Protein Concentration Dependence Research Articles

Intermolecular Electrostatic Interactions in Cytochrome c Protein Monolayer on Montmorillonite Alumosilicate Surface: A Positive Cooperative Effect.

Montmorillonite (MM) crystal nanoplates acquire anticancer properties when coated with the mitochondrial protein cytochrome c (cytC) due to the cancer cells' capability to phagocytize cytC-MM colloid particles. The introduced exogenous cytC initiates apoptosis: an irreversible cascade of biochemical reactions leading to cell death. In the present research, we investigate the organization of the cytC layer on the MM surface by employing physicochemical and computer methods-microelectrophoresis, static, and electric light scattering-to study cytC adsorption on the MM surface, and protein electrostatics and docking to calculate the local electric potential and Gibbs free energy of interacting protein globules. The found protein concentration dependence of the adsorbed cytC quantity is nonlinear, manifesting a positive cooperative effect that emerges when the adsorbed cytC globules occupy more than one-third of the MM surface. Computer analysis reveals that the cooperative effect is caused by the formation of protein associates in which the cytC globules are oriented with oppositely charged surfaces. The formation of dimers and trimers is accompanied by a strong reduction in the electrostatic component of the Gibbs free energy of protein association, while the van der Waals component plays a secondary role.

Protein photodegradation in the visible range? Insights into protein photooxidation with respect to protein concentration

Visible light (400–800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.

Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant.

Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.

Contributions to the \u2018noise floor\u2019 in gene expression in a population of dividing cells

Experiments with cells reveal the existence of a lower bound for protein noise, the noise floor, in highly expressed genes. Its origins are still debated. We propose a minimal model of gene expression in a proliferating bacterial cell population. The model predicts the existence of a noise floor and it semi-quantitatively reproduces the curved shape of the experimental noise vs. mean protein concentration plots. When the cell volume increases in a different manner than does the mean protein copy number, the noise floor level is determined by the cell population’s age structure and by the dependence of the mean protein concentration on cell age. Additionally, the noise floor level may depend on a biological limit for the mean number of bursts in the cell cycle. In that case, the noise floor level depends on the burst size distribution width but it is insensitive to the mean burst size. Our model quantifies the contributions of each of these mechanisms to gene expression noise.

Micro- and macro-viscosity relations in high concentration antibody solutions

Molecular crowding in highly concentrated monoclonal antibody (mAb) solutions results in significant increases in viscosity, which complicates fill-finish steps and patient administration by subcutaneous injection. As viscosity measurements for optimization of the mAb formulation require significant amounts of material not always available in early development, fluorescence correlation spectroscopy (FCS) is evaluated as a potential ultra-low volume technique for viscosity measurement of high concentration protein solutions assuming the Generalised Stokes Einstein relation (GSE) remains valid. Using like-charge fluorescent tracers of different sizes, FCS provided measurements of microviscosities which were compared to the macroviscosity. After parametrising the protein concentration dependence of the viscosity by the exponential coefficient (k) of a simple exponential model, FCS derived k-values of like-size tracer to the crowder followed the same ordering as the macroviscosity derived k-values with respect to solvent conditions. Furthermore, k and the diffusion-derived protein-protein interaction parameter, kD, are linked, and, attractive conditions for mAbs result in a stronger concentration dependence of the viscosity. For tracers and crowders of like-size, a key result is negative deviations from the GSE relation are observed in presence of strong attractive interactions between crowder molecules. These data demonstrate that FCS has application to the screening of high concentration mAb solutions for formulation selection.

Electrochemical Studies on the Binding of Antibody—Aptamer Hybrid Receptor Layers to HER2 Protein

One of the possibilities of early detection of cancer disease is the analysis of the presence of protein biomarkers. Herein, we present the studies on the hybrid antibody—aptamer receptor layers for electrochemical detection of HER2 protein that is known as a biomarker of e.g. breast cancer. The application of MB-labelled aptamer did not allow to evidence the interaction with HER2 protein. Moreover, the use of unlabelled aptamer as a capture element and polyclonal antibody as a reporter probe did not show a dependence of current signal change vs HER2 protein concentration as well. On the contrary, the application of monoclonal antibody as a capture probe and aptamer labelled at 3′ end with MB as reporter probe enabled the determination of HER2 within 0.01–10 ng·ml−1 concentration range. The elaborated affinity assay also showed good selectivity towards HER2 biomarker and was used for HER2 determination in spiked serum sample.

Thermodynamics of the Thermal Denaturation of Acid Molten Globule State of Cytochrome c Indicate a Reversible High-Temperature Oligomerization Process.

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ 1/2 I2 ⇄ 1/3 I3 ⇄ 1/4 I4) including three new oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible oligomerization.

Differences in Protein Concentration Dependence for Nucleation and Elongation in Light Chain Amyloid Formation.

Light chain (AL) amyloidosis is a lethal disease characterized by the deposition of the immunoglobulin light chain into amyloid fibrils, resulting in organ dysfunction and failure. Amyloid fibrils have the ability to self-propagate, recruiting soluble protein into the fibril by a nucleation-polymerization mechanism, characteristic of autocatalytic reactions. Experimental data suggest the existence of a critical concentration for initiation of fibril formation. As such, the initial concentration of soluble amyloidogenic protein is expected to have a profound effect on the rate of fibril formation. In this work, we present in vitro evidence that fibril formation rates for AL light chains are affected by the protein concentration in a differential manner. De novo reactions of the proteins with the fastest amyloid kinetics (AL-09, AL-T05, and AL-103) do not present protein concentration dependence. Seeded reactions, however, exhibited weak protein concentration dependence. For AL-12, seeded and protein concentration dependence data suggest a synergistic effect for recruitment and elongation at low protein concentrations, while reactions of κI exhibited poor efficiency in nucleating and elongating preformed fibrils. Additionally, co-aggregation and cross seeding of κI variable domain (VL) and the κI full length (FL) light chain indicate that the presence of the constant domain in κI FL modulates fibril formation, facilitating the recruitment of κI VL. Together, these results indicate that the dominant process in fibril formation varies among the AL proteins tested with a differential dependence of the protein concentration.

Long-term stability of sodium caseinate-stabilized nanoemulsions.

Oil-in-water (5 wt%) nanoemulsions were prepared with different concentration (2.5-10wt%) of sodium caseinate as a sole emulsifier and their long-term storage stability was investigated for 6months. Previous studies associated with sodium caseinate looked only into nanoemulsion formation; hence the challenges with long-term stability were not addressed. All nanoemulsions displayed an average droplet size <200nm, which remained unchanged over 6months. However, all of them displayed rapid creaming due to unabsorbed protein induced depletion flocculation, whose extent increased with protein concentration, although the cream layer formed was weak and re-dispersible upon gentle mixing. Microstructural analysis of the cream layer showed compaction of flocculated nanodroplet network with time leaving the aqueous phase out. Calculation of depletion interaction energy showed an increase in inter-droplet attraction with protein concentration and decrease with a reduction in droplet size, making the nanoemulsions more resistant to flocculation than conventional emulsions. This work aids in understanding the dependence of protein concentration on long-term stability of sodium caseinate-stabilized nanoemulsions.

Effects of ionic strength on inclusion body refolding at high concentration

For commercial applications refolding process must be fast, inexpensive and highly efficient. In the past many strategies for protein refolding were introduced. Still, simple refolding methods with high product concentrations are still rare. Refolding experiments were performed with fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 produced as inclusion bodies. Solubilizates were refolded with batch dialysis or by continuous exchange of dialysis buffers with variable ionic strength. By employing dialysis with gentle removal of denaturant the dependence of protein concentration and decreasing refolding yields could be overcome compared to batch dialysis and yields were enhanced by 52% at protein concentrations of approx. 10 mg/mL. The average specific activity of refolded FTF was 123 U/mg, 83% relative to standard FTF. Rising ionic strength of refolding buffers to 600 mM leads to complete renaturation of solubilized protein at equal protein concentration. Buffer composition plays a less significant role on renaturation output. The effect might be correlated with ion charge density of co-solvents.

An ultrasensitive assay format for detecting ULK1 inhibition by monitoring the phosphorylation status of Atg13

A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer318) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer318-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer318-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer318-Atg13 upon ULK1 inhibition.

Prediction of salt effects on protein phase behavior by HIC retention and thermal stability

In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim®2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range.

Quantifying Protein Concentration using Designed DNA Carriers and Solid-State Nanopores

Designed “DNA carriers” have been proposed as a new method for nanopore based specific protein detection. Target proteins bind to the DNA carrier at defined positions creating a second level transient current drop against the background DNA translocation. In this work, we investigate the ability of this method to quantify protein concentrations. After incubation with target proteins of different concentrations, the fraction of DNA translocations showing a secondary current spike allows for quantification of the corresponding protein concentration. The protein concentration dependence is measured with two standard systems: biotin-streptavidin and digoxigenin-antibody system, at nanomolar or sub-nanomolar protein concentrations. The results demonstrate a dynamic range of ∼0.1 to 0.9 occupied fraction and show the potential for quantitative and specific protein detection using the DNA carrier method.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

Relationship between concentration of crude protein in spring barley (Hordeum vulgare L.), white goosefoot (Chenopodium album L.), charlock (Sinapis arvensis L.) and prickly grass (Echinochloa crus-galli (L.) Beauv.)

Laboratory bioassay was carried out at the Laboratory of Agronomic and Zootechnical Research of the Lithuanian University of Agriculture (Aleksandras Stulginskis University) at 54°89’N and 23°83’E during 2004–2005. The field experiment from which plant samples were taken for the laboratorial bioassay was performed in 2003–2004 in spring barley Aura crop at the Experimental Station of the Lithuanian University of Agriculture at 54°52’N and 23°49’E. The Lithuanian territory occupies an intermediate geographical position between the west European oceanic climate and the Eurasian continental climate. Lithuania belongs to the western region of the Atlantic Ocean continental climatic area with the average annual precipitation of 675 mm (572–978 mm) and temperature of 6–7 °C. The change of crude protein concentration in weeds affects accumulation of crude protein in spring barley. The aim of the experiment was to establish the dependence of spring barley biomass crude protein concentration on crude protein concentration in white goosefoot, charlock and prickly grass biomass from the same agrophytocenosis. The plant biomass taken from organic and conventional agriculture crops was evaluated for crude protein concentration by the Kjeldahl method. The highest concentration of crude protein in the biomass was established in the conventional agriculture system: 20.5% in spring barley and 21.7% in white goosefoot, 20.5% in spring barley and 20.4% in charlock. In the organic agriculture system, the highest concentration of crude protein in the biomass was established in prickly grass (18.9%) and spring barley (12.8%). The increase of crude protein concentration in white goosefoot, charlock and prickly grass induced the crude protein concentration increase in spring barley. The following relationship between the crude protein concentration was described by a linear correlation-regression analysis: in spring barley and white goosefoot r = 0.898 (p

Adsorption of cytochrome c on montmorillonite nanoplates: Protein concentration dependence

Cytochrome c [cytC] is a mitochondrial hemoprotein functioning as electron carrier in the respiratory chain of the biological cells. Being adsorbed on colloid particles cytC can be introduced in the cells by phagocytoses. In the present work we study the adsorption of cytC on montmorillonite (MM) particles combining the electro-optic and electrophoretic techniques. MM particles were chosen as nanoplates having negative pH-independent charge and high ratio surface/mass. The measurements were done at pH 6.5 where cytC globule is positively charged. The main employed method is the electric light scattering based on orientation of colloid particles in sinusoidal electric field. Interfacial electric polarizability was obtained from the degree of orientation at steady-state and the particle size – from the relaxation time after the field switching off. Microelectrophoresis was used to monitor the alteration of the surface charge at protein adsorption. The cytC-concentration dependence of the polarizability and the mobility shows out that the total (net) charge of cytC-MM complex turns its sign from negative to positive, the isoelectric point appears at 5:3mg/mg (0.135mol/kg) cytC/MM and saturated protein adsorption is reached at additional twofold increasing of cytC/MM ratio. The suspension is stable at low and high protein concentrations, at intermediate ones aggregation arises.

Intradimer/Intermolecular Interactions Suggest Autoinhibition Mechanism in Endophilin A1

Endophilin A1 is a homodimeric membrane-binding endocytic accessory protein with a high dimerization affinity. Its function has been hypothesized to involve autoinhibition. However, the autoinhibition mechanism, as well as the physicochemical basis for the high dimerization affinity of endophilin in solution, have remained unclear. In this contribution, we use a Förster resonance energy transfer (FRET) method to investigate the homodimerization mechanism and intradimer molecular interactions in endophilin. For the endophilin N-BAR domain (which lacks the SH3 domain including a linker region of the full length protein), we observe a large temperature dependence of the dimerization affinity and dimer dissociation kinetics, implying large dimerization enthalpy and dissociation activation enthalpy, respectively. Our evaluation of the protein concentration dependence of dimer dissociation kinetics implies that endophilin reversibly forms monomers via a dissociation/reassociation mechanism. Furthermore, we use a kinetic method that allows us to compare the dissociation kinetics of full-length endophilin to that of truncated mutants. We find that mutants that lack either H0 helix or SH3 domain show significantly faster dissociation kinetics relative to full-length endophilin. This observation supports the presence of an intradimer, intermonomer cross-interaction between H0 helix and SH3 domain from different subunits within a homodimer. Because the H0 helix is known to play a significant role in endophilin’s membrane interactions, our measurements support a syngergistic model where these interactions are inhibited in the absence of SH3 domain binding ligands such as dynamin’s prolin rich domains, and where the binding of these ligands may be suppressed for non-membrane-bound endophilin.

Interaction of silver nanoparticles with proteins: A characteristic protein concentration dependent profile of SPR signal

Silver nanoparticles are finding increasing applications in biological systems, for example as antimicrobial agents and potential candidates for control drug release systems. In all such applications, silver nanoparticles interact with proteins and other biomolecules. Hence, the study of such interactions is of considerable importance. While BSA has been extensively used as a model protein for the study of interaction with the silver nanoparticles, studies using other proteins are rather limited. The interaction of silver nanoparticles with light leads to collective oscillation of the conducting electrons giving rise to surface plasmon resonance (SPR). Here, we have studied the protein concentration dependence of the SPR band profiles for a number of proteins. We found that for all the proteins, with increase in concentration, the SPR band intensity initially decreased, reaching minima and then increased again leading to a characteristic “dip and rise” pattern. Minimum point of the pattern appeared to be related to the isoelectric point of the proteins. Detailed dynamic light scattering and transmission electron microscopy studies revealed that the consistency of SPR profile was dependent on the average particle size and state of association of the silver nanoparticles with the change in the protein concentration. Fluorescence spectroscopic studies showed the binding constants of the proteins with the silver nanoparticles were in the nano molar range with more than one nanoparticle binding to protein molecule. Structural studies demonstrate that protein retains its native-like structure on the nanoparticle surface unless the molar ratio of silver nanoparticles to protein exceeds 10. Our study reveals that nature of the protein concentration dependent profile of SPR signal is a general phenomena and mostly independent of the size and structure of the proteins.

Self-association of the galectin-9 C-terminal domain via the opposite surface of the sugar-binding site

Galectin-9 is a lectin, which has various biological functions such as T-cell differentiation and apoptosis. Multivalency of carbohydrate binding is required for galectin-9 to function. Although galectin-1 (a proto-type galectin) forms an oligomer to obtain its multivalency, galectin-9 (a tandem-repeat-type one) has two carbohydrate recognition domains (CRD) in one polypeptide. However, a single CRD of galectin-9, especially the C-terminal one, exhibited pro-apoptotic activity suggesting oligomer formation capability. In this study, we monitored the nuclear magnetic resonance (NMR) signals of the backbone atoms of the galectin-9 C-terminal CRD (G9CCRD). Protein concentration dependence of the signals suggested that a region (F1-F4 strands) opposite to the ligand-binding site was involved in the self-association of G9CCRD. Site-directed mutagenesis in this region (Leu210, Trp277 and Leu279 to Thr; G9CCRD-3T) inhibited the self-association of G9CCRD, and improved the solubility, whereas it reduced its pro-apoptotic activity towards T cells. The high pro-apoptotic activity of G9CCRD seems to be due to the ability to form an oligomer. In addition, the same substitution in two-CRD-containing galectin-9 (G9Null-3T) also diminished the self-association and improved its solubility, although it hardly reduced the anti-proliferative and pro-apoptotic activities. G9CCRD contributes the self-association of full-length galectin-9 at high protein concentrations.

Gelation behaviour and rheological properties of acid-induced soy protein-stabilized emulsion gels

The protein concentration dependence on the rheological properties of acid-induced gels formed with unheated and heated soy protein-stabilized emulsions (UHSPE and HSPE) was investigated at different acidification temperatures. Pre-heat treatment on soy protein solutions resulted in a higher storage modulus (G′) and a shorter gelation time (tgel) of acid-induced emulsion gels. A maximum in tan δ was observed in the UHSPE gels but no maximum was detected in the HSPE gels. Increasing the acidification temperature decreased the G′ and tgel. The dependence of the G′ on the protein concentration (c) can be scaled with a power law: G′ ∼ cA. The exponent (A) increased with pre-heat treatment and acidification temperature. The experimental data.fitted the fractal scaling model (G′ ∼ φA) and the simple time- scaling model above very well for the acid-induced soy protein-stabilized HSPE gels with varying oil volume fraction. The large deformation and fracture properties were significantly affected by soy protein concentration, pre-heat treatment, acidification temperature and volume fraction of oil droplets (p < 0.05).