Synthetic T-Cell Receptor-like Protein Behaves as a Janus Particle in Solution.

Protein engineering enables the creation of tailor-made proteins for a variety of applications. ImmTACs stand out as promising therapeutics for cancer and other treatments while also presenting unique challenges for stability, formulation, and delivery. We have shown that ImmTACs behave as Janus particles in solution, leading to self-association at low concentrations, even when the average protein-protein interactions suggest that the molecule should be stable.

Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations.

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.

Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme.

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR).

ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism.

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering.

Measuring Nonspecific Protein-Protein Interactions by Dynamic Light Scattering.

Dynamic light scattering has become a method of choice for measuring and quantifying weak, nonspecific protein-protein interactions due to its ease of use, minimal sample consumption, and amenability to high-throughput screening via plate readers. A procedure is given on how to prepare protein samples, carry out measurements by commonly used experimental setups including flow through systems, plate readers, and cuvettes, and analyze the correlation functions to obtain diffusion coefficient data. The chapter concludes by a theoretical section that derives and rationalizes the correlation between diffusion coefficient measurements and protein-protein interactions.

Controlling Phase Separation of Lysozyme with Polyvalent Anions.

The ability of polyvalent anions to influence protein-protein interactions and protein net charge was investigated through solubility and turbidity experiments, determination of osmotic second virial coefficients ( B), and ζ-potential values for lysozyme solutions.  B values showed that all anions reduce protein-protein repulsion between positively charged lysozyme molecules, and those anions with higher net valencies are more effective. The polyvalent anions pyrophosphate and tripolyphosphate were observed to induce protein reentrant condensation, which has been previously observed with negatively charged proteins in the presence of trivalent cations.

Analysis of Mesoscopic Structured 2-Propanol/Water Mixtures Using Pressure Perturbation Calorimetry and Molecular Dynamic Simulation.

In this paper we demonstrate the application of pressure perturbation calorimetry (PPC) to the characterization of 2-propanol/water mixtures. PPC of different 2-propanol/water mixtures provides two useful measurements: (i) the change in heat (Δ); and (ii) the [Formula: see text] value. The results demonstrate that the Δ values of the mixtures deviate from that expected for a random mixture, with a maximum at ~20-25 mol% 2-propanol.

Molecular Mechanism for the Hofmeister Effect Derived from NMR and DSC Measurements on Barnase.

The effects of sodium thiocyanate, sodium chloride, and sodium sulfate on the ribonuclease barnase were studied using differential scanning calorimetry (DSC) and NMR. Both measurements reveal specific and saturable binding at low anion concentrations (up to 250 mM), which produces localized conformational and energetic effects that are unrelated to the Hofmeister series. The binding of sulfate slows intramolecular motions, as revealed by peak broadening in C heteronuclear single quantum coherence spectroscopy.

A study of the relationship between water and anions of the Hofmeister series using pressure perturbation calorimetry.

Pressure perturbation calorimetry (PPC) was used to study the relationship between water and sodium salts with a range of different anions. At temperatures around 25 °C the heat on pressurisation (ΔQ) from 1 to 5 bar was negative for all solutions relative to pure water. The raw data showed that as the temperature rose, the gradient was positive relative to pure water and the transition temperature where ΔQ was zero was related to anion surface charge density and was more pronounced for the low-charge density anions.

Three stages of lysozyme thermal stabilization by high and medium charge density anions.

Addition of high and medium charge density anions (phosphate, sulfate, and chloride) to lysozyme in pure water demonstrates three stages for stabilization of the protein structure. The first two stages have a minor impact on lysozyme stability and are probably associated with direct interaction of the ions with charged and partial charges on the protein's surface. There is a clear transition between the second and third stages; in the case of sodium chloride, disodium sulfate and disodium hydrogen phosphate this is at 550, 210, and 120 mM, respectively.

Biopharmaceutical liquid formulation: a review of the science of protein stability and solubility in aqueous environments.

Formulation scientists employed in the biopharmaceutical industry face the challenge of creating liquid aqueous formulations for proteins that never had evolutionary pressure to be exceptionally stable or soluble. Yet commercial products usually need a shelf life of 2 years to be economically viable. The research done in this field is dominated by physical chemists who have developed theories like preferential interaction, preferential hydration and excluded volume to explain the mechanisms for the interaction between salt, small organic molecules and proteins.

Analysis of the hydration water around bovine serum albumin using terahertz coherent synchrotron radiation.

Terahertz spectroscopy was used to study the absorption of bovine serum albumin (BSA) in water. The Diamond Light Source operating in a low alpha mode generated coherent synchrotron radiation that covered a useable spectral bandwidth of 0.3-3.

Thermal stability of lysozyme as a function of ion concentration: a reappraisal of the relationship between the Hofmeister series and protein stability.

Anion and cation effects on the structural stability of lysozyme were investigated using differential scanning calorimetry. At low concentrations (<5 mM) anions and cations alter the stability of lysozyme but they do not follow the Hofmeister (or inverse Hofmeister) series. At higher concentrations protein stabilization follows the well-established Hofmeister series.

Dual effects of sodium phytate on the structural stability and solubility of proteins.

The interaction between sodium phytate and three proteins was studied using solubility experiments and differential scanning calorimetry (DSC) to assess structural stability. Lysozyme, which is positively charged at neutral pH, bound phytate by an electrostatic interaction. There was evidence that phytate cross-linked lysozyme molecules forcing them out of solution.

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella.

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site.