Proteins In Aqueous Solution Research Articles

Electrode Surface Potential-Driven Protein Adsorption and Desorption through Modulation of Electrostatic, van der Waals, and Hydration Interactions.

When proteins in aqueous solutions are exposed to solid substrates, they adsorb due to the dynamic interplay of electrostatic, van der Waals, and hydration interactions and do so in a rather irreversible fashion, which makes protein recovery troublesome. Here, we use a gold electrode as the solid substrate and modulate the surface potential to systematically induce protein adsorption as well as partial desorption. We use different methods such as surface plasmon resonance, atomic force microscopy, and electrowetting and show that biasing the electrode to more negative potentials (by −0.4 V compared to the open-circuit potential at pH 6) results in an increased adsorption barrier of 6 kJ mol–1 for the negatively charged protein β-lactoglobulin. Further, we clearly demonstrate that this is due to an increased double layer potential of −0.06 V and an increase in hydration repulsion. This indicates that an electric potential can directly influence surface interactions and thus induce partial β-lactoglobulin desorption. These observations can be the basis for biosensors as well as separation technologies that use only one trigger to steer protein ad- and desorption, which is low in energy requirement and does not generate large waste streams, as is the case for standard protein separation technologies.

Hierarchical build-up of bio-based nanofibrous materials with tunable metal–organic framework biofunctionality

Multifunctional, light-weight, responsive materials show promise in a range of applications including soft robotics, therapeutic delivery, advanced diagnostics and charge storage. This paper presents a novel, scalable, efficient and sustainable approach for the preparation of cellulose nanofibril-based aerogels via a facile ice-templating, solvent exchange and air-drying procedure, which could replace existing inefficient drying processes. These ambient-dried aerogels (∼99% porosity) exhibit a high specific compressive modulus (26.8 ± 6.1 kPa m3 kg−1, approaching equivalence of carbon-nanotube-reinforced aerogels), wet stability and shape recovery (80–90%), favorable specific surface area (90 m2 g−1) and tunable densities (2–20 kg m−3). The aerogels provide an ideal nanofibrillar substrate for in-situ growth of metal–organic frameworks (MOFs), via co-assembly of MOF precursors with proteins in aqueous solutions. The resulting hybrid aerogels show a nine-fold increase in surface area (810 m2g−1), with preserved wet stability and additional protein biofunctionality. The hybrid aerogels facilitate a pH-controlled release of immobilized proteins, following a concomitant disassembly of the surface grown MOFs, demonstrating their use in controlled delivery systems. The colorimetric protein binding assay of the biofunctionalized hybrid aerogel also demonstrates the potential of the material as a novel 3D bioassay platform, which could potentially be an alternative to plate-based enzyme-linked immunosorbent assay.

On the use of multiple-time-step algorithms to save computing effort in molecular dynamics simulations of proteins.

Computer simulation of proteins in aqueous solution at the atomic level of resolution is still limited in time span and system size due to limited computing power available and thus employs a variety of time‐saving techniques that trade some accuracy against computational effort. Examples of such time‐saving techniques are the application of constraints to particular degrees of freedom or the use of a multiple‐time‐step (MTS) algorithm distinguishing between particular forces when integrating Newton's equations of motion. The application of two types of MTS algorithms to bond‐stretching forces versus the remaining forces in molecular dynamics (MD) simulations of a protein in aqueous solution or of liquid water is investigated and the results in terms of total energy conservation and the influence on various other properties are compared to those of MD simulations of the same systems using bond‐length, and for water bond‐angle, constraints. At comparable computational effort, the use of bond‐length constraints in proteins leads to better energy conservation and less distorted properties than the two MTS algorithms investigated.

Conformationally flexible core-bearing detergents with a hydrophobic or hydrophilic pendant: Effect of pendant polarity on detergent conformation and membrane protein stability

Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. Statement of significanceThis study introduces new amphiphiles for use as biochemical tools in membrane protein studies. We identified a few hydrophilic pendant-bearing amphiphiles such as M-GTM-O11 and M-GTM-O12 that show remarkable efficacy for membrane protein solubilization and stabilization compared to a gold standard DDM, the hydrophobic counterparts (E-GTMs) and a significantly optimized detergent LMNG. In addition, detergent results obtained in the current study reveals the effect of detergent pendant polarity on protein solubility and stability. Thus, the current study represents both significant chemical and conceptual advance. The detergent tools and design principle introduced here advance protein science and facilitate structure-based drug design and development.

Universal Relation between Instantaneous Diffusivity and Radius of Gyration of Proteins in Aqueous Solution.

Protein conformational fluctuations are highly complex and exhibit long-term correlations. Here, molecular dynamics simulations of small proteins demonstrate that these conformational fluctuations directly affect the protein's instantaneous diffusivity D_{I}. We find that the radius of gyration R_{g} of the proteins exhibits 1/f fluctuations that are synchronous with the fluctuations of D_{I}. Our analysis demonstrates the validity of the local Stokes-Einstein-type relation D_{I}∝1/(R_{g}+R_{0}), where R_{0}∼0.3 nm is assumed to be a hydration layer around the protein. From the analysis of different protein types with both strong and weak conformational fluctuations, the validity of the Stokes-Einstein-type relation appears to be a general property.

Insights into Solution Structures of Photosynthetic Protein Complexes from Small-Angle Scattering Methods

High-resolution structures of photosynthetic pigment–protein complexes are often determined using crystallography or cryo-electron microscopy (cryo-EM), which are restricted to the use of protein crystals or to low temperatures, respectively. However, functional studies and biotechnological applications of photosystems necessitate the use of proteins isolated in aqueous solution, so that the relevance of high-resolution structures has to be independently verified. In this regard, small-angle neutron and X-ray scattering (SANS and SAXS, respectively) can serve as the missing link because of their capability to provide structural information for proteins in aqueous solution at physiological temperatures. In the present review, we discuss the principles and prototypical applications of SANS and SAXS using the photosynthetic pigment–protein complexes phycocyanin (PC) and Photosystem I (PSI) as model systems for a water-soluble and for a membrane protein, respectively. For example, the solution structure of PSI was studied using SAXS and SANS with contrast matching. A Guinier analysis reveals that PSI in solution is virtually free of aggregation and characterized by a radius of gyration of about 75 Å. The latter value is about 10% larger than expected from the crystal structure. This is corroborated by an ab initio structure reconstitution, which also shows a slight expansion of Photosystem I in buffer solution at room temperature. In part, this may be due to conformational states accessible by thermally activated protein dynamics in solution at physiological temperatures. The size of the detergent belt is derived by comparison with SANS measurements without detergent match, revealing a monolayer of detergent molecules under proper solubilization conditions.

Ice Growth Suppression in the Solution Flows of Antifreeze Protein and Sodium Chloride in a Mini-Channel

The control of ice growth inside channels of aqueous solution flows is important in numerous fields, including (a) cold-energy transportation plants and (b) the preservation of supercooled human organs for transplantation. A promising method for this control is to add a substance that influences ice growth in the flows. However, limited results have been reported on the effects of such additives. Using a microscope, we measured the growth of ice from one sidewall toward the opposite sidewall of a mini-channel, where aqueous solutions of sodium chloride and antifreeze protein flowed. Our aim was to considerably suppress ice growth by mixing the two solutes. Inclined interfaces, the overlapping of serrated interfaces, and interfaces with sharp and flat tips were observed in the cases of the protein-solution, salt-solution, and mixed-solution flows, respectively. In addition, it was found that the average interface velocity in the case of the mixed-solution flow was the lowest and decreased by 64% compared with that of pure water. This significant suppression of the ice-layer growth can be attributed to the synergistic effects of the ions and antifreeze protein on the diffusion of protein.

Probing the Viscosity of Aqueous Solutions Containing Ribosome-Bound Nascent Proteins

Understanding protein folding and metabolism within cells requires a fair evaluation of the complex solution conditions of the intracellular milieu. Viscosity is one important cellular property that affects the rotational and translational motion of biomolecules. Previous studies yielded information on the viscosity of a variety of aqueous solutions of protein, DNA, and RNA, and demonstrated that these solutions typically show Newtonian behavior at shear rates less than 10,000 s−1. However, it is not known how ribosomes carrying nascent protein chains (RNCs) affect solution viscosity.

Affinity Diversification of a Polymer Probe for Pattern-recognition-based Biosensing Using Chemical Additives.

Pattern-recognition-based sensing has attracted attention as a promising alternative to conventional sensing methods that rely on selective recognition. Here, we report on novel strategy using chemical additives with the ability to modulate probe/analyte interactions to more easily construct pattern-recognition-based sensing systems for proteins and cells. The fluorescence of dansyl-modified cationic poly-L-lysine (PLL-Dnc) is enhanced upon binding to proteins in aqueous solution, while the addition of salts, inert polymers, or alcohols modulates the protein/PLL-Dnc interactions via a variety of mechanisms. Subsequent readout of the fluorescence changes produces response patterns that reflect the characteristics of the analytes. Multivariate analysis of the response patterns allowed for accurate identification of not only eight structurally similar albumin homologues, but also four mammalian cells. This strategy, which uses inexpensive and common additives, significantly improves the accessibility of pattern-recognition-based sensing, which will offer new opportunities for the detection of various bioanalytes.

Analysis of Fractal Structures in Dehydrated Films of Protein Solutions

The article deals with dendritic structures resulting from self-organization processes in aqueous solutions of albumin proteins. The methods for obtaining the structures and experimental results are presented. It is shown that dendrites are fractal structures that are symmetric under certain conditions of their formation and can have different characteristics depending on the isothermal dehydration of liquid samples. The fractal dimension of the structures in films of the albumin protein solution has been calculated. Dependences of the fractal dimension on the concentrations of salts and protein in the initial solutions and also on the dehydration temperature have been revealed. It has been shown that as the protein concentration in the solution grows, the salt concentration for the initiation of the dendritic structure formation increases. It has been found that the temperature dependences of the fractal dimension of the structures become smoother with increasing protein concentration in solutions. The relationship between geometric characteristics of dendrites and self-organization parameters during drying is discussed.

Isothermal Titration Calorimetry of Membrane Proteins.

The ability to quantify protein-protein interactions without adding labels to protein has made isothermal titration calorimetry (ITC) a preferred technique to study proteins in aqueous solution. Here, we describe the application of ITC to the study of protein-protein interactions in membrane mimics using the association of integrin αIIb and β3 transmembrane domains in phospholipid bicelles as an example. A higher conceptual and experimental effort compared to water-soluble proteins is required for membrane proteins and rewarded with rare thermodynamic insight into this central class of proteins.

Studying the mechanism of phase separation in aqueous solutions of globular proteins via molecular dynamics computer simulations.

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.

Cocoa melanoidins reduce the formation of dietary advanced glycation end-products in dairy mimicking system

The control of Maillard reaction in foods is important to preserve protein nutritional quality. In this study, we investigated the effects of melanoidins obtained from different roasted cocoa beans toward the formation of dietary advanced glycation end-products (d-AGEs) in aqueous solution of whey protein (WP) and glucose, glyoxal and methylglyoxal at 35 °C and pH 7.0. Cocoa melanoidins (4 mg/mL) were more effective to inhibit glyoxal-derived d-AGEs than methylglyoxal-derived d-AGEs, with 74.4% and 48% reduction of N-ε-carboxymethyllysine and methylglyoxal-hydroimidazolone formation in WP/glyoxal and WP/methylglyoxal system, respectively. Furthermore, protein-bound N-ε-fructosyllysine, measured through furosine, decreased down to 57.2% in presence of cocoa melanoidins in WP/glucose model system suggesting an effective control of the Maillard reaction in an early stage. These findings highlighted that cocoa melanoidins are functional ingredients able to mitigate protein glycation in dairy products during storage.

The Effect of Lactation Number on Physico-Chemical and Microbiological Parameters of Milk from Small and Medium Bovine Farms located in Transilvania Region

Milk is a biological fluid where fats are distributed in microglobules (2-10 million/cm3), in an aqueous solution of proteins, carbohydrates and other substances. The main physico-chemical properties of milk are: density, viscosity, specific heat, boiling point, freezing point, pH and total acidity. Microbiological quality is represented by the presence or absence of pathogens or alterations, which can contaminate milk as a raw material. The comparative nutritional and microbiological quality of milk from three counties, from Transilvania Region, were fat percentage, total protein, casein content, lactose, pH, urea, total germ count (TGC) and somatic cell count (SCC). The best percentages of fat were obtained in Bihor County, with mean values of all lactations of 4.00%. This was followed by Hunedoara County with 3.97% and Alba County with 3.95%. The highest protein percentages were obtained in samples collected in Bihor County, with mean values of 3.57%, followed by Hunedoara County with 3.52% and Alba County with 3.51%. Most physicochemical indices fall within the normal limits, unanimously accepted by existing standards, but microbiological indices in some cases do not meet the requirements for the admission of raw milk.

Probing the response of poly (N-isopropylacrylamide) microgels to solutions of various salts using etalons

The Hofmeister series is a qualitative ordering of ions according to their ability to precipitate proteins in aqueous solution and is extremely important to consider when trying to understand materials and biomolecular structure and function. Herein, we utilized optical devices (etalons) composed of poly(N-isopropylacrylamide) (pNIPAm)-co-10% acrylic acid (AAc) or pNIPAm-based microgels to investigate how various salts in the Hofmeister series influenced the microgel hydration state. Etalons were exposed to a series of salts solutions at different concentrations and the position of the peaks in the reflectance spectra monitored using reflectance spectroscopy. As expected, pNIPAm-co-10%AAc microgel-based etalons responded to the presence of ions, although in this case the response to cations deviated from the Hofmeister series. However, when using etalons prepared with pNIPAm-based microgels, the responses followed the Hofmeister series for both cation and anions. Finally, we observed that the sensitivity of etalons prepared with pNIPAm microgels was significantly higher than the response obtained from etalons composed of pNIPAm-co-10%AAc microgels. This was explained by considering the charge on the pNIPAm-co-10%AAc microgels that influences how osmotic and Hofmeister effects impacts hydration state.

Hydrophilic Interactions Dominate the Inverse Temperature Dependence of Polypeptide Hydration Free Energies Attributed to Hydrophobicity.

We address the association of the hydrophobic driving forces in protein folding with the inverse temperature dependence of protein hydration, wherein stabilizing hydration effects strengthen with increasing temperature in a physiological range. All-atom calculations of the free energy of hydration of aqueous deca-alanine conformers, holistically including backbone and side-chain interactions together, show that attractive peptide-solvent interactions and the thermal expansion of the solvent dominate the inverse temperature signatures that have been interpreted traditionally as the hydrophobic stabilization of proteins in aqueous solution. Equivalent calculations on a methane solute are also presented as a benchmark for comparison. The present study calls for a reassessment of the forces that stabilize folded protein conformations in aqueous solutions and of the additivity of hydrophobic/hydrophilic contributions.

Array-Based Discriminative Optical Biosensors for Identifying Multiple Proteins in Aqueous Solution and Biofluids.

Identification of proteins is an important issue both in medical research and in clinical practice as a large number of proteins are closely related to various diseases. Optical sensor arrays with recognition ability have been flourished to apply for distinguishing multiple chemically or structurally similar analytes and analyzing unknown or mixed samples. This review gives an overview of the recent development of array-based discriminative optical biosensors for recognizing proteins and their applications in real samples. Based on the number of sensor elements and the complexity of constructing array-based discriminative systems, these biosensors can be divided into three categories, which include multi-element-based sensor arrays, environment-sensitive sensor arrays and multi-wavelength-based single sensing systems. For each strategy, the construction of sensing platform and detection mechanism are particularly introduced. Meanwhile, the differences and connections between different strategies were discussed. An understanding of these aspects may help to facilitate the development of novel discriminative biosensors and expand their application prospects.

ANS fluorescence: Potential to discriminate hydrophobic sites of proteins in solid states

In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.

The Clogging Effect in the Process of Protein Separation by Ultrafiltration

In this study, five ultrafiltration membranes (polysulfone, cellulose acetate and polyethe-rsulfone) were tested in the treatment of aqueous protein solutions similar to wastewater from fermentation industries. The experiments were made in tangential flow filtration. The permeate flux for the five membranes tested at the optimum pressure of 3 bar decreased due to the effect of clogging the pores by the macromolecular protein solutions. Cellulose acetate membranes showed the lowest permeate flux (Ac-Cel1=152.4 L/m2.h and Ac-Cel2=40.3 L/m2.h) which doesn�t recommend them for the ultrafiltration process of bovine serum albumin. When a polysulfone membrane was used in several cycles of protein-containing wastewater ultrafiltration, the permeate flow decreased progressively from one cycle to another due to the internal clogging of the membrane (501.6 L/m2.h up to 444.0 L/m2.h). Regarding the ultrafiltration of protein solutions with a suspended yeast content, the clogging was predominant on the membrane�s surface, which results in a decrease of the permeate flux by over 50%.

Effect of Ethanol on the Textural Properties of Whey Protein and Egg White Protein Hydrogels during Water-Ethanol Solvent Exchange.

This study aims at investigating the effect of ethanol (EtOH) on the textural properties of whey protein and egg white protein hydrogels. The hydrogels were produced by thermally induced gel formation of aqueous protein solutions. The water contained in the gel network was subsequently exchanged by EtOH to assess structural changes upon exposure of hydrogels to ethanolic aqueous phases. The textural properties of the hydrogel and alcogel samples were analyzed by uniaxial compression tests. For both protein sources, the hardness increased exponentially when pH and EtOH concentration were increased. This increase correlated with a shrinkage of the gel samples. The gel texture was found to be elastic at low EtOH concentrations and became stiff and hard at higher EtOH concentrations. It was found that the solvent exchange influences the ion concentration within the gels and, therefore, the interactions between molecules in the gel structure. Non-covalent bonds were identified as substantially responsible for the gel structure.