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

From MDPI via Jisc Publications RouterHistory: accepted 2021-11-03, pub-electronic 2021-11-09Publication status: PublishedA 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. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo

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 0.3-3.3 THz (10-110 cm(-1)). As the BSA concentration was raised, there was a nonlinear change in absorption inconsistent with Beer's law. At low BSA concentrations (0-1 mM), the absorption remained constant or rose slightly. Above a concentration of 1 mM BSA, a steady decrease in absorption was observed, which was followed by a plateau that started at 2.5 mM. Using a overlapping hydration layer model, the hydration layer was estimated to extend 15 Å from the protein. Calculation of the corrected absorption coefficient (αcorr) for the water around BSA by subtracting the excluded volume of the protein provides an alternative approach to studying the hydration layer that provides evidence for complexity in the population of water around BSA.This is the accepted version of an article first published in The Journal of Physical Chemistry A. The version of record is available from ACS Publications at http://pubs.acs.org/doi/abs/10.1021/jp407410

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/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.</jats:p

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 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day-1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L-1 h-1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of -1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7-9 °C, relative to those at 1-4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14-18% at 25-100 mg/mL, relative to those at 1-4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh

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. Stabilization of lysozyme can be explained by the free energy required to hydrate the protein as it unfolds. At low ion concentrations, the protein’s hydration layer is at equilibrium with the bulk water. After the transition, bulk water is depleted and the protein is competing for water with the ions. With competition for water between the protein and the ions at higher salt concentrations, the free energy required to hydrate the interior of the protein rises and it is this that stabilizes the protein structure

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy

Interaction between Macromolecules, Inorganic Salts and Small Organic Molecules

Solutes are present within aqueous systems in almost every area of research. The term solute can include salts, small organic molecules, proteins and any other chemical that can be dissolved. Understanding how solutes influence the properties of each other and medium they are dissolved in is of paramount importance if a system is to be fully understood, but the extent to which solutes perturb their medium is often overlooked. The aim of this research project was to use analytical techniques such as differential scanning calorimetry, pressure perturbation calorimetry and terahertz spectroscopy to better understand the mechanism by which water and solutes influence protein stability. Experimental results suggest that Hofmeister ions influence protein stability at higher salt concentrations by modulating the free energy required to hydrate the newly exposed core of the protein. Destabilising ions reduce the free energy required to hydrate the protein core and stabilising ions increase the free energy. At low salt concentrations salts influence protein stability to a small degree by interacting electrostatically with proteins. Pressure perturbation calorimetry studies suggested that Hofmeister ions are able to influence water dynamics at elevated temperatures through their electric field. These findings support the hypothesis that ions are able to stabilise proteins by competing for water with the unfolding protein through an electrostatic interaction. Coherent synchrotron radiation in the terahertz region of the electromagnetic spectrum was able to detect an extended hydration layer around bovine serum albumin. These findings supported other terahertz spectroscopy experiments that also detected extended hydration layers around proteins and suggests that water around protein molecules is more complex than a single layer of water molecules

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. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo

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 (B22), and ζ-potential values for lysozyme solutions. B22 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. Reentrant condensation is a phenomenon in which low concentrations of polyvalent ions induce protein precipitation, but further increasing polyvalent ion concentration causes the protein precipitate to resolubilize. Interestingly, citrate does not induce lysozyme reentrant condensation despite having a similar charge, size, and shape to pyrophosphate. We observe qualitative differences in protein behavior when compared against negatively charged proteins in solutions of trivalent cations. The polyphosphate ions induce a much stronger protein–protein attraction, which correlates with the occurrence of a liquid–gel transition that replaces the liquid–liquid transition observed with trivalent cations. The results indicate that solutions of polyphosphate ions provide a model system for exploring the link between the protein-phase diagram and model interaction potentials and also highlight the importance that ion-specific effects can have on protein solubility