Biological Molecules In Solution Research Articles

Multifunctional cyanoaryl porphyrazine pigments with push-pull structure of macrocycle framing: Photophysics and possible applications

We report the experimental study of fluorescence properties of three porphyrazine derivatives, distinct in the number of fused aromatic rings in aryl groups contained in the macrocycle frame and/or presence of Pd in the macrocycle, in solutions with biological molecules and in methanol-glycerol mixtures. Spectral and lifetime fluorescence analysis has revealed that all three pigments demonstrated two fluorescence transition bands from the first S1 and the second S2 electronic excited states to the ground state S0, and thus followed the anti-Kasha’s rule. Strong dependence of the quantum yield and lifetimes in the S1 → S0 fluorescence band on solution viscosity was observed. A significant increase of the fluorescence decay times and fluorescence intensity with albumin concentration and with presence of other biological molecules in solution was observed as well. A model of the low-laying energy states of porphyrazine derivatives has been developed for elucidation of the fluorescence properties observed, that took into account several radiative and non-radiative relaxation channels in molecular excited states. In particular, the model suggests that the first electronic excited state consists of planar and bent conformations separated by a potential energy barrier and shows a typical molecular-rotor behavior as a function of solution viscosity. Potential applications of the porphyrazine pigments as viscosity sensors and fluorescence probes were considered.

Nanoscale analysis of frozen honey by atom probe tomography

Three-dimensional reconstruction of the analysed volume is one of the main goals of atom probe tomography (APT) and can deliver nearly atomic resolution (~ 0.2 nm spatial resolution) and chemical information with a mass sensitivity down to the ppm range. Extending this technique to frozen biological systems would have an enormous impact on the structural analysis of biomolecules. In previous works, we have shown that it is possible to measure frozen liquids with APT. In this paper, we demonstrate the ability of APT to trace nanoscale precipitation in frozen natural honey. While the mass signals of the common sugar fragments CxHy and CxOyHz overlap with (H2O)nH from water, we achieved correct stoichiometric values via different interpretation approaches for the peaks and thus determined the water content reliably. Next, we use honey to investigate the spatial resolution capabilities as a step toward the measurement of biological molecules in solution in 3D with sub-nanometer resolution. This may take analytical techniques to a new level, since methods of chemical characterization for cryogenic samples, especially biological samples, are still limited.

A Low-Cost Exercise Relating Glutamate Solubility to pH & Buffering Capacity

The concept of solubility, and its relation to molecular structure, is important for understanding the properties of biological molecules in solution as they interact with the solvent and with each other. To demonstrate the relation between structure, solubility, and buffering capacity, we report a low-cost laboratory exercise using monosodium glutamate (MSG) as a model solute. Students observe how glutamate solution initially resists acidification and how glutamate solubility is first decreased and then restored with acidification. Students should interpret these observations relative to the structure of the solute. This exercise has been employed in introductory level courses for both biology majors and nonmajors and would also be appropriate for AP Biology.

Cellular effects of terahertz waves.

.Significance: An increasing interest in the area of biological effects at exposure of tissues and cells to the terahertz (THz) radiation is driven by a rapid progress in THz biophotonics, observed during the past decades. Despite the attractiveness of THz technology for medical diagnosis and therapy, there is still quite limited knowledge about safe limits of THz exposure. Different modes of THz exposure of tissues and cells, including continuous-wave versus pulsed radiation, various powers, and number and duration of exposure cycles, ought to be systematically studied.Aim: We provide an overview of recent research results in the area of biological effects at exposure of tissues and cells to THz waves.Approach: We start with a brief overview of general features of the THz-wave–tissue interactions, as well as modern THz emitters, with an emphasis on those that are reliable for studying the biological effects of THz waves. Then, we consider three levels of biological system organization, at which the exposure effects are considered: (i) solutions of biological molecules; (ii) cultures of cells, individual cells, and cell structures; and (iii) entire organs or organisms; special attention is devoted to the cellular level. We distinguish thermal and nonthermal mechanisms of THz-wave–cell interactions and discuss a problem of adequate estimation of the THz biological effects’ specificity. The problem of experimental data reproducibility, caused by rareness of the THz experimental setups and an absence of unitary protocols, is also considered.Results: The summarized data demonstrate the current stage of the research activity and knowledge about the THz exposure on living objects.Conclusions: This review helps the biomedical optics community to summarize up-to-date knowledge in the area of cell exposure to THz radiation, and paves the ways for the development of THz safety standards and THz therapeutic applications.

Size-Exclusion Particle Separation Driven by Micro-Flows in a Quasi-Spherical Droplet: Modelling and Experimental Results.

Aqueous solution droplets are supported quasi contact-free by superhydrophobic surfaces. The convective flow in evaporating droplets allows the manipulation and control of biological molecules in solution. In previous works, super-hydrophobic drops on nano-patterned substrates have been used to analyze otherwise undetectable species in extremely low concentration ranges. Here, we used particle image velocimetry (PIV) for studying the flow field in water droplets containing polystyrene particles on a pillared silicon super-hydrophobic chip. The particles describe vortex-like motions around the droplet center as long as the evaporating droplet maintains a spherical shape. Simulations by a Finite Element Method (FEM) suggest that the recirculating flow is due to the temperature gradient along the droplet rim, generating a shear stress. Notably, the characteristics of the internal flow can be modulated by varying the intensity of the temperature gradient along the drop. We then used the flow-field determined by experiments and an approximate form of the Langevin equation to examine how particles are transported in the drop as a function of particle size. We found that larger particles with an average size of are preferentially transported toward the center of the substrate, differently from smaller particles with a 10-fold lower size that are distributed more uniformly in the drop. Results suggest that solutions of spherical particles on a super-hydrophobic chip can be used to separate soft matter and biological molecules based on their size, similarly to the working principle of a time-of-flight (ToF) mass analyzer, except that the separation takes place in a micro-sphere, with less space, less time, and less solution required for the separation compared to conventional ToF systems.

Investigating How Protein Mixtures Interact with Gold Nanoparticles

Gold nanoparticles (AuNP) have numerous biological applications, including drug and gene delivery, chemo- and photo-therapy, biosensing, and bioimaging. When exposed to biological fluids, AuNPs will interact with proteins in solution, and these proteins will compete to bind to the AuNP surface. It remains impossible to predict the outcome of competitive binding to AuNPs, but this knowledge could potentially aid scientists hoping to target nanoparticles to specific cells in the body. NMR has been used extensively to study interactions between biological molecules in solution. Here, we explore using 2D 1H-15N HSQC and CPMG NMR methods to study protein interaction kinetics with various nanoparticles. Specifically, we used a 1H-15N HSQC technique to quantify AuNP binding versus time for a mixture of GB3 and Ubiquitin, two small model proteins. GB3 and Ubiquitin signals were sampled at 15 min time points. An external standard was used to quantify absolute binding to the AuNPs. Binding was observed to be extremely fast, with a majority of binding occurring in the dead time (3 min) of the experiment. We also used CPMG relaxation experiments to monitor the timescale of protein exchange with a pretreated, protein-functionalized AuNP surface. Our results suggest that competition between proteins can lead to increased surface exchange rates (∼ms). This differs slightly from a previous study, where surface exchange was extremely slow (18 hr). Together, our results suggest a mechanism by which nanoparticle surface character may change over time, and this may be an important consideration in the design of nanoparticle-based therapeutics.

Rotating magnetic particles for lab-on-chip applications - a comprehensive review.

Magnetic particles are widely used in lab-on-chip and biosensing applications, because they have a high surface-to-volume ratio, they can be actuated with magnetic fields and many biofunctionalization options are available. The most well-known actuation method is to apply a magnetic field gradient which generates a translational force on the particles and allows separation of the particles from a suspension. A more recently developed magnetic actuation method is to exert torque on magnetic particles by a rotating magnetic field. Rotational actuation can be achieved with a field that is uniform in space and it allows for a precise control of torque, orientation, and angular velocity of magnetic particles in lab-on-chip devices. A wide range of studies have been performed with rotating MPs, demonstrating fluid mixing, concentration determination of biological molecules in solution, and characterization of structure and function of biomolecules at the single-molecule level. In this paper we give a comprehensive review of the historical development of MP rotation studies, including configurations for field generation, physical model descriptions, and biological applications. We conclude by sketching the scientific and technological developments that can be expected in the future in the field of rotating magnetic particles for lab-on-chip applications.

Resonant Soft X-Ray Scattering Provides Protein Structure with Chemical Specificity

We introduce resonant soft X-ray scattering (RSoXS) as an approach to study the structure of proteins and other biological molecules in solution. Scattering contrast calculations suggest that RSoXS has comparable or even higher sensitivity than hard X-ray scattering because of contrast generated at the absorption edges of constituent elements, such as carbon and oxygen. Here, we demonstrate that working near the carbon edge reveals the envelope function of bovine serum albumin, using scattering volumes of 10-5 μL that are multiple orders of magnitude lower than traditional scattering experiments. Furthermore, tuning the X-ray energy within the carbon absorption edge provides different signatures of the size and shape of the protein by revealing the density of different types of bonding motifs within the protein. The combination of chemical specificity, smaller sample size, and enhanced X-ray contrast will propel RSoXS as a complementary tool to existing techniques for the study of biomolecular structure.

New Basis Set for the Evaluation of Specific Rotation in Flexible Biological Molecules in Solution.

A detailed theoretical investigation of specific rotation is carried out in solution for nine flexible molecules of biological importance. Systematic search for the main conformers is followed by time-dependent density functional theory (TD-DFT) calculations of specific rotation employing a wide range of basis sets. Due to conformational flexibility of the compounds under study, the possibility of basis set size reduction without deterioration of the results is investigated. The increasing size (d-)aug-cc-pVXZ (X = D, T, Q) bases of Dunning et al., and the ORP basis set, recently developed to efficiently provide molecular specific rotation, are used for this purpose. The polarizable continuum model is employed at all steps of the investigation. Comparison of the present results with the available data obtained in a vacuum reveals considerable differences, the values in solution being much closer to the experimental specific rotation data available. The ORP basis set proves to be competitive with the d-aug-cc-pVDZ set of Dunning in specific rotation calculations carried out in solution. While having the same number of functions, the former yields, in general, results considerably closer to the reference triple-ζ values. We can thus recommend the ORP basis set to study the optical rotation in conformationally flexible molecules in solution.

Fixed Path Length Sample Holders Enable Robust Cryosaxs Measurements from Sub-Microliter Sample Volumes

Small angle x-ray scattering (SAXS) gives structural information about biological molecules in solution. However, large (∼30 microliter) sample volumes are needed to mitigate radiation damage, limiting the use of SAXS in studying rare molecules. By cryocooling SAXS samples, radiation damage and required sample volumes are reduced by orders of magnitude [1], but challenges in creating identically-sized frozen samples complicate background subtraction. Here we present microfabricated silicon sample holders for cryoSAXS. These rigid sample holders have a fixed x-ray path length, simplifying background subtraction. Less than 800 nL of sample are required, facilitating measurements on expensive or hard-to-express molecules. These fixed path length, low volume sample holders make cryoSAXS a more accessible technique capable of probing a wide range of biological molecules.1. S. P. Meisburger et al. Biophys. J. 104, 227 (2013).

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers for the measurement of twist and torque.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Observing Real-Time Molecular Event Dynamics of Apoptosis in Living Cancer Cells using Nuclear-Targeted Plasmonically Enhanced Raman Nanoprobes

Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.

How the ESRF helps industry and how they help the ESRF.

The ESRF has worked with, and provided services for, the pharmaceutical industry since the construction of its first protein crystallography beamline in the mid-1990s. In more recent times, industrial clients have benefited from a portfolio of beamlines which offer a wide range of functionality and beam characteristics, including tunability, microfocus and micro-aperture. Included in this portfolio is a small-angle X-ray scattering beamline dedicated to the study of biological molecules in solution. The high demands on throughput and efficiency made by the ESRF's industrial clients have been a major driving force in the evolution of the ESRF's macromolecular crystallography resources, which now include remote access, the automation of crystal screening and data collection, and a beamline database allowing sample tracking, experiment reporting and real-time at-a-distance monitoring of experiments. This paper describes the key features of the functionality put in place on the ESRF structural biology beamlines and outlines the major advantages of the interaction of the ESRF with the pharmaceutical industry.

Separation of Polypeptides by Isoelectric Point Focusing in Electrospray-Friendly Solution Using a Multiple-Junction Capillary Fractionator

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.

Small angle scattering – moving forward

School of Biological Sciences, University of Auckland, Macromolecular , Private Bag 92-019, Auckland, New Zealand, and Argonne National Laboratory, Biosciences Division, Bldg 202, Room Q142, Argonne, IL 60439, USA One of the great strengths of crystallographic research has always been the willingness of crystallographers to work together to develop standards that will enhance both the quality and the impact of our science. This attitude, facilitated by the International Union of Crystallography (IUCr) though its various Commissions, has led to advances that have benefited much wider constituencies. A good example is the policies developed, through the IUCr, relating to the deposition and release of biological structural data (Baker et al., 1996; Baker & Saenger, 1999; Commission on Biological Macromolecules, 2000). The present issue of Acta Cryst. D highlights another such initiative in a rapidly developing area of importance to structural biology: small-angle scattering (Jacques et al., 2012). Small-angle scattering (SAS), whether of X-rays or neutrons, has been used for many years to gain structural insights into biological molecules in solution. One key advantage is obvious – you do not need crystals! In the past few years, a number of factors have come together to generate an explosion of interest in the use of SAS. New, high-quality, SAS beamlines have been developed at synchrotrons. Powerful and easy-to-use software has been developed for the interpretation of SAS data. As structural biologists tackle problems of everincreasing complexity, SAS data can be used in a variety of ways: to help interpret crystal structures by relating them to solution, to map substructures on to larger assemblies or to develop de novo information on large systems. At the same time, there are potential hazards. SAS experiments are easy to perform, and with powerful, easy-to-use software available, models are easily generated. The question then becomes one of how valid the interpretations of a particular experiment may be. On p. 620 of this issue, David Jacques, Mitchell Guss, Dmitri Svergun and Jill Trewhella take a very important step forward. They present a set of guidelines that should go a long way towards ensuring that conclusions drawn from SAS experiments can be properly assessed by readers and reviewers. These guidelines, developed through the IUCr Commission on Small-angle Scattering, consider issues such as the nature and quality of the sample, the solvent used, SAS data collection and processing protocols, the parameterization of models and the validation of results. These guidelines and the associated discussion should be read and thought about by anyone planning an SAS experiment, and will undoubtedly be of great value to researchers as the use of SAS continues to expand. It is also noteworthy that, in parallel, the Protein Data Bank has established an SAS Task Force to advise on whether models based on SAS analysis should be deposited, and, if so, with what accompanying data and what forms of validation. Meanwhile, the stage is set for continued expansion of this important technique. As Editors of a journal dedicated to biological structure and structural methods we welcome this initiative.

Biomolecular electrostatics using a fast multipole BEM on up to 512 gpus and a billion unknowns

We present teraflop-scale calculations of biomolecular electrostatics enabled by the combination of algorithmic and hardware acceleration. The algorithmic acceleration is achieved with the fast multipole method ( fmm) in conjunction with a boundary element method ( bem) formulation of the continuum electrostatic model, as well as the bibee approximation to bem. The hardware acceleration is achieved through graphics processors, gpus. We demonstrate the power of our algorithms and software for the calculation of the electrostatic interactions between biological molecules in solution. The applications demonstrated include the electrostatics of protein–drug binding and several multi-million atom systems consisting of hundreds to thousands of copies of lysozyme molecules. The parallel scalability of the software was studied in a cluster at the Nagasaki Advanced Computing Center, using 128 nodes, each with 4 gpus. Delicate tuning has resulted in strong scaling with parallel efficiency of 0.8 for 256 and 0.5 for 512 gpus. The largest application run, with over 20 million atoms and one billion unknowns, required only one minute on 512 gpus. We are currently adapting our bem software to solve the linearized Poisson–Boltzmann equation for dilute ionic solutions, and it is also designed to be flexible enough to be extended for a variety of integral equation problems, ranging from Poisson problems to Helmholtz problems in electromagnetics and acoustics to high Reynolds number flow.

Using optical resonance of whispering gallery modes in microspheres for real-time detection and identification of biological compounds

We have experimentally developed a technique for obtaining optical resonance of whispering gallery modes in quartz microspheres in solutions of biological molecules. We have estimated the sensitivity of the method with respect to the change in the refractive index of the medium surrounding the microsphere, and have also estimated the change in the concentration of the dissolved molecules using ethanol, glucose, ascorbic acid, and biotin as examples. According to this estimate, we found that a 1% change in the ethanol concentration in water is comparable with a change in refractive index by 0.00058 RIU (refractive index units), while with a 3% increase in glucose and ascorbic acid concentration in deionized water, a resonance shift of 170 nm and 200 nm was achieved.

Relaxation of Polymer Coated ${\rm Fe}_{3}{\rm O}_{4}$ Magnetic Nanoparticles in Aqueous Solution

Magnetic nanoparticles have shown great capability of sensing biological molecules in solution by correlating the magnetic susceptibility with the nanoparticle surface coatings. Here, we report on a new experimental approach of measuring magnetic nanoparticle relaxation. It uses a broadband ferromagnetic resonance setup specifically designed for samples in solution, enabling measurements of nanoparticles at variable frequencies up to 40 GHz. This design enables precise determination of the g-factor of the particles, by determining the resonance field for each microwave frequency. In addition this also enables us to determine the frequency dependence of the resonance linewidth, which provides information about the relaxation mechanisms of the magnetic nanoparticles. Polymer coated Fe <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> nanoparticles were synthesized using a modified co-precipitation method by introducing the polymer coatings (e.g., polyacrylic acid) during the synthesis. Thereby water soluble nanoparticles with a narrow size distribution were produced in a single step. The relaxation measurement of these nanoparticles showed only a slight increase in linewidth over the entire frequency range suggesting that extrinsic relaxation mechanisms are dominant.

Multiple extrema in the intermolecular potential and the phase diagram of protein solutions

Recent experiments have revealed several surprising features of the phase equilibria in protein solutions: liquid-liquid phase separation which is, in some cases, metastable with respect to the liquid-solid equilibrium, and in others-unobservable; widely varying crystallization enthalpies, including completely athermal crystallization; the co-existence of several crystalline polymorphs; and others. Other studies have shown that the solvent molecules at the hydrophobic and polar patches on the protein molecular surfaces are structured, introducing repulsive forces at surface separations equal to several water molecule sizes. In search of a causal link between the latter and former findings, we apply Monte Carlo simulation techniques in the investigation of phase diagrams associated with globular biological molecules in solution. We account for the solvent structuring via short-range isotropic two-body intermolecular potentials exhibiting multiple extrema. We show that the introduction of a repulsive maximum or a secondary attractive minimum at separations longer than the primary attractive minimum has dramatic effects on the phase diagram: liquid-liquid separation curves are driven to lower or higher temperatures, the sensitivity of the solubility curve (liquidus) to temperature, i.e., the enthalpy of crystallization, is significantly reduced or enhanced, metastable liquid-liquid separation may become stable and vice versa, and both low- and high-density crystalline phases are observed. The similarity of these features of the simulated phase behavior to those observed experimentally suggests that at least some of the mysteries of the protein phase equilibria may be due to the structuring of the solvent around the protein molecular surfaces. Another conclusion is that at least some of the dense liquids seen in protein solutions may be stable and not metastable with respect to a solid phase.