Depression of T-cell Epitope Generation by Stabilizing Hen Lysozyme

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.

Intact and proteolytically modified human serpins, alpha 1-proteinase inhibitor, antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor, were compared by circular dichroism, fluorescence spectroscopy, and resistance against unfolding by guanidine HCl. The modified proteins were prepared from the intact and active inhibitors by selective proteolytic cleavage in their reactive site loops and tested for complete loss of activity. Significant differences in the spectral properties between intact and modified inhibitors indicate that a major conformational rearrangement is triggered by the cleavage. This leads to a large increase in conformational stability as demonstrated by large shifts of the transition profiles recorded as a function of guanidine HCl concentration at 20 degrees C by circular dichroism at 220 nm. Intact inhibitors were unfolded in two steps of about equal size centered at 0.8-1.7 and 2.5-3.5 M concentrations of the denaturant, respectively. Under identical conditions modified inhibitors are completely stable, and their denaturation occurs only well above 4 M guanidine HCl in one or two steep transition steps. From the similarity of the spectral changes and shifts in transition profiles for all four serpins studied it is concluded that the conformational changes and stabilization triggered by the modification hit is an important common mechanistic feature of this class of inhibitors. This is supported by the observation that ovalbumin, which is homologous with the serpins but apparently lacks inhibitory activity, exhibits neither spectral changes nor a significant change in stability upon proteolytic modification.

The molecular thermodynamic model studied is based on the two‐state mechanism of inactivation, in which only native folded and polymorphous unfolded protein forms are present at equilibrium. The influence of solvent on protein stability is described in terms of perturbation of the protein distribution between the two conformational states. An expression derived for the chemical potential of the protein accounts for conformational changes, ideal mixing effects, and interaction of the protein with the surrounding medium.Thermal unfolding of lysozyme was then studied in the absence or presence of hydroxylic compounds. Ultraviolet difference spectroscopy was used to monitor the conformational changes induced by heating and to determine the melting temperature of the protein. The additives investigated are ethanol, glycols, and natural osmolytes. Media containing ethanol and glycols destabilized lysozyme, whereas sugars increased the conformational stability of the protein. For all of the systems examined the melting temperature was linearly related to the surface tension of the mixed solvent, supporting the ability of the model to describe the influence of the solvent and composition on lysozyme unfolding. Model predictions agreed fairly well with published differential scanning calorimetric data. The influence of hydroxylic additives on protein's conformational stability does not extend to any special property of these components, but to their ability to perturb the surface tension of water. This model can be used to interpret and correlate thermal unfolding data and to solve the problem of protein stabilization.

Increasing Protein Conformational Stability by Optimizing β-Turn Sequence

Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry.

The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes was investigated employing alpha- and gamma-chymotrypsin. As a measure of the conformational stability the melting temperature T(m) was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier-transform infrared spectroscopy employing two regions sensitive to protein structure, the amide-I (1600-1700 cm(-1)) and amide-III (1215-1335 cm(-1)). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the alpha-helix and a significant increase in the beta-sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization-induced structural changes by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization-induced structural alterations, we conducted co-lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air-drying experiment, mainly causes protein structural alterations.

Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.

Apparently contradictory statements about the thermodynamics of aqueous protein solutions and of hydrophobic effect are quoted and discussed. Some credibility is found in the divergent points of view and it is pointed out that they focus attention on different aspects of the complicated conditions in aqueous solutions, some of which are more important than others for the stability of protein conformations.

Polymeric carriers designed to encapsulate protein antigens have great potential for improving the efficacy of vaccines and immunotherapeutics for diseases such as cancer. We recently developed a carrier system based on polyacrylamide hydrogel microparticles cross-linked with acid-labile moieties. After being phagocytosed by antigen-presenting cells, the protein encapsulated within the carrier is released and processed for subsequent presentation of antigenic epitopes. To understand the impact of particle size on the activation of T-cells following uptake by antigen-presenting cells, particles with mean diameters of 3.5 microm and 35 nm encapsulating a model protein antigen were synthesized by emulsion and microemulsion based polymerization techniques, respectively. In vivo tests demonstrated that both sizes of particles were effective at stimulating the proliferation of T-cells and were capable of generating an antigen-specific cytotoxic T-cell response when coadministered with immunostimulatory DNA. Contrary to previous reports in the literature, our results suggest that there is no significant difference in the magnitude of T-cell activation for the two sizes of particles used in these experiments. This disparity in findings may be related to fundamental differences in material properties of the carriers used in these studies, such as the hydrophilicity of the polyacrylamide particles described here versus the hydrophobic nature of carriers investigated by other groups.

Interpolyelectrolyte complexes of lysozyme with short poly[di(carboxylatophenoxy)phosphazene]. Binding energetics and protein conformational stability

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Proteins must fold to a globular conformation to carry out the most important tasks in living organisms. The folded, biologically active conformation of a protein is only marginally more stable than the unfolded, inactive conformations. Thus, making proteins more stable is important in medicine and basic research. The major destabilising force that must be overcome is conformational entropy. The major stabilising forces are the hydrophobic effect and hydrogen bonding. The ionisable side chains of amino acid residues may also contribute favourably to protein stability through attractive charge–charge interactions, ion pair formation or the formation of hydrogen bonds when such groups are buried in the protein interior. Replacing a nonproline or nonglycine residue in a β‐turn can also significantly increase protein stability. Key Concepts: Under physiological conditions, a folded protein is ∼20 to 60 kJ mol –1 more stable than unfolded forms. The major destabilising force to protein folding is conformational entropy, which contributes ∼7 kJ mol −1 per residue. The major stabilising forces are the hydrophobic effect, where the burying of each –CH 2 – contributes ∼−5 kJ mol −1 , and hydrogen bonding, especially buried intramolecular hydrogen bonds, which may contribute ∼−7 kJ mol −1 per bond. The ionisable side chains of amino acid residues may contribute favourably to protein stability through attractive charge–charge interactions, ion pair formation and hydrogen bonding when the ionisable group is buried in the protein interior. Replacing a nonproline or nonglycine residue in a β‐turn may increase protein stability.