This communication summarizes findings from the earliest encounters with extreme enthalpy‒entropy compensation, a phenomenon first detected in the 1950s by a reappraisal of isopiestic and calorimetric measurements on aqueous urea solutions in terms of solute self-association. Because concurrent studies of carboxylic acid association were confined to measurement of the equilibrium constant by conductance, IR spectrophotometry or potentiometric titration measurements, temperature-independence of the dimerization constant was mistakenly taken to signify a value of zero for Δ H^o instead of (Δ H^o ‒ TΔ S^o ). In those studies of small-solute self-association the extreme enthalpy‒entropy compensation was reflecting the action of water as a reactant whose hydroxyl groups were competing for the solute carbonyl involved in self-association. Such action gives rise to a positive temperature dependence of Δ H^o that could well be operating in concert with that responsible for the commonly observed negative dependence for protein‒ligand interactions exhibiting extreme enthalpy‒entropy compensation, where the solvent contribution to the energetics reflects changes in the extent of ordered water structure in hydrophobic environments.

Abstract Most albumin in blood plasma is thought to be monomeric with some 5% covalently dimerized. However, many reports in the recent biophysics literature find that albumin is reversibly dimerized or even oligomerized. We review data on this from X‐ray crystallography and diverse biophysical techniques. The number‐average molecular weight of albumin would be increased by dimerization, affecting size‐dependent filtration processes of albumin such as at the glycocalyx of the capillary endothelium and the podocyte slit‐diaphragm of the renal glomerulus. If correct, and depending on characteristics of the process, such as Kd, reversible dimerization of albumin in plasma would have major implications for normal physiology and medicine. We present quantitative models of the impact of dimerization on albumin molecular forms, on the number‐average molecular weight of albumin, and estimate the effect on the colloid osmotic pressure of albumin. Dimerization reduces colloid osmotic pressure as total albumin concentration increases below that expected in the absence of dimerization. Current models of albumin filtration by the renal glomerulus would need revision to account for the dynamic size of albumin molecules filtered. More robust biophysical data are needed to give a definitive answer to the questions posed and we suggest possible approaches to this.

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-β1 (TGF-β1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-β1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5 and αvβ6 and to the TGFβRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Galectin-3 binding to β1 integrin was validated in vitro by co-immunoprecipitation in HLFs. Proximity ligation assays indicated galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface, that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 stimulation, colocalization was detectable only in HLFs from IPF patients suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients reduced Col1a1, TIMP1 and HA secretion to a similar degree as TGF-β type I receptor inhibitor. These data suggest galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.

IEDB screen for MHC class II binding epitopes

Overview of the kinetic binding parameters of the parental 88 mAbs

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

Individual tumor growth curves from mice included in the in vivo COLO205 xenograft model

Negligible direct cell killing ability by i129G1 on low to moderate binding cancer cell lines compared to the high-binding COLO205

This study establishes the existence of substantial agreement between published results from traditional boundary spreading measurements (including synthetic boundary measurements in the analytical ultracenrifuge) on two globular proteins (bovine serum albumin, ovalbumin) and the concentration dependence of diffusion coefficient predicted for experiments conducted under the operative thermodynamic constraints of constant temperature and solvent chemical potential. Although slight negative concentration dependence of the translational diffusion coefficient is the experimentally observed as well as theoretically predicted, the extent of the concentration dependence is within the limits of experimental uncertainty inherent in diffusion coefficient measurement. Attention is then directed toward the ionic strength dependence of the concentration dependence coefficient (k(D)) describing diffusion coefficients obtained by dynamic light scattering, where, in principle, the operative thermodynamic constraints of constant temperature and pressure preclude consideration of results in terms of single-solute theory. Nevertheless, good agreement between predicted and published experimental ionic strength dependencies of k(D) for lysozyme and an immunoglobulin is observed by a minor adaptation of the theoretical treatment to accommodate the fact that thermodynamic activity is monitored on the molal concentration scale because of the constraint of constant pressure that pertains in dynamic light scattering experiments.

ABSTRACT The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

This investigation examines the source of the disparity between experimental values of the light scattering second virial coefficient A_2 (mL.mol/g 2 ) for proteins and those predicted on the statistical mechanical basis of excluded volume. A much better theoretical description of published results for lysozyme is obtained by considering the experimental parameters to monitor the difference between the thermodynamic excluded volume term and its hydrodynamic counterpart. This involves a combination of parameters quantifying concentration dependence of the translational diffusion coefficient obtained from dynamic light scattering measurements. That finding is shown to account for observations of a strong correlation between A_2M_2 (mL/g), where M 2 is the molar mass (molecular weight) of the macromolecule and the diffusion concentration parameter k_D (mL/g). On the grounds that k_D is regarded as a hydrodynamic parameter, the same status should be accorded the light scattering second virial coefficient rather than its current incorrect thermodynamic designation as B_2 (mL.mol/g 2 ), or just B , the osmotic second virial coefficient for protein self-interaction.

Cartoon representation of the key Fc-engineered constructs

This study aims to understand possible effects of flavour compounds on the structure and conformation of endogenous proteins. Using methyl anthranilate (a grape flavour compound added to drinks, confectionery, and vape-liquids) and bovine serum albumin (BSA, a model serum protein) we designed experimental investigations using analytical ultracentrifugation, size exclusion chromatography small angle X-ray scattering, and fluorescence spectroscopy to reveal that methyl anthranilate spontaneously binds to BSA (ΔG°, ca. -21 KJ mol-1) which induces a conformational compactness (ca. 10 %) in the monomer structure. Complementary molecular modelling and dynamics simulations suggested the binding occurs at Sudlow II of BSA via establishment of hydrogen bonds with arginine409, lysine413 and serine488 leading to an increased conformational order in domains IA, IIB and IIIB. This work aims to set the foundation for future research on flavour-protein interactions and offer new sets of opportunities for understanding the effects of small compounds on protein structure.

Oncogenic translational programmes underpin cancer development and are often driven by dysregulation of oncogenic signalling pathways that converge on the eukaryotic translation initiation (eIF) 4F complex. Altered eIF4F activity promotes translation of oncogene mRNAs that typically contain highly structured 5’UTRs rendering their translation strongly dependent on RNA unwinding by the DEAD-box helicase eIF4A1 subunit of the eIF4F complex. While eIF4A1-dependent mRNAs have been widely investigated, it is still unclear how highly structured mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that RNA sequence motifs regulate eIF4A1 unwinding activity in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which recruit and stimulate eIF4A1 unwinding of localised RNA structure to facilitate mRNA translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA substrate resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex region that supports efficient unwinding. Hence, we conclude a model in which mRNAs utilise AG-rich sequences to specifically recruit eIF4A1, enabling assembly of the helicase-active multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

AbstractMurine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved (“i”) chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286–306 and 339–378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa–targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential.Significance:Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Background: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3'-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. Methods: Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. Results: AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed beta-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14-3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. Conclusions: This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection. Significance: The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14-3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14-3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy.

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

Isothermal titration calorimetry (ITC) has become one of the mainstays of determining the affinity of small molecules with macromolecules. As such there has been a resurgence in interest in the underlying processes that contribute to a ‘good’ ITC experiment. In this article we discuss the thermodynamic origins of the data seen in an ITC experiment, some of the background theory and exemplars of what good data look like. We also discuss the origins of more complex phenomena seen with ITC, and how these may be interpreted.

This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions—the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20–100 g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein–cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory $$A_{2}^{\text{app}}$$ still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source–protein self-interactions and/or protein–cosolute contributions.

The feasibility of employing molecular crowding cosolutes to facilitate the detection of protein self-association by zonal size exclusion chromatography is investigated. Theoretical considerations have established that although the cosolute-induced displacement of a self-association equilibrium towards the oligomeric state invariably occurs in the mobile phase of the column, that displacement is only manifested as a decreased protein elution volume for cosolutes sufficiently small to partition between the mobile and stationary phases. Indeed, the use of a crowding agent sufficiently large to be confined to the mobile phase gives rise to an increased elution volume that could be misconstrued as evidence of cosolute-induced protein dissociation. Those theoretical considerations are reinforced by experimental studies of α-chymotrypsin (a reversibly dimerizing enzyme) on Superdex 200. The use of cosolutes such as sucrose and small polyethylene glycol fractions such as PEG-2000 is therefore recommended for the detection of protein self-association by molecular crowding effects in size exclusion chromatography.