Function Of Protein Concentration Research Articles

Stacked graphene nanoplatelet paper sensor for protein detection

Carbon-based sensors have shown great potential to revolutionize protein diagnostic tools, including the ability to detect pathogens and biomarkers. Many different microdevices have been fabricated using carbon nanotubes and graphene nanoplatelets. However, creating devices on the nanoscale can be difficult and expensive. Stacked graphene nanoparticle composites (hereafter called graphene paper) are a convenient and novel technology that has not been previously reported as a carbon-based sensor platform. Graphene paper is inexpensive and available on a macroscale, making device construction simple. Additional advantages include that paper composition and additives can be manipulated to increase the sensitivity and eventually the selectivity of proteins. We have created a microdevice sensor that detects proteins in solution by measuring the surface electrical resistivity of graphene/cellulose composite paper as a function of protein concentration. Four different proteins were tested for their ability to change the surface resistivity of the graphene paper and there was a clear correlation between the molecular weight of the protein and the equilibrium dissociation constant calculated by fitting the protein adsorption data to the Langmuir isotherm model. This result implies that the dissociation constant is likely a function of the size of the protein.

66 Architectural role of HMO1 in bending, bridging, and compacting DNA

HMO1 proteins are abundant Saccharomyces cerevisiae (yeast) High Mobility Group Box (HMGB) protein (Kamau, Bauerla & Grove, 2004). HMGB proteins are nuclear proteins which are known to be architectural proteins (Travers, 2003). HMO1 possesses two HMGB box domains. It has been reported that double box HMGB proteins induce strong bends upon binding to DNA. It is also believed that they play an essential role in reorganizing chromatin and, therefore, are likely to be involved in gene activation. To characterize DNA binding we combine single molecule stretching experiments and AFM imaging of HMO1 proteins bound to DNA. By stretching DNA bound to HMO1, we determine the dissociation constant, measure protein induced average DNA bending angles, and determine the rate at which torsional constraint of the DNA is released by the protein. To further investigate the local nature of the binding, AFM images of HMO1-DNA complexes are imaged, and we probe the behavior of these complexes as a function of protein concentration. The results show that at lower concentrations, HMO1 preferentially binds to the ends of the double helix and links to the separate DNA strands. At higher concentrations HMO1 induces formation of a complex network that reorganizes DNA. Although HMG nuclear proteins are under intense investigation, little is known about HMO1. Our studies suggest that HMO1 proteins may facilitate interactions between multiple DNA molecules.

A simple rapid method for protein determination

A simple, rapid method is described for determination of protein in the range of 20-200 µg. The procedure is based on the reaction between protein and amido black 10B. An insoluble residue is precipitated and the dye concentration in solution decreases. The difference between the optical density of the blank (without protein) and sample with protein is a linear function of protein content.

Lysozyme Hydration in Concentrated Aqueous Solutions. Effect of an Equilibrium Cluster Phase

Water close to proteins plays a key role in determining their structural and functional properties. Despite being a subject of considerable interest, the characterization of hydration water, as far as its total amount is concerned, is still controversial and its influence on protein structure and folding is not yet fully understood. In this work, we have investigated the dielectric properties of lysozyme aqueous solutions over the frequency range where the orientational polarization relaxation of the aqueous phase occurs (from 500 MHz to 50 GHz). Measurements extend over a wide concentration range, up to 300 mg/mL, corresponding to a volume fraction of the order of 0.4. The analysis of the dielectric spectra, based on the decrease of the dielectric increment of the γ-dispersion as a function of protein concentration, allows us to estimate the total amount of hydration water (both bound water and loosely bound water) present in the system investigated. We observe a decrease of the hydration number as a function of the protein concentration. This behavior is well accounted for by considering the formation of small equilibrium clusters with aggregation number of some units, as recently reported by Stradner et al. (1) on the basis of small-angle X-ray and neutron scattering measurements.

Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein–DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.

Interpretation of Protein Adsorption through Its Intrinsic Electric Charges: A Comparative Study Using a Field-Effect Transistor, Surface Plasmon Resonance, and Quartz Crystal Microbalance

We describe the highly sensitive detection of the nonspecific adsorption of proteins onto a 1-undecanethiol self-assembled monolayer (SAM)-formed gold electrode by parallel analysis using field effect transistor (FET), surface plasmon resonance (SPR), and quartz crystal microbalance (QCM) sensors. The FET sensor detects the innate electric charges of the adsorbed protein at the electrode/solution interface, transforming the change in charge density into a potentiometric signal in real time, without the requirement for labels. In particular, using the Debye-Huckel model, the degree of potential shift was proportional to the dry mass of adsorbed albumin and β-casein. A comparison of the FET signal with SPR and QCM data provided information on the conformation and orientation of the surface-bound protein by observing characteristic break points in the correlation slopes between the signals. These slope transitions reflect a multistage process that occurs upon protein adsorption as a function of protein concentration, including interim coverage, film dehydration, and monolayer condensation. The FET biosensor, in combination with SPR and QCM, represents a new technology for interrogating protein-material interactions both quantitatively and qualitatively.

Lactose Repressor Experimental Folding Landscape: Fundamental Functional Unit and Tetramer Folding Mechanisms

The fundamental principles that govern monomer folding are believed to be congruent with those of protein oligomers. However, the effects of protein assembly during the folding reaction can result in a series of complex transitions that are considerably more challenging to deconvolute. Here we developed the experimental protein folding mechanism for the lactose repressor (LacI), for both the dimeric and the tetrameric states, using equilibrium unfolding and kinetic experiments, and by leveraging the previously reported monomer folding landscape. Reaction details for LacI oligomers were observed by way of circular dichroism, intrinsic fluorescence, and Förster resonance energy transfer (FRET) and as a function of protein concentration. In general, the dimer and tetramer are four-phase folding reactions in which the first three transitions are tantamount to the folding of constituent monomers. The final reaction phase of the LacI dimer can be attributed to protein assembly, based on the concentration dependence of the observed folding rates and intermolecular FRET measurements. Unlike the dimer, the latter reaction phase of the LacI tetramer is not dependent on protein concentration, likely because of a strong tethering of the monomers, which simplifies the folding reaction by eliminating an explicit protein assembly phase. Finally, folding of the LacI dimer and tetramer was assessed in the presence of polyethylene glycol to rule out inert molecular crowding as the driving force for the protein folding reaction; in addition, these data provide insight into the folding mechanism in vivo.

Nanoscale adhesion, friction and wear of proteins on polystyrene

Protein layers are routinely deployed on biomaterials and biological micro/nanoelectromechanical systems (bioMEMS/NEMS) as a functional layer allowing for specific molecular recognition, binding properties or to facilitate biocompatibility. In addition, uncoated biomaterial surfaces will have uncontrolled protein layers adsorbing to the surface within seconds of implantation, so a pre-defined protein layer will improve the host response. Implanted biomaterials also experience micromotion over time which may degrade any surface protein layers. Degradation of these protein layers may lead to system failure or an unwanted immune response. Therefore, it is important to characterize the interfacial properties of proteins on biomaterial surfaces. In this study, the nanoscale adhesion, friction and wear properties of proteins adsorbed to a spin coated polystyrene surface were measured using atomic force microscopy (AFM) in deionized (DI) water and phosphate buffered saline. Adhesion, friction and wear have been measured for bovine serum albumin (BSA), collagen, fibronectin and streptavidin (STA) in DI water and PBS as a function of protein concentration. These proteins were chosen due to their importance and widespread application in the biotechnology field. Adhesion and friction were also measured for BSA and STA at two different temperatures and different pH values to simulate a biological environment. Based on this study, adhesion, friction and wear mechanisms of the different proteins are discussed.

An Application of Ultraviolet Spectroscopy to Study Interactions in Proteins Solutions at High Concentrations

Studies of protein-protein interactions (PPIs), especially in high-concentration solutions, have become increasingly important from a pharmaceutical perspective. Analytical methods used to study protein interactions, however, rely primarily on the detection of nonideality in relatively dilute (<50 mg/mL) solutions. We present here an application of variable-pathlength ultraviolet (UV)-visible absorption spectroscopy to examine and better understand such interactions over a wide concentration range (5-240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (ΔAbs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ΔAbs, corrected for light scattering, was found to increase with protein concentration for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody). Because PPIs influence solution viscosity, we studied the correlation between ΔAbs measurements and viscosity as a function of protein concentration. The magnitude of ΔAbs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ΔAbs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.

Behaviour of oil droplets during spray drying of milk-protein-stabilised oil-in-water emulsions

The effects of spray drying on the behaviour of oil droplets in oil-in-water emulsions (12.0%, w/w, maltodextrin; 20.0%, w/w, soya oil) stabilised with either sodium caseinate or whey protein isolate (WPI) were examined as a function of protein concentration (0.5–5.0%, w/w). Spray drying and redispersion caused a shift in the droplet size distribution to larger values for all emulsions made using low protein concentrations (0.5–2.0%, w/w), in comparison with their respective parent emulsions. However, the droplet size distribution was affected only very slightly by spray drying when the protein concentration was above 2.0% (w/w). The effects of maltodextrin concentration (1.0–25.0%, w/w) on the behaviour of WPI-stabilised emulsions (0.5–10.0%, w/w, WPI, 20.0%, w/w, soya oil) were also examined. Emulsions containing low levels of maltodextrin showed marked re-coalescence during spray drying and redispersion even at a WPI concentration of 10.0% (w/w).

How actin network dynamics control the onset of actin-based motility

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including "primer" contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Micromechanical PDGF recognition via lab-on-a-disc aptasensor arrays

A plug-and-play CD-like platform is used to perform a statistical detection of platelet derived growth factor (PDGF) proteins through aptamer-based surface functionalization of multiple microcantilever arrays. When PDGF proteins bind to aptamer coatings, the cantilevers deflect. The deflection response is monitored by optical read-out units from a commercial DVD-ROM device. We report on the use of an improved sensing platform which facilitates measurements under continuous liquid flow and with temperature control. Also, the mechanical wobbling of the DVD-ROM platform has been minimized and the scanning system has been optimized in order to detect cantilever deflections in liquid with nanometer scale resolution. The capability of the sensing platform is demonstrated by detection of clinically relevant concentrations of PDGF proteins. We present statistical measurements on 100 microcantilevers at different concentrations of PDGF, ranging from 10nM to 400nM. Hereby it is possible to reliably characterize the averaged mechanical response of cantilevers as a function of protein concentration.

Specific Domains in Yeast Translation Initiation Factor eIF4G Strongly Bias RNA Unwinding Activity of the eIF4F Complex toward Duplexes with 5′-Overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

Potential role of the binding of whey proteins to human buccal cells on the perception of astringency in whey protein beverages

Whey protein beverages have been shown to be astringent, which means that they are not appealing to consumers. The exact mechanism of astringency in whey protein beverages is yet to be fully elucidated. In this preliminary study, the binding between β-lactoglobulin (β-LG), lactoferrin (LF) and human oral epithelial cells (HSC-2 and NO-1–N-1 cells) at pH 3.5 and pH 7.4 was assayed as a function of protein concentration using an enzyme-linked immunosorbent assay. The binding of β-LG and LF to HSC-2 and NO-1-N-1 cells was dependent on protein type, protein concentration, pH and time. The intensity of the binding to HSC-2 and NO-1–N-1 cells was much greater for LF than for β-LG and was protein concentration dependent, which was consistent with the in vivo astringency perception of LF and β-LG. The findings demonstrated that the binding interaction between whey proteins and human oral epithelial cells may play an important role in the perception of astringency in whey protein beverages.

The energetic contributions of scaffolding and coat proteins to the assembly of bacteriophage procapsids

In vitro assembly of bacteriophage P22 procapsids requires coat protein and sub-stoichiometric concentrations of the internal scaffolding protein. If there is no scaffolding protein, coat protein assembles aberrantly, but only at higher concentrations. Too much scaffolding protein results in partial procapsids. By treating the procapsid as a lattice that can bind and be stabilized by scaffolding protein we dissect procapsid assembly as a function of protein concentration and scaffolding/coat protein ratio. We observe that (i) the coat–coat association is weaker for procapsids than for aberrant polymer formation, (ii) scaffolding protein makes a small but sufficient contribution to stability to favor the procapsid form, and (iii) there are multiple classes of scaffolding protein binding sites. This approach should be applicable to other heterogeneous virus assembly reactions and will facilitate our ability to manipulate such in vitro reactions to probe assembly, and for development of nanoparticles.

Influence of the presence of monoglyceride on the interfacial properties of soy protein isolate

This study focused on the contribution of soy protein isolate (SPI), in the absence or presence of monostearin (ME), to surface and interfacial properties as a function of protein concentration and pH, which is relevant to the physical stability of a variety of food systems. An increase in protein content always yielded a rapid decrease in surface tension followed by an evolution towards an asymptotic value. Addition of ME gave rise to mixed SPI/ME films, although the interface became dominated by SPI above the concentration for interfacial saturation. The relative interfacial shear viscosity of SPI films showed a marked dependence on: aging time, which may be attributed to a reorganisation of protein species at the interface with some penetration of hydrophobic parts into the oil phase; shear forces, which may partially reverse this reorganisation, leading to shear-thickening behaviour; and pH, which is the key factor controlling which SPI species is predominant at the interface. The effect of adding ME also depends on pH, favouring a reinforcement of SPI/ME films only at low pH, at which 3S and 7S fractions are dominant. The results obtained indicate that SPI shows excellent potential to favour stabilisation of air/water and oil/water interfaces in food systems.

Single-Molecule DNA Repair in Live Bacteria

Cells rely on sophisticated DNA repair mechanisms to maintain integrity of a genome that is under constant attack by mutagenic chemicals, ionizing radiation, or UV light. Single-molecule fluorescence microscopy allows, for the first time, direct observation of DNA repair in living bacteria. This in vivo approach enables a quantitative description of the chain of events within an entire repair pathway, studying control, timing, and coordination during the initial damage response, the search for damaged sites, and the repair protein function.Using photoactivation, super-resolution localization, and tracking we follow the movement of individual molecules of DNA Polymerase I, a protein essential for DNA repair synthesis and lagging strand replication in E.coli. Consistent with early reports, we measure 300 copies of Pol1 per cell; the molecules have a spatial distribution reflecting that of the bacterial chromosome. In cells without DNA damage, most Pol1 molecules move with an apparent diffusion coefficient of 1.75 um2/s, consistent with simulations of unhindered Brownian motion within a cell. We observe significant displacement of Pol1 molecules between consecutive 15-ms frames, setting an upper limit on the DNA residence time in a facilitated diffusion model that combines 1-D diffusion on DNA with a 3-D random walk in the cytoplasm.In contrast, in cells with DNA damage by mutagen methyl-methanesulfonate (MMS), we clearly observe stationary Pol1 molecules, and individual tracks showing binding and dissociation events; the fraction of bound-to-free Pol1 scales with the damage extent. These observations suggest that stationary Pol1 molecules are performing repair synthesis to fill short single-stranded DNA gaps created by the base excision repair (BER) machinery. Current work focuses on the real-time DNA damage response as a function of protein concentration and binding activity, and examining additional repair proteins to build a comprehensive model of the BER pathway in vivo.

COMPARISON OF ADSORPTION BEHAVIOR OF BOVINE SERUM ALBUMIN AND OSTEOPONTIN ON HYDROXYAPATITE AND ALUMINA

Adsorption behavior of bovine serum albumin (BSA) and osteopontin (OPN) on osteoconductive hydroxyapatite (HA) and non-osteoconductive alpha-type alumina (-Al2O3) was studied as function of protein concentration and pH, using Bradford dye binding assay. BSA showed a much larger binding capacity on -Al2O3 than that on HA irrespective of BSA solution concentration and pH. Furthermore, OPN is likely to show the same adsorption characteristic as BSA. Definite correlation was not observed between the albumin or OPN adsorption capacity and the osteoconductivity of materials, suggesting that other factors (e.g., orientation, arrangement, etc. of albumin and/or OPN) likely govern expression of the osteoconductivity. (Received February 2, 2012; Accepted March 30, 2012)

New routes to food gels and glasses

We describe the possibility to create solid-like protein samples whose structural and mechanical properties can be varied and tailored over an extremely large range in a very controlled way through an arrested spinodal decomposition process. We use aqueous lysozyme solutions as a model globular protein system. A combination of video microscopy, small-angle neutron and X-ray scattering and reverse Monte Carlo modeling is used to characterize the structure of the bicontinuous network with two coexisting phases of a dilute protein solution and a glassy or arrested dense protein backbone at all relevant length scales. Rheological measurements are then used to determine the complex mechanical response of these protein gels as a function of protein concentration and quench temperature. While in particular the origin of the dependence of the mechanical properties on quench depth and concentration is not well understood currently, it seems ultimately connected to the particular bicontinuous structure of the arrested spinodal network created by the interplay between the early stage of a spinodal decomposition and the position of the glass line. We then generalize this behavior and discuss how this could open up new routes to prepare gel-like food systems with adjustable structural and mechanical properties. We present results from a first feasibility study where we use a depletion interaction caused by the addition of small non-adsorbing polymers to suspensions of casein micelles in order to create food gels with tunable structural and mechanical properties through an arrested spinodal decomposition process.