Comparing Bulk Aerosol Profiles in the Mixed Layer in Coastal Los Angeles and the Inland Empire

Characteristic westerly sea breeze carries air over the Los Angeles Basin in Southern California to the Inland Empire approximately 50 miles inland, directly impacting air quality in both of these two highly polluted regions. As particles play a critical role in air quality and human health, this study compares the bulk aerosol profiles of the Los Angeles pollution source and Inland Empire receptor regions during the 2013 and 2014 NASA Student Airborne Research Program (SARP) campaigns onboard the NASA DC-8 airborne laboratory. The source and receptor regions were characterized by a series of missed approaches at the Los Angeles International Airport, Long Beach Airport, and Los Alamitos Army Airfield (coastal sources) as well as the Ontario International Airport, San Bernardino International Airport, and March Air Reserve Base (inland receptors). The aerosol populations in each region were compared, and the changes evolved were analyzed alongside volatile organic compound (VOC) concentrations from Whole Air Samples. Particle size distributions were collected using a Droplet Measurement Technologies Ultra High Sensitivity Aerosol Spectrometer (DMT-UHSAS). Aerosol concentration, mass, and mode diameter increased significantly between coastal pollution source and inland pollution receptor regions in all cases, along with an increase in mode diameter. The observed changes cannot be accounted for by aerosol aging over the Los Angeles basin alone, suggesting new particle emission/formation over this region could be a dominating factor in the changes. Positive correlations between particle increases at receptor sites and anthropogenic VOC tracers will be discussed

Protein-adaptive Differential Scanning Fluorimetry (paDSF): dyes and analyses for flexible in vitro measurements of protein state

As the focus of biological research expands beyond traditional enzymatic activities, protein apparent thermal stability has become an important generalizable read-out for protein biochemical states. Differential Scanning Fluorimetry (DSF) is a method which uses an environmentally sensitive dye, typically SYPRO Orange, to determine the apparent thermal melting temperature (Tma) of a purified protein. DSF is used widely across disciplines, from high throughput ligand discovery to buffer optimizations for structural biology. However, widespread protein incompatibilities with both the dyes and analyses for DSF left applications severely limited. Here, we share the discovery of over 100 novel dyes, updated theoretical models, and improved analyses methods for DSF. These discoveries achieve the flexibility necessary to adapt DSF to diverse proteins on demand, increasing protein compatibility from 29 to 94% of proteins. By providing the dye library and analyses alongside efficient approaches to select among them for a given protein, this work expands the functionality of DSF without compromising its ease and efficiency. The dye library is termed “Aurora”, the updated analyses “DSFworld”, and the expanded version of DSF made possible using them “protein-adaptive DSF” (paDSF). paDSF can support new understanding of protein functions, and greater ability to modulate these functions with small molecules

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's-related Tau protein

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides

Chemical validation of a druggable site on Hsp27/HSPB1 using in silico solvent mapping and biophysical methods

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library