Protein Binding Strength Research Articles

Desorption properties of BSA and LSZ on carbonate ion-modified apatite

Desorption behavior of two proteins; bovine serum albumin (BSA) and egg white lysozyme (LSZ) was followed on hydroxyapatite (HAp) after 72 h-adsorption in a static system, focusing modification effect of carbonate ion on the HAp surface. Desorbed amounts in a 5 mM - phosphate buffer solution (PBS) showed maxima in 2 h for BSA and 1 h for LSZ. The ratio of the maximal desorbed amount to the initially adsorbed one: the desorption rate was chosen as an index of the binding strength of protein to the surface. For LSZ, the desorption rate decreased as increase of the carbonate content, indicating stronger binding. This was ascribed to the increase of the strongly-adsorption site of LSZ: the P site of the c face because the increase of the carbonate in HAp inhibits crystal growth along the c axis and hence increases the c face. On the other hand, any clear dependency was not found for BSA. However, four samples of the various carbonate contents were divided into two groups; one with the higher desorption rate with nearly full and more than full surface coverages and the other with the lower one of 0.42-0.60, assuming to be side-on (multiple-site) adsorption. This suggested that more numbers of weaker binding sites were involved in BSA adsorption on HAp with the higher desorption rate than the lower one.

Quantifying Protein Microstructure and Electrostatic Effects on the Change in Gibbs Free Energy of Binding in Immobilized Metal Affinity Chromatography

Electrostatic forces play a major role in maintaining both structural and functional properties of proteins. A major component of protein electrostatics is the interactions between the charged or titratable amino acid residues (e.g., Glu, Lys, and His), whose pK(a) (or the change of the pK(a)) value could be used to study protein electrostatics. Here, we report the study of electrostatic forces through experiments using a well-controlled model protein (T4 lysozyme) and its variants. We generated 10 T4 lysozyme variants, in which the electrostatic environment of the histidine residue was perturbed by altering charged and neutral amino acid residues at various distances from the histidine (probe) residue. The electrostatic perturbations were theoretically quantified by calculating the change in free energy (DeltaDeltaG(E)) using Coulomb's law. On the other hand, immobilized metal affinity chromatography (IMAC) was used to quantify these perturbations in terms of protein binding strength or change in free energy of binding (DeltaDeltaG(B)), which varies from -0.53 to 0.99 kcal/mol. For most of the variants, there is a good correlation (R(2) = 0.97) between the theoretical DeltaDeltaG(E) and experimental DeltaDeltaG(B) values. However, there are three deviant variants, whose histidine residue was found to be involved in site-specific interactions (e.g., ion pair and steric hindrance), which were further investigated by molecular dynamics simulation. This report demonstrates that the electrostatic (DeltaDeltaG(Elec)) and microstructural effects (DeltaDeltaG(Micro)) in a protein can be quantified by IMAC through surface histidine mediated protein-metal ion interaction and that the unique microstructure around a histidine residue can be identified by identifying the abnormal binding behaviors during IMAC.

Correlation between Protein Binding Strength on Immobilized Metal Affinity Chromatography and the Histidine-Related Protein Surface Structure

Immobilized metal affinity chromatography (IMAC) was investigated for its ability to characterize the histidine-related surface structure of a protein, that is, a histidine residue's surface accessibility and its potential involvement in intramolecular interactions. T4 lysozyme was chosen as the model protein. Seven amino acid sites were selected on the basis of their relative surface accessibility, and they were substituted with histidine via site-directed protein mutagenesis to generate seven T4 lysozyme variants, each containing only one histidine residue on its surface, with various surface accessibility. IMAC was then used to experimentally quantify the interaction of each lysozyme variant with immobilized copper ions. A direct correlation was shown between the protein binding affinity and the surface accessibility of the histidine residue. Of all the lysozyme variants, K83H and K147H showed unusually low binding strength, as compared with variants having a histidine residue with a similar surface accessibility. However, with the aid of molecular modeling, their relatively low binding affinities were predicted to be the result of the involvement of the histidine residue in intramolecular interactions. In contrast to previously reported results, our results showed that lysozyme still binds to the IMAC column, even if its histidine residue is involved in intramolecular bonding, such as a hydrogen bond, albeit at reduced strength, as compared with the variant containing a histidine residue with a similar surface accessibility.

Increasing the binding strength of proteins to PEI coated supports by immobilizing at high ionic strength

The immobilization of proteins was performed by ionic adsorption on agarose supports coated with polyethyleneimine of high molecular size (PEI) (600 kDa). The immobilization processes were carried out at different ionic strengths (5, 50, 100, 300, 500 and 700 mM NaCl). At NaCl concentrations lower than 100 mM, the immobilization proceeded very rapidly in all cases, while in some cases at concentrations over this value; the adsorption rate was significantly decreased. In all cases, the activity of the enzymes remained over 90% during the immobilization process. Thus, considering the immobilization rate, it seems to be better to use low ionic strength. However, desorption of proteins that had been adsorbed at low ionic strength started when using only moderately high ionic strength. When the enzymes were adsorbed at higher NaCl concentrations the strength of the adsorption was in all cases much higher than when it was performed under the usual low ionic strength. This may be explained assuming that at high ionic strength, proteins can only become immobilized on areas of the protein which are able to simultaneously yield a very intense multipoint adsorption, even permitting the penetration in the polymeric bed structure, while using low ionic strength any area of the protein with negative charges may be involved in the adsorption.

ANION EXCHANGE CHROMATOGRAPHY OF PROTEINS. EFFECT OF CHARGED GROUPS OF THE STATIONARY PHASE ON PROTEIN BINDING

The purpose of this work was to study how the basicity and density of charged groups of anion exchangers affect protein binding strength and chromatographic performance at different sample loading. For this study, several cellulose-based anion exchangers with different ligand basicity and quantity were synthesized. The binding strength of protein by the ion exchangers, and the chromatographic performance were evaluated. Investigations showed that a significant part of the protein adsorbed in the high loading mode may be eluted with 0.05–0.1 M NaCl in Tris-HCl buffer pH 7.5. This indicates very weak binding. It may be the result of multi-layer binding of protein molecules to that already adsorbed on the surface of stationary phase. The amount of weakly bound protein decreases with decreasing of sample loading. Results of gradient elution of BSA adsorbed at low loading mode showed that the binding strength of protein is dependent on the basicity of ionic groups of the stationary phase. A tendency towards improved chromatographic performance was observed with increasing density of charged groups on the surface of the stationary phase.

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers.

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.

Static protein adsorption, ultrafiltration behavior and cleanability of hydrophilized polysulfone membranes

A systematic study on the influence of incrementally increased hydrophilicity of UF membranes made from chemically modified polysulfone on static protein adsorption, protein ultrafiltration performance and cleanability was performed. Hydrophilicity was imparted to the polymeric backbone either by carboxylation or sulfonation. Membranes with 100% retention towards sulfhydryl modified bovine serum albumin (cys-BSA) were made by the phase inversion process from unmodified polysulfone, five carboxylated polysulfones (degrees of carboxylation ranging from 0.26 to 1.74) and two sulfonated polysulfones (degrees of sulfonation of 0.24 and 0.58). 1000 ppm aqueous solutions of cys-BSA at pH values 3, 4.8 and 9 were used in KCl medium at either low or high concentration. Static adsorption, ultrafiltration (UF) flux reduction and protein binding strength were lower at pH 3 than at pH 9 but reached an expected maximum at the protein's isoelectric point at pH 4.8. Hydrophilized polysulfone membranes show less static adsorption and lower UF flux reduction than hydrophobic unmodified membranes. In all experiments we found that the higher the degree of carboxylation or sulfonation, the greater this effect. High ionic strengths reduced these advantages at pH values above or below the IEP but had a positive effect at the IEP.

Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity

We report measurements of the relative binding affinity of CAP for DNA sequences which have been systematically mutated in the region flanking the consensus binding site. Our experiments focus on the locus one helical turn from the dyad axis where DNA bending toward the minor groove is induced upon C-AP binding. The binding free energy and extent of bending are moderately well correlated for the set of 56 sequences. Changes in binding affinity spanning a factor of about 50 could be accounted for by additive contributions of dinucleotides; with a few exceptions, the relative ranking of dinucleotide contributions to binding and bending are similar. We conclude that dinucleotides are the smallest independent unit required for quantitative interpretation of CAP-induced DNA bending and binding in the distal domains of the CAP consensus binding site. The imperfect correlation between binding strength and extent of bending implies that sequence changes affect protein binding strength not only by altering the DNA deformation energy required to form the complex, but also by affecting directly the free energy of interaction between protein and DNA.