Effect Of Non-specific Interactions Research Articles

Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry.

Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein–stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein–stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.

Quantitative characterization of temperature-independent polymer-polymer interaction and temperature-dependent protein-protein and protein-polymer interactions in concentrated polymer solutions.

To study the effect of non-specific interactions arising from proteins being in a crowded environment on physiological processes, the self-interaction of concentrated Dextran T70 and Ficoll 70 and the interactions between a dilute protein and these polymeric macromolecules were quantified using non-ideal tracer sedimentation equilibrium. Sedimentation equilibria of each polymer were measured between 5 and 37°C, and sedimentation equilibria of 2mgcm-3 superoxide dismutase (SOD) in 0-0.1gcm-3 of each polymer was also measured. Results were analyzed using a model-free thermodynamic virial expression of activity coefficients in terms of the concentration of polymer and a structural model using a statistical thermodynamics approximation. The equilibrium gradients of each of the polymers suggest repulsive interaction, which is independent of temperature. However, the net repulsive interaction between superoxide dismutase (SOD) species and the polymers is dependent on temperature. The ratio of the solvation energy of SOD in Dextran T70 to that in Ficoll 70, lnγSOD(Dex)/lnγSOD(Fic) at the same w/v concentration was about 1.8 at 37°C, 1.6 at the intermediate temperature, and ranges from 1.2 to 1.6 at 5°C over the entire concentration range. The enthalpy and entropy of interaction of SOD with dilute Dextran T70 are - 14kJmol-1 and - 5.6JK-1 mol-1, respectively. For SOD in dilute Ficoll 70, the enthalpy and entropy are - 8.1kJmol-1 and 12.9JK-1mol-1, respectively. Thus, Dextran T70 contributes more to the attractive protein-polymer interaction and to self-association of protein than Ficoll 70 and reasons for this are discussed.

Effect of non-specific interactions on formation and stability of specific complexes

We introduce a simple model to describe the interplay between specific and non-specific interactions. We study the influence of various physical factors on the static and dynamic properties of the specific interactions of our model and show that contrary to intuitive expectations, non-specific interactions can assist in the formation of specific complexes and increase their stability. We then discuss the relevance of these results for biological systems.

Protein interaction patterns in different cellular environments are revealed by in-cell NMR

In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins.

The effect of non-specific interactions on cellular adhesion using model surfaces

The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH2), and methyl (CH3) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH3 < COOH ≈ epoxide ≪ NH2. The NH2 SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.

Investigation of Interactions Between Drug Enantiomers and Flavoprotein as a Chiral Selector by Affinity Capillary Electrophoresis

Interactions between drug enantiomers and flavoprotein as a pseudo chiral stationary phase were investigated by using affinity capillary electrophoresis (CE) in order to avoid the effects of nonspecific interactions that occur in chiral HPLC. Circular dichroism (CD) measurement was used to monitor changes of the secondary structure of flavoprotein under various analysis conditions in affinity CE. The chiral discrimination region for ketoprofen on the flavoprotein surface was concluded to consist of α-helix structure, and the decrease of chiral separation ability with increase of methanol content in the electrophoretic buffer was directly related to conformational change of the α-helix. Studies with chemically modified flavoprotein indicated that two types of interaction at the chiral discrimination region are required for chiral separation: π-π interaction of a tryptophan residue with the aromatic ring of ketoprofen, and ionic interaction of the carboxyl group of ketoprofen with an amino group and a carboxyl group of the protein.

Retention Behavior of Amino Acids Using Immobilized Ag(I) Chromatography

AbstractA mathematical description of retention times for several amino acids on a polyacrylamide resin containing immobilized Ag(I) is presented in this paper. Immobilization of Ag(I) ions onto a chromatography support allows for the expansion of immobilized metal affinity chromatography (IMAC) to include soft acid/soft base interactions. For the retention of methionine, histidine, and tryptophan, a mathematical model is presented that rationalizes the retention behavior of these amino acids on a Ag(I) column as a function of silver ion loading and pH. The pH effect is important in that methionine is retained longer than histidine at pH values below 5, while at pH 7 histidine is retained longer than methionine. The effects of nonspecific interactions with the modified polymer, electrostatic interactions, and soft acid/soft base interactions are also taken into account by the model. This approach may also be useful for modeling the retention times of amino acids on other types of IMAC columns.

Biospecific adsorption of lysozyme onto monoclonal antibody ligand immobilized on nonporous silica particles

It is very important to understand the equilibrium and dynamic characteristics of biospecific adsorption (affinity chromatography) for both scientific and application purposes. Experimental equilibrium and dynamic column data are presented on the adsorption of lysozyme onto antibody immobilized on nonporous silica particles. The Langmuir model is found to represent the equilibrium experimental data satisfactorily, and the equilibrium association constants and heats of adsorption have been estimated for two systems with different ligand densities. The effects of nonspecific interactions are more pronounced in the system with low-density ligand. The dynamic interaction kinetic parameters are estimated by matching the predictions of a fixed-bed model with the experimental breakthrough curves. The agreement between theory and experiment is good for the initial phases of breakthrough, where the mechanism of biospecific adsorption is dominant. In the later phase (saturation neighborhood) of breakthrough, the effects of nonspecific interactions appear to be greater in the low-density ligand system. The kinetics of the nonspecific interactions were estimated from the data of the later phase of breakthrough and were found to be considerably slower than those attributed to biospecific adsorption.

RNA-DNA hybridization: demonstration in a mammalian system of competition by preincubation.

Nonspecific intermolecular interactions obscure the interpretation of competition experiments involving hybridization of DNA and RNA in mammalian systems when the labeled RNA/DNA ratio is low. The effect of these interactions can be minimized by choosing a sufficiently high ratio of labeled RNA/DNA. No serious effect of nonspecific interactions is observed in bacteriophage systems, even at very low labeled RNA/DNA ratios.