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Abstract Here, we present a Python based software that allows for the rapid visualization, data mining, and basic model applications of quartz crystal microbalance with dissipation data. Our implementation begins with a Tkinter GUI to prompt the user for all required information, such as file name/location, selection of baseline time, and overtones for visualization (with customization capabilities). These inputs are then fed to a workflow that will use the baseline time to scrub and temporally shift data using the Pandas and Numpy libraries and carry out the plot options for visualization. The last stage consists of an interactive plot, that presents the data and allows the user to select ranges in MatPlotLib-generated panels, followed by application of data models, including Sauerbrey, thin films in liquid, among others, that are carried out with NumPy and SciPy. The implementation of this software allows for simple and expedited data analysis,in lieuof time consuming and labor-intensive spreadsheet analysis. Metadata 

Abstract Intrinsically disordered proteins and protein regions (IDPs) are prevalent in all proteomes and are essential to cellular function. Unlike folded proteins, IDPs exist in an ensemble of dissimilar conformations. Despite this structural plasticity, intramolecular interactions create sequence-specific structural biases that determine an IDP ensemble’s three-dimensional shape. Such structural biases can be key to IDP function and are often measured in vitro, but whether those biases are preserved inside the cell is unclear. Here we show that structural biases in IDP ensembles found in vitro are recapitulated inside human-derived cells. We further reveal that structural biases can change in a sequence-dependent manner due to changes in the intracellular milieu, subcellular localization, and intramolecular interactions with tethered well-folded domains. We propose that the structural sensitivity of IDP ensembles can be leveraged for biological function, can be the underlying cause of IDP-driven pathology or can be used to design disorder-based biosensors and actuators. 

Abstract Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti‐inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti‐chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion‐tagged chemokine is produced inEscherichia coli, then efficient cleavage of the N‐terminal fusion partner is carried out with lab‐produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab‐produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP‐fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti‐inflammatory therapeutic, showing a binding constant for vCCI:vMIP‐fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP‐fluor) can be used in competition assays with other chemokines and we report aK_dfor vCCI:CCL17 of 14 μM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations. 

Abstract Pathogenic dsDNA prompts AIM2 assembly leading to the formation of the inflammasome, a multimeric complex that triggers the inflammatory response. The recognition of foreign dsDNA involves AIM2 self-assembly concomitant with dsDNA binding. However, we lack mechanistic and kinetic information on the formation and propagation of the assembly, which can shed light on innate immunity’s time response and specificity. Combining optical traps and confocal fluorescence microscopy, we determine here the association and dissociation rates of the AIM2-DNA complex at the single molecule level. We identify distinct mechanisms for oligomer growth via the binding of incoming AIM2 molecules to adjacent dsDNA or direct interaction with bound AIM2 assemblies, resembling primary and secondary nucleation. Through these mechanisms, the size of AIM2 oligomers can increase fourfold in seconds. Finally, our data indicate that single AIM2 molecules do not diffuse/scan along the DNA, suggesting that oligomerization depends on stochastic encounters with DNA and/or DNA-bound AIM2. 

Abstract The costs of foraging can be high while also carrying significant risks, especially for consumers feeding at the top of the food chain.To mitigate these risks, many predators supplement active hunting with scavenging and kleptoparasitic behaviours, in some cases specializing in these alternative modes of predation.The factors that drive differential utilization of these tactics from species to species are not well understood.Here, we use an energetics approach to investigate the survival advantages of hunting, scavenging and kleptoparasitism as a function of predator, prey and potential competitor body sizes for terrestrial mammalian carnivores.The results of our framework reveal that predator tactics become more diverse closer to starvation, while the deployment of scavenging and kleptoparasitism is strongly constrained by the ratio of predator to prey body size.Our model accurately predicts a behavioural transition away from hunting towards alternative modes of predation with increasing prey size for predators spanning an order of magnitude in body size, closely matching observational data across a range of species.We then show that this behavioural boundary follows an allometric power‐law scaling relationship where the predator size scales with an exponent nearing 3/4 with prey size, meaning that this behavioural switch occurs at relatively larger threshold prey body size for larger carnivores.We suggest that our approach may provide a holistic framework for guiding future observational efforts exploring the diverse array of predator foraging behaviours. 

DNA scanning proteins slide on the DNA assisted by a clamping interface and uniquely recognize their cognate sequence motif. The transcription factors that control cell fate in eukaryotes must forgo these elements to gain access to both naked DNA and chromatin, so whether or how they scan DNA is unknown. Here we use single-molecule techniques to investigate DNA scanning by the Engrailed homeodomain (enHD) as paradigm of promiscuous recognition and open DNA interaction. We find that enHD scans DNA as fast and extensively as conventional scanners and 10,000,000 fold faster than expected for a continuous promiscuous slide. Our results indicate that such supercharged scanning involves stochastic alternants between local sequence sweeps of ∼85 bp and very rapid deployments to locations ∼500 bp afar. The scanning mechanism of enHD reveals a strategy perfectly suited for the highly complex environments of eukaryotic cells that might be generally used by pioneer transcription factors. TeaserEukaryotic transcription factors can efficiently scan DNA using a rather special mechanism based on promiscuous recognition. 

ABSTRACT The protein ASC polymerizes into intricate filament networks to assemble the inflammasome, a filamentous multiprotein complex that triggers the inflammatory response. ASC carries two Death Domains integrally involved in protein self-association for filament assembly. We have leveraged this behavior to create non-covalent, pH-responsive hydrogels of full-length, folded ASC by carefully controlling the pH as a critical factor in the polymerization process. We show that natural variants of ASC (ASC isoforms) involved in inflammasome regulation also undergo hydrogelation. To further demonstrate this general capability, we engineered proteins inspired in the ASC structure that successfully form hydrogels. We analyzed the structural network of the natural and engineered protein hydrogels using transmission and scanning electron microscopy, and studied their viscoelastic behavior by shear rheology. Our results reveal one of the very few examples of hydrogels created by the self-assembly of globular proteins and domains in their native conformation and show that Death Domains can be used alone or as building blocks to engineer bioinspired hydrogels. 

Like their prokaryotic counterparts, eukaryotic transcription factors must recognize specific DNA sites, search for them efficiently, and bind to them to help recruit or block the transcription machinery. For eukaryotic factors, however, the genetic signals are extremely complex and scattered over vast, multichromosome genomes, while the DNA interplay occurs in a varying landscape defined by chromatin remodeling events and epigenetic modifications. Eukaryotic factors are rich in intrinsically disordered regions and are also distinct in their recognition of short DNA motifs and utilization of open DNA interaction interfaces as ways to gain access to DNA on nucleosomes. Recent findings are revealing the profound, unforeseen implications of such characteristics for the mechanisms of DNA interplay. In this review we discuss these implications and how they are shaping the eukaryotic transcription control paradigm into one of promiscuous signal recognition, highly dynamic interactions, heterogeneous DNA scanning, and multiprong conformational control. 

Metamorphic proteins switch reversibly between two differently folded states under a variety of environmental conditions. Their identification and prediction are gaining attention, but the fundamental physicochemical basis for fold switching remains poorly understood. In this Perspective article, we address this problem by surveying the landscape of well-characterized metamorphic proteins and noting that a significant fraction of them display temperature sensitivity. We then make the case that the dependence on temperature, in particular cold-denaturation effects, is likely to be an underlying property of many metamorphic proteins regardless of their ultimate triggering mechanisms, especially those with a single domain. The argument is supported by rigorous analysis of hydrophobic effects in each well-characterized metamorphic protein pair and a description of how these parameters relate to temperature. The conclusion discusses the relevance of these insights to a better understanding of prediction, evolution, and de novo design strategies for metamorphic proteins.