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We present a carboxylate-targeting reactive fragment screening platform using 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. This work will provide a simple accessible method to rapidly discover tool molecules to interrogate important biological targets.

Background The relationship between environmentally transmitted tick parasites, Ixodes spp., and their main reproductive host, deer, is generally thought to be positive. However, measuring host abundance and density directly can be challenging and indirect methods are often used. The observed relationship between the parasite and host may be affected by sampling scale and season, which could lead to different inferences being made. Here, we aimed to test the effect of sampling scale and season on the relationship between density of deer and the density of questing Ixodes ricinus nymphs. Methods The density of deer (primarily Dama dama) was estimated using line transect distance sampling of deer dung quantified in different seasons (winter and summer) and measured at three different nested scales (site, transect and observation level). Questing nymph density was measured using blanket drag methods and estimates were calculated at the same scales as deer density estimates. General linear models were used to evaluate the relationship between questing nymphs, deer density and other environmental variables at each sampling scale and each season deer density was measured at. Results While a positive relationship between deer density and questing nymph density was detected at the site and transect scale, no relationship was apparent at the observation level. This was likely due to increased variation and reduced precision of deer dung counts at the finest sampling scale. Seasonal changes in deer populations were observed likely reflecting seasonal shifts in habitat usage. The summer estimates of deer density explained questing nymph density whereas winter estimates did not. Conclusions Our results show that the scale of sampling can affect the detectability of the positive association between host and vector species. Furthermore, such associations can be obscured if hosts exhibit seasonal changes in habitat use. Thus, both sampling scale and season are important to consider when investigating the relationship between host and vector species.

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising strategy. However, high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance.Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which blocks both p53/MDM2 and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA and protoporphyrin IX (PpIX). Ion-mobility mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.Contribution to the fieldGiven the immense importance of the p53 tumor suppressor for cancer, efforts have been made to identify p53 activators, which sterically inhibit MDM2. Because high-affinity MDM2 inhibitors are facing problems with considerable side effects, other approaches are needed to reactivate p53 for improved cancer therapy. The allosteric mechanism of action of p53 activator RITA, which we discovered, and its dependence on the oncogenic switch, is an unexpected turn in the p53 story. Our findings provide a basis for the development of p53 activators with a similar mode of functioning, either through the classical drug discovery route or through the drug repurposing approach. Allosteric modulators might have great potential as single agents, or in combination with the standard of care. Further, p53 modulators could serve as invaluable tools to better understand its biology.

Background Animal-attached devices can be used on cryptic species to measure their movement and behaviour, enabling unprecedented insights into fundamental aspects of animal ecology and behaviour. However, direct observations of subjects are often still necessary to translate biologging data accurately into meaningful behaviours. As many elusive species cannot easily be observed in the wild, captive or domestic surrogates are typically used to calibrate data from devices. However, the utility of this approach remains equivocal. Methods Here, we assess the validity of using captive conspecifics, and phylogenetically-similar domesticated counterparts (surrogate species) for calibrating behaviour classification. Tri-axial accelerometers and tri-axial magnetometers were used with behavioural observations to build random forest models to predict the behaviours. We applied these methods using captive Alpine ibex (Capra ibex) and a domestic counterpart, pygmy goats (Capra aegagrus hircus), to predict the behaviour including terrain slope for locomotion behaviours of captive Alpine ibex. Results Behavioural classification of captive Alpine ibex and domestic pygmy goats was highly accurate (> 98%). Model performance was reduced when using data split per individual, i.e., classifying behaviour of individuals not used to train models (mean ± sd = 56.1 ± 11%). Behavioural classifications using domestic counterparts, i.e., pygmy goat observations to predict ibex behaviour, however, were not sufficient to predict all behaviours of a phylogenetically similar species accurately (> 55%). Conclusions We demonstrate methods to refine the use of random forest models to classify behaviours of both captive and free-living animal species. We suggest there are two main reasons for reduced accuracy when using a domestic counterpart to predict the behaviour of a wild species in captivity; domestication leading to morphological differences and the terrain of the environment in which the animals were observed. We also identify limitations when behaviour is predicted in individuals that are not used to train models. Our results demonstrate that biologging device calibration needs to be conducted using: (i) with similar conspecifics, and (ii) in an area where they can perform behaviours on terrain that reflects that of species in the wild.

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design., Graphical Abstract, Highlights • HDX-MS reveals complex formation impact on conformational dynamics of CysK and CysE. • CysK binding ensures conformational symmetry within the CysE trimer. • Long-range allostery propagates through CysE stabilizing the inter-trimer interface. • Insights into the allostery of CS complex could help guide antibacterial drug design., In Brief We have used hydrogen/deuterium exchange MS to unveil the allosteric changes occurring during complex formation of CysE and CysK, the last two enzymes of cysteine biosynthetic pathway in bacteria. Significant changes in conformation and dynamics occur in each protein upon complex formation, including long distance intraprotein and interprotein communication. Being absent in mammals, both CysE and CysK represent potential targets for new antibacterial drugs, and our results on the formation and allostery of the complex could help guide drug development.

Background Tri-axial accelerometers are frequently deployed on terrestrial quadrupedal mammals using collars, because they are easy to fit and are thought to have minimal impact on the subject. Collar-attached devices are not fixed to the body and can move independently of the body. This may result in inaccurate measures of acceleration, reducing the accuracy of measured body movement. We determined the effect of collar size and collar weight on acceleration measured by a collar-mounted accelerometer on a quadruped mammal. The aim was to suggest best practice for sizes and weights of collars on which to deploy tri-axial accelerometers. Using pygmy goats, Capra aegagrus hircus, which were trained to walk at different speeds (0.8–3.0 km/h) on a treadmill, we measured body acceleration using a collar-mounted tri-axial accelerometer, with different collar sizes (individual neck circumference + 1 cm to + 9 cm) and collar weight (0.4% to 1.2% of individual weight). Results There was a significant effect of collar size, collar weight and walking speed on measured acceleration. Measured acceleration was less accurate and more variable when collars were looser and heavier. To measure body acceleration more accurately, we found that collar size should be within 5 cm or 16% of an individual’s neck circumference when it was heavy (up to 1.2% of animal’s body weight) or within 7 cm (33%) of neck circumference if the collar was light (up to 0.6% of animal body weight). Conclusion We suggest that not only reporting collar size and weight for welfare purposes, but it is also important to consider these aspects for scientific rigour, to ensure data are collected as accurately as possible. We provide guidelines for researchers fitting collar-attached devices to ensure a higher degree of accuracy of recorded body acceleration.

A novel mass spectrometry approach is reported which investigate how ion-molecule charge reduction reactions between radical anions and protein cations modulate protein conformation. An electron transfer reagent (1,3-dicyanobenzene) transfers electrons to positively charged proteins and there are no observable products of dissociation. ETnoD product ions are detected as charged-reduced species with the same molecular weight as the precursor ion, and no significant evidence for proton transfer. We present collision cross section distributions of precursor and product ions before and after exposure to radical ions. Cytochrome c and myoglobin are examined as exemplar systems under both aqueous salt and denaturing conditions before and after exposure to radical anions. We consistently observe depletion of the more compact precursor ion conformers on exposure to the ETD reagent. Remarkably, by examining the collision cross section distributions of the product ions it can be seen that the addition of a single electron can cause a dramatic rearrangement in protein conformation for charge states that are highly populated when sprayed from salty aqueous conditions. Furthermore, a given net charge on an exposed precursor and product ion favours a preferred collision cross section distribution, indicating that the distribution of charge on proteins in the gas phase dictate their conformation. An exception is reported for the low charge state of cytochrome c where compaction was seen in the radical formed post reduction compared to the electrospray generated ion under ETnoD optimised conditions. We propose a model that postulates how electron transfer to conformation stabilising salt bridges may explain our observations.

Weir construction has fragmented many rivers, resulting in the exclusion of some fish populations from suitable habitat. A cheap retrofit fishway for small, sloping weirs is the Low Cost Baffle (LCB) solution – A series of notched baffles perpendicular to flow on the downstream weir face, generating an angled passage route across the weir face. To test the degree to which LCBs can pass upstream-moving, lowland-river fish at steep weirs, LCBs were fitted onto a 1:3.3-sloping gauging-weir face, in an urban tributary of the River Thames, England. The study also compared the passage of wild and stocked fish (the latter are employed to facilitate population recovery in restored English rivers). Passive Integrated Transponder (PIT) antennas were positioned on the weir to record the upstream movement of PIT-tagged barbel (Barbus barbus; nstock = 120), chub (Squalius cephalus; nstock = 119; nwild = 194), dace (Leuciscus leuciscus; nwild = 50), and roach (Rutilus rutilus; nwild = 30). Over six months, more stocked fish attempted passage (58.9%) than wild (14.6%; χ21 = 26.7, p

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14–1.1 s time frame. The HDX of Hb at 0.14–1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14–1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14–1.1 s time frame. The HDX of Hb at 0.14–1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14–1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.

Given the immense significance of p53 restoration for anti-cancer therapy and that p53-activating molecules are in clinical trials, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of a novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in allosteric regulation of p53, whose targeting by small molecules RITA, PpIX and licofelone blocks the binding of two p53 inhibitors, MDM2 and MDMX, thereby restoring p53 function. Deletion and mutation analysis followed by molecular modeling and its thorough validation, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site in p53 induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators as targeted drugs. Our study provides a basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacopeia.

Given the immense significance of p53 restoration for anti-cancer therapy, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of a novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in p53 regulation, whose targeting by RITA, PpIX and licofelone blocks the binding of p53 inhibitors, MDM2 and MDMX. Deletion and mutation analysis followed by molecular modelling, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators. Our study provides basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacological potential.

The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.

Developing drug-like molecules to inhibit the interactions formed by disordered proteins is desirable due to the high correlation of disorder with protein implicated in disease, but is challenging due in part to the lack of atomistically resolved and resolvable structures from conformationally dynamic systems. Ion mobility mass spectrometry (IM-MS) is well-positioned to assess protein ligand interactions along with the effect of a given inhibitor on conformation. Here we demonstrate the use of IM-MS to characterize the effect of two inhibitors RITA and Nutlin-3 on their respective binding partners: p53 and MDM2. RITA binds N-terminal transactivation domain of p53 (Np53) weakly, preventing direct observation of the complex in the gas phase. Nonetheless, upon incubation with RITA, we observe an alteration in the charge state distribution and in the conformational distributions adopted by Np53 in the gas phase. This finding supports the hypothesis that RITAs mode of action proceeds via a conformational change in p53. Circular dichroism corroborates our gas phase findings, showing a slight increase in secondary structure content on ligand incubation, and HDX-MS experiments also highlight the dynamic properties of this protein. Using the same approach we present data to show the effect of Nutlin-3 binding to the N-terminal domain of MDM2 (N-MDM2), N-MDM2 presents as at least two conformational families in the absence of Nutlin-3. Upon Nutlin-3 binding, the protein undergoes a compaction event similar to that exhibited by RITA on Np53. This multi-technique approach highlights the inherent disorder in these systems; and in particular exemplifies the power of IM-MS as a technique to study transient interactions between small molecule inhibitors and intrinsically disordered proteins.

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.