Protein Molecules In Solution Research Articles

An accelerated antibody aggregation test based on time sequenced dynamic light scattering

A rapid method for determination of the colloidal stability of protein molecules in solution is reported as an efficient tool for evaluating the stability of antibody formulations. Using human polyclonal immunoglobulin G (IgG) as a model protein and dynamic light scattering (DLS) as a technique to determine the size of particles in solution, the rate of aggregation is investigated at different temperatures and antibody concentrations. To reduce the observation period while increasing precision, a new approach to DLS analysis is developed that comprises: (i) a distribution analysis of high-resolution data, and fitting for multiple particle sizes present in a solution, (ii) a temperature ramp to an intermediate temperature followed by a stress test at constant temperature over several hours, and (iii) 3-D plotting to reveal the time-dependent evolution of the particle size distribution at the selected temperature. The resulting 3-D plots enable robust identification of the onset of aggregation with different dispersion conditions. This method enables rapid evaluation of the effects of parameters such as temperature and concentration on the stability of antibody solutions.

Properties of nano protein particle in solutions of myofibrillar protein extracted from giant squid (Dosidicusgigas).

This work studied the quantitation of myofibrillar protein isolated from giant squid by three methods (Bradford, biuret, and direct ultraviolet absorbance). The results were examined in relation to compositional size exclusion chromatography, static light scattering, zeta-potential and thermal analysis; comparisons between the apparent vs. the true protein concentration revealed the existence of disk-like nano protein particles (NPP) with a height of 2-3nm, diameters ranging from several tens nm to 140nm, and fractal dimensions df of less than 1.3. In order to probe the heterogeneity of NPP particles, their properties were studied under consecutive dilutions: the df decreased from 1.265 to 1.087, the zeta-potential increased from -4.55mV to -1.83mV, the denaturation temperature reduced from 63.9°C to 58.6°C, and the endothermic enthalpy reduced from 0.529Js-1 to 0.362Js-1. In addition, ca. 90% protein molecules in solution were aggregated to form NPP.

Mathematical modeling the formation of a histone octamer

A physical model of the interaction of protein molecules and their ability to form complex biological systems for the in vitro case in a solution of monovalent salt has been developed. Their reactive abilities using the methods of electrostatics based on the example of the step-by-step formation of the histone octamer from the H2A, H2B, H3, and H4 proteins have been studied. To analyze the ability of protein molecules to form compounds the matrix of potential energy of interactions between protein molecules in solutions with different concentrations of monovalent salt has been examined.

Real-time shape approximation and fingerprinting of single proteins using a nanopore

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

Surface-Induced Protein Unfolding in Submicron Electrospray Emitters.

The charging of protein ions formed by nanoelectrospray ionization (nanoESI) with tips that are between 1.5 μm and 250 nm in outer diameter is compared. More charging is obtained with the smaller tip sizes for proteins that have a net positive charge in solution, and additional high-charge-state distributions are often observed. A single charge-state distribution of holo-myoglobin ions is produced by nanoESI from a slightly acidified aqueous solution with the micron outer diameter tips, but some apo-myoglobin ions are produced with the submicron tips. In contrast, the charge-state distributions for proteins with a net negative charge in solution do not depend on tip size. Both the formation of high charge states and the appearance of higher-charge-state distributions, as well as the loss of the heme group from myoglobin, indicate that a fraction of the protein population is unfolding with the smaller tips. The increased charging with the smaller tip sizes for proteins with a net positive charge but not for proteins with a net negative charge indicates that the unfolding occurs prior to nanoelectrospray ionization as a result of Coulombic attraction between positively charged protein molecules in solution and the glass surfaces of the emitter tips that are negatively charged. These results demonstrate a novel method for producing highly charged protein ions that does not require exposing the proteins to additional chemicals either in solution or in the gas phase.

Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding.

Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm-Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm-Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm-Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm-Bragg theory by introducing the concept of 'folded' and 'unfolded' peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, , but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm-Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.

In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

The molecular state of hen egg white lysozyme in solution has been studied by small-angle X-ray scattering (SAXS) combined with molecular simulation. The addition of a precipitant is shown to change the state of the protein molecules in solution. The SAXS data were processed using the constructed models of different oligomers. Under the crystallization conditions, lysozyme is shown to be present in solution as monomers (96.0%), dimers (1.9%), and octamers (2.1%), whereas tetramers and hexamers are not found. The modeled structure of the octamer is not consistent with the commonly accepted unit cell containing eight lysozyme molecules. Meanwhile, the modeled octamers are well-fitted to the crystal structure and can serve as building blocks in the course of crystal growth.

Staying Together: Protein Molecules in Mesoscopic Clusters

Collective behavior of protein molecules in solutions is critically important for understanding their biological functions, as well as for efficient control of various technological processes. A number of recent experimental studies have observed the formation of various clusters in protein solutions in different systems (1–5). While the existence of small molecular complexes in solutions is a common phenomenon, a surprising thing in protein solutions is a variety and complexity of such aggregates.

Real-Time Shape Determination and 5-D Fingerprinting of Single Proteins

This talk describes the use of electrolyte-filled nanopores to determine, simultaneously and in real time, the shape, volume, charge, rotational diffusion coefficient, and dipole moment of individual proteins. It introduces the main concepts for a quantitative understanding and analysis of modulations in ionic current that arise from rotational dynamics of single proteins as they move through the electric field inside a nanopore. The resulting multi-parametric information raises the possibility to characterize, identify, and quantify individual proteins and protein complexes in a mixture. This approach interrogates single proteins and determines parameters such as the shape and dipole moment, which are excellent protein descriptors and cannot be obtained otherwise from single protein molecules in solution. Therefore, this five-dimensional characterization of proteins at the single particle level has the potential for instantaneous protein identification, quantification, and possibly sorting with implications for structural biology, proteomics, biomarker detection, and routine protein analysis.

A model-free method for extracting interaction potential between protein molecules using small-angle X-ray scattering

A small-angle X-ray scattering has been used to probe protein–protein interaction in solution. Conventional methods need to input modeled potentials with variable/invariable parameters to reproduce the experimental structure factor. In the present study, a model-free method for extracting the excess part of effective interaction potential between protein molecules in solutions over an introduced hard-sphere potential by using experimental data of small-angle X-ray scattering is presented on the basis of liquid-state integral equation theory. The reliability of the model-free method is tested by the application to experimentally derived structure factors for dense lysozyme solutions with different solution conditions [Javid et al., Phys. Rev. Lett. 99, 028101 (2007), Schroer et al., Phys. Rev. Lett. 106, 178102 (2011)]. The structure factors calculated from the model-free method agree well with the experimental ones. The model-free method provides the following picture of the lysozyme solution: these are the stabilization of contact-pair configurations, large activation barrier against their formations, and screened Coulomb repulsion between the charged proteins. In addition, the model-free method will be useful to verify whether or not a model for colloidal system is acceptable to describing protein–protein interaction.

Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor

Detection of proteins and nucleic acids is dominantly performed using optical fluorescence based techniques, which are more costly and timely than electrical detection due to the need for expensive and bulky optical equipment and the process of fluorescent tagging. In this paper, we discuss our study of the electrical properties of nucleic acids and proteins at the nanoscale using a nanoelectronic probe we have developed, which we refer to as the Nanoneedle biosensor. The nanoneedle consists of four thin film layers: a conductive layer at the bottom acting as an electrode, an oxide layer on top, and another conductive layer on top of that, with a protective oxide above. The presence of proteins and nucleic acids near the tip results in a decrease in impedance across the sensing electrodes. There are three basic mechanisms behind the electrical response of DNA and protein molecules in solution under an applied alternating electrical field. The first change stems from modulation of the relative permittivity at the interface. The second mechanism is the formation and relaxation of the induced dipole moment. The third mechanism is the tunneling of electrons through the biomolecules. The results presented in this paper can be extended to develop low cost point-of-care diagnostic assays for the clinical setting.

Exploring Light and Life

Exploring Light and Life

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

To assess the effect of sugar molecules on solution viscosity at high protein concentrations. A high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity. The addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations. Disaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.

Anisotropic Picosecond X-ray Solution Scattering from Photoselectively Aligned Protein Molecules

Anisotropic X-ray scattering patterns of transiently aligned protein molecules in solution are measured by using pump-probe X-ray solution scattering. When a linearly polarized laser pulse interacts with an ensemble of molecules, the population of excited molecules is created with their transition dipoles preferentially aligned along the laser polarization direction. We measured the X-ray scattering from the myoglobin protein molecules excited by a linearly polarized, short laser pulse and obtained anisotropic scattering patterns on 100 ps time scale. An anisotropic scattering pattern contains higher structural information content than a typical isotropic pattern available from randomly oriented molecules. In addition, multiple independent diffraction patterns measured by using various laser polarization orientations will give substantially increased amount of structural information compared with a single isotropic pattern. By monitoring the temporal change of the anisotropic scattering pattern from 100 ps to 1 μs, we observed the orientational dynamics of photo-generated myoglobin with the rotational diffusion time of ∼15 ns.

Proteins in dynamic equilibrium

Protein molecules in solution exist as an equilibrium of different conformations, but the sizes and shifts of these populations cannot be determined from static structures. A report now shows how they can be measured in solution.

The Influence of Secondary Emulsifiers on Lipid Oxidation within Sodium Caseinate‐Stabilized Oil‐in‐Water Emulsions

AbstractThe effect of protein displacement at the interface by a secondary emulsifier on the oxidative stability of sodium caseinate‐stabilized tuna oil‐in‐water emulsion systems was determined. Emulsions were prepared with a selection of anionic and non‐ionic emulsifiers and stored at both 25 and 50 °C with no added prooxidant, and at 4 °C in the presence of ferrous sulfate. The progress of oxidation during storage was monitored through solid phase microextraction headspace analysis. Metal ion catalyzed oxidation was enhanced for the emulsions stabilized with an anionic emulsifier in comparison to emulsion systems stabilized with non‐ionic emulsifiers and sodium caseinate alone. The increased oxidation observed for the emulsion with the anionic surfactant is due to electrostatic interactions between divalent metal ions and the negatively charged surfactant at the oil‐water interface. The sodium caseinate interfacial layer had little prooxidant effect at the droplet surface, most likely due to the ability of free protein molecules in solution to sequester metal ions, which may have provided some protection against oxidative deterioration.

Thematic Minireview Series: Single-molecule Measurements in Biochemistry and Molecular Biology

For many years, one of the fundamental barriers to defining the mechanisms of molecular and cellular processes was our inability to observe individual molecules because most conventional experimental approaches reveal only the average properties of ensembles of molecules existing in different states. For example, although individual protein molecules in solution are likely to differ at any given time in their conformation and the ions and molecules with which they are interacting, most experimental approaches monitor only their average conformation and average binding properties.

Phase transitions of folded proteins

Proteins exhibit a rich phase behavior. Even omitting the amyloid structures formed after partial protein unfolding, proteins form crystals, polymers, and other solid aggregates, as well as dense liquids and gels. Some of these condensed phases underlie pathological conditions, others play a crucial role in the biological function of the respective protein or are an essential part of its laboratory or industrial processing. In this review, we first discuss the thermodynamic characteristics of the solution and the interactions between protein molecules in solution, which underlie the protein phase behavior. We highlight the role of water structured at the protein molecular surface as a main contributor to the free energy of the phase transition. We define the driving force for phase transformations. We then summarize the fundamentals and review recent findings on the kinetics of nucleation of dense liquid droplets and crystals. We define the transition from nucleation to spinodal decomposition for these two phase transitions. We review the two-step mechanism of protein crystal nucleation, in which mesoscopic metastable protein clusters serve as precursors to the ordered crystal nuclei. Lastly, we discuss the mechanisms of growth of crystals: the generation of new crystal layers, the pathways of the molecules from the solution into the growth sites, the density of the growth sites and the factors, which determine the activation barriers for association of the molecules to the growth sites.

Fabrication of Si Nanowire Field-Effect Transistor for Highly Sensitive, Label-Free Biosensing

We fabricated a biosensor based on a silicon nanowire field-effect transistor (SiNW FET) with a Si3N4 gate insulator for highly sensitive detection of target biomolecules. The fabricated SiNW FET acted as an ion-sensitive FET that could detect the charge density in solutions flowing along the gate surface by responding to the pH of the solutions. The SiNW FET also detected charged protein molecules in solution, suggesting that our device can be used in highly sensitive, label-free biosensing.