increase in the amplitude of the torsion action does not lead to an automatic increase in the amplitude of rotational movements and an increase in the zones’ open states. Calculations with a constant torsion moment demonstrate that open states zones are more often formed at the boundaries of the gen and in regions with a predominance of A–T pairs. It is shown, that for the occurrence of open states in the part of the gene that contains a small number of A–T pairs, a large amount of torque is required. When the torque is applied to a certain region of the gene, the probability of the formation of the open state depends on the content of A–T pairs in this region, the size of this region, and on the exposure time. For this mathematical model, open states zones can be closed when the torsion action stops. The simulation results showed that the values of the torsion moment required for the appearance of open states zones, in some cases, are close to experimentally measured (13–15 pN·nm).

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176€, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.

Remote Exploration of Experimental Biophysical Instrumentation in Core Facilities

ABSTRACT Biophysics is defined by the experimental data that are collected on an extensive array of powerful and elegant tools. To solve important problems in biophysics, an understanding of the capabilities and limitations of the current instrumental methods is needed. Although lecture-based courses can instruct students on the physical principles of biophysical instrumentation, the actual practical use of instrumentation can seem far from the concepts taught through presentations or books. Traditionally, laboratory courses can expose students to hands-on use and understanding of experimental methods. During the COVID-19 pandemic, laboratory-based courses were challenging or, at times, prohibited. The educational aim of this article is to connect the instrumental concepts learned in lecture to the use of instruments for experiments when students are unable to go into laboratory environments. I present a low-stakes assignment for students to explore the biophysical instrumentation at core facilities. Prompts were provided to guide students through methods and challenges when using an instrument in a laboratory. These were then shared in a group environment so students could learn about multiple instruments in a single class and further benefit from social interactions with their peers, combating isolation during remote courses. Beyond remote instruction during COVID-19, this assignment can be applicable to future courses where laboratory work is cost-, time-, or location-prohibitive. Adaptations for in-person instruction are also discussed.

Inhibitory Effects of Sulfated Polysaccharides from the Sea Cucumber Cucumaria Frondosa against Aβ40 Aggregation and Cytotoxicity.

Abnormal aggregation and deposition of Aβ is one of the causative agents for Alzheimer's disease. The development of inhibitors for Aβ aggregation has been considered a possible method to prevent and treat Alzheimer's disease. Edible sea cucumbers contain many bioactive molecules, including saponins, phospholipids, peptides, and polysaccharides. Herein, we report that polysaccharides extracted from sea cucumber Cucumaria frondosa could reduce the aggregation and cytotoxicity of Aβ40. By utilizing multiple biochemical and biophysical instruments, we found that the polysaccharides could inhibit the aggregation of Aβ40. A chemical kinetics analysis further suggested that the major inhibitory effects of the polysaccharides were achieved by disassembling mature fibrils, which in turn reduced the cytotoxicity of Aβ. These results suggested that the polysaccharides extracted from sea cucumber could be used as an effective inhibitor for Aβ.

Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques.

Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3-4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.

Vertical Motions and Their Effects on a Biogeochemical Tracer in a Cyclonic Structure Finely Observed in the Ligurian Sea

AbstractVertical velocities can be estimated indirectly from in situ observations by theoretical frameworks like the ω‐equation. Direct measures of vertical exchanges are challenging due to their typically ephemeral spatiotemporal scales. In this study we address this problem with an adaptive sampling strategy coupling various biophysical instruments. We analyze the 3‐D organization of a cyclonic mesoscale structure finely sampled during the Observing Submesoscale Coupling At High Resolution cruise in the Ligurian Sea during fall 2015. The observations, acquired with a moving vessel profiler, highlight a subsurface low‐salinity layer (≃50 m), as well as rising isopycnals, generated by geostrophic cyclonic circulation, in the structure's center. Reconstructed 3‐D fields of density and horizontal velocities are used to estimate the vertical velocity field down to 250 m by applying the adiabatic QG ω‐equation, for the first time in this region. The vertical motions are characterized by multipolar patterns of downward and upward velocities on the edges of the structure and significantly smaller vertical velocities in its center. Both the 3‐D distribution of particles (size ≥100 μm), measured with a laser optical plankton counter, and the Synechococcus and Prochlorococcus abundances (cell per cubic meter) measured by flow cytometry are consistent with the 3‐D velocity field. In particular, a secondary vertical recirculation is identified that upwells particles (from 250 to 100 m) along isohalines to the structure's center. Besides demonstrating the effect of vertical patterns on biogeochemical distributions, this case study suggests to use particle matter as a tracer to assess physical dynamics.

Mechanobiology of Cancer Stem Cells and Their Niche.

Though the existence of cancer stem cells remained enigmatic initially, over the time their participation in tumorigenesis and tumor progression has become highly evident. Today, they are also appreciated as the causal element for tumor heterogeneity and drug-resistance. Cancer stem cells activate a set of molecular pathways some of which are triggered by the unique mechanical properties of the tumor tissue stroma. A relatively new field called mechanobiology has emerged, which aims to critically evaluate the mechanical properties associated with biological events like tissue morphogenesis, cell-cell or cell-matrix interactions, cellular migration and also the development and progression of cancer. Development of more realistic model systems and biophysical instrumentation for observation and manipulation of cell-dynamics in real-time has invoked a hope for some novel therapeutic modalities against cancer in the future. This review discusses the fundamental concepts of cancer stem cells from an intriguing viewpoint of mechanobiology and some important breakthroughs to date.

Novel Regulatory System in Plants and the Necessity of a Breeding Phytotron in the Russian Federation

A novel plant epigenetic regulatory system is reported. This is a change in product spectra of genes determining a productivity trait caused by a change in a limiting factor of the environment. Based on this discovery, the Theory of Ecogenetic Organization of Plant Quantitative Traits (TEGOPQT) was elaborated over the period from 1984 to 2014. The Theory gave rise to 24 prognostic corollaries and 10 know-hows. They are able to increase sharply the effectiveness of plant breeding aimed at higher yields. The prerequisite of this success is a creation of a priority breeding phytotron in the Russian Federation. The paper lists the necessary biophysical instruments and other devices that are needed to equip the growth chambers of the phytotron.

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

A high throughput array microscope for the mechanical characterization of biomaterials.

In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

Optimisation of a dosing regime for a topical skin protectant (barrier cream)

Context: Topical skin protectants (barrier creams) have the potential to reduce or enhance the severity of dermal lesions following exposure to allergens or irritants. Therefore, it is essential that such products are subject to appropriate clinical evaluation prior to marketing. Consequently, it is important to accurately define a dosing regime in order to assess test products under appropriate conditions.Objective: In this study, we extended the use of a standard rubefacient (methyl nicotinate; MN) assay to establish the optimum thickness and duration of action of a novel barrier cream (RD1433). White petroleum jelly (Vaseline®) was used as a comparator product.Methods: The dermal response to MN was measured on the volar forearm skin of volunteers (n = 12; average age 47.5 years) using an array of biophysical instruments and visual scoring. When applied at a nominal thickness of 0.1 mm, RD1433 retained effectiveness against MN for up to six hours. In contrast, Vaseline® was relatively ineffective. Moreover, RD1433 provoked no measurable signs of irritation and so can be considered acceptable for further clinical evaluation.Conclusion: Future clinical studies using RD1433 should be based on topical application of a 0.1 mm thickness layer every six hours.

Upgrade of MacCHESS facility for X-ray scattering of biological macromolecules in solution.

X-ray scattering of biological macromolecules in solution is an increasingly popular tool for structural biology and benefits greatly from modern high-brightness synchrotron sources. The upgraded MacCHESS BioSAXS station is now located at the 49-pole wiggler beamline G1. The 20-fold improved flux over the previous beamline F2 provides higher sample throughput and autonomous X-ray scattering data collection using a unique SAXS/WAXS dual detectors configuration. This setup achieves a combined q-range from 0.007 to 0.7 Å(-1), enabling better characterization of smaller molecules, while opening opportunities for emerging wide-angle scattering methods. In addition, a facility upgrade of the positron storage ring to continuous top-up mode has improved beam stability and eliminated beam drift over the course of typical BioSAXS experiments. Single exposure times have been reduced to 2 s for 3.560 mg ml(-1) lysozyme with an average quality factor I/σ of 20 in the Guinier region. A novel disposable plastic sample cell design that incorporates lower background X-ray window material provides users with a more pristine sample environment than previously available. Systematic comparisons of common X-ray window materials bonded to the cell have also been extended to the wide-angle regime, offering new insight into best choices for various q-space ranges. In addition, a quantitative assessment of signal-to-noise levels has been performed on the station to allow users to estimate necessary exposure times for obtaining usable signals in the Guinier regime. Users also have access to a new BioSAXS sample preparation laboratory which houses essential wet-chemistry equipment and biophysical instrumentation. User experiments at the upgraded BioSAXS station have been on-going since commissioning of the beamline in Summer 2013. A planned upgrade of the G1 insertion device to an undulator for the Winter 2014 cycle is expected to further improve flux by an order of magnitude.

Ominous: Playfulness and emergence in a performance for biophysical music

This article tackles the issue of playfulness in biosignal-based performance with digital musical instruments. The practical context is that of an interactive sound sculpture performance, entitled Ominous, for the Xth Sense, a biophysical musical instrument. This work posits that a prominent quality of the body physiology is its emergence. Developed by cultural theorist Brian Massumi, the notion of bodily emergence is useful to inform playfulness in musical performance for it can be used to understand a performer's body as being continuously changed by physiological and autonomic processes. This article presents a strategy for musical interaction that uses the relations in time and intensity of two biosignals to make the musical instrument adapt to the performer's physiological states. This strategy is put into practice by implementing a system that, drawing upon findings in biomedical engineering, brings together biosignal feature extraction, multidimensional mapping and digital neural networks.

Sedimentation Velocity Analysis of the EGFPs in E. coli Whole Cell Extracts using Fluorescence Detection System

The analytical ultracentrifugation (AUC) is a classic biophysical instrument to analyze protein interactions in solutions. A recently introduced fluorescence detection system (FDS) improves the specificity and sensitivity of the AUC, and offers the potential to analyze the protein interactions in biological fluids. We explore challenges posed by the application of FDS-AUC to the study of protein interactions in E.coli whole cell extracts, using fluorescent proteins (EGFPs) as model system. At experimentally feasible concentrations of cell extracts, we find no discernable effects of hydrodynamic nonideality on the sedimentation of EGFPs. However, at high concentrations E.coli whole cell extracts produce significant signals from auto fluorescence with complicated quenching patterns. Goal of this work is to establish detection limits and develop procedures to improve specificity.

Optimizing buffer conditions to improve crystallizability and SAXS measurement

The Sample Preparation and Characterization (SPC) facility at EMBL Hamburg is situated next to the PETRA3 beamlines for protein crystallography and small angle scattering (SAXS) that are operated by EMBL. The facility is equipped with molecular biology and biophysical instrumentation to carry out purification and characterization of macromolecular samples. It serves a mixed community of local EMBL scientists, beamline visitors and scientists from the European Union research area. Most incoming samples are destined for high-throughput crystallization at the facility or characterization by SAXS. The facility offers standardized quality control reports on each incoming sample, including characterization by mass spectrometry and Thermofluor. Based on this report, local staff can suggest and perform optimization protocols that increase the stability of the sample. For instance, Thermofluor screens(1) were developed that probe the effect of buffers and additives that are commonly used in sample preparation. These systematic screens provide a high-throughput method to identify stabilizing conditions for sample purification, storage and structural characterization. This technique has been also a valuable asset providing a high-throughput method for assessing the crystallizability of proteins by screening for conditions which contribute to the protein sample homogeneity, stability and solubility. The home-made screens have been tested on more than 200 different protein constructs at SPC facility. The aim of the SPC facility is to integrate off-line biophysical techniques with synchrotron beamlines, to offer the European user community a full package for sample characterization. The facility is especially geared towards cell biologists with little experience in structure determination. Expert staff is available to help to plan, perform and interpret biophysical experiments. It is possible for users to book SPC equipment together with their synchrotron beamtime, for protein purification, circular dichroism and isothermal calorimetry. Funded access to the facility is currently provided by the European Community's Seventh Framework Programme Biostruct-X. For further information, please contact spc@embl-hamburg.de.

Comparative Signature Diagrams to Evaluate Biophysical Data for Differences in Protein Structure Across Various Formulations

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.

Silicon nanotweezers for biomechanical and bioelectrical assays

In modern life, technologies enabling detection of biological molecules at a low threshold, for health and ecological concerns, are in high demand. Directly interrogating the molecules is a promising direction to clarify the noisy response of conventional assays arising from simultaneous different reactions. Besides sophisticated biophysical instrument such as the atomic force microscope, this paper proposes silicon nanotweezers (SNT) as a new microsystem for molecular manipulation. SNT can trap molecules and sense their biomechanical and bioelectrical response in minute operations. In this review SNT characteristics are overviewed; their operation modes are illustrated by molecule and cell trapping, manipulation and characterization in air and in solution. As they are tiny and can be mass produced by highly parallel microsystem technology, SNT can be seen as a potential molecular and cellular probe for routine analysis and bio detection.

Reactions of non‐immunologic contact urticaria on scalp, face, and back

This study compared the reactivity of scalp, face, and back to nonimmunologic contact urticants (NICU) to ascertain relative responsiveness. Model urticants, benzoic acid (BA) and hexyl nicotinate (HN) with 3 concentrations of each were applied to marked skin of 10 bald males during 6 weeks. One urticant was applied to one side of nasolabial fold, back, and scalp and the other applied to the contralateral side. Reactivity was assessed by visual scores (VS) and biophysical instruments. Subjects ranked skin sensation with a 10-point visual analogue scale. With 0.25% HN application, upper back VS significantly (p<0.05) exceeded scalp and back VS also showed significantly (p<0.05) stronger reaction than face at 60 min post-application; however, at 2.5% BA site, VS of face exhibited significantly (p<0.05) higher than back at 15 min post-application but with 0.625% BA site, VS of back was significantly (p<0.05) higher than face. The a* value was significantly (p<0.05) higher on back than scalp with 0.625% BA treatment. Thus symptoms and measurements vary among sites. Differences may be related to solubility related percutaneous penetration. We encourage investigation into this relatively neglected but clinically important arena, to help explain difference in consumer/patient acceptance of topical formulations.

Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system

Information about the stability of proteins is paramount to determine their optimal storage or reaction conditions. It is also essential to determine protein stability in high-throughput when screening for new or improved functions of proteins obtained from large mutant libraries. In drug discovery programs, monitoring of ligand-induced stabilization effects can be used to identify lead compounds in high-throughput. These studies require expensive biophysical instrumentation and large quantities of purified proteins. To address these issues, we developed a new method, using GFP as a reporter system to quantify the stability of a protein and its ligand-associated stabilization effects that requires neither special equipment nor extensive purification steps. Here, GFP is fused to a protein of interest (POI) through a linker and is used as a reporter system for protein unfolding and aggregation. The three POIs used in this study include the Ter-binding protein Tus, glycerol kinase and chloramphenicol acetyl transferase. The fluorescent fusion protein is subjected to irreversible thermal denaturation leading to formation of aggregates, which are eliminated by a centrifugation step. The residual fluorescence of the soluble fraction can be directly related to the stability of the POI and can be quantitatively monitored using a fluorescence plate reader. The GFP-based stability assay (GFP-Basta) was able to identify stabilizing compounds and afforded a new quantitative method for the screening and ranking of ligands for three different proteins. These applications are particularly useful for drug discovery, directed evolution, structural and functional genomics.