A genetically-encoded biosensor for direct detection of perfluorooctanoic acid.

Determination of per- and polyfluoroalkyl substances (PFAS) in drinking water at the low levels set by regulatory officials has been a major focus for sensor developing researchers. However, it is becoming more apparent that detection of these contaminants in soils, foods and consumer products is relevant and necessary at part per billion and even part per million levels. Here, a fluorescent biosensor for the rapid detection of PFOA was engineered based on human liver fatty acid binding protein (hLFABP). By conjugating circularly permuted green fluorescent protein (cp.GFP) to a split hLFABP construct, the biosensor was able to detect perfluorooctanoic acid PFOA in PBS as well as environmental water samples with LODs of 236 and 330 ppb respectively. Furthermore, E. coli cells cytosolically expressing the protein-based sensor were demonstrated to quickly detect PFOA, demonstrating feasibility of whole-cell sensing. Overall, this work demonstrates a platform technology utilizing a circularly permuted GFP and split hLFABP conjugate as a label-free optical biosensor for PFOA.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

Engineering human liver fatty acid binding protein for detection of poly- and perfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are a large group of synthetic fluorinated chemicals with surface active and water-repellent properties. The combination of wide-spread use in numerous consumer and industrial products and extended biological half-lives arising from strong carbon-fluorine bonds has led to significant accumulation of PFAS in humans. As most human interaction with PFAS comes from ingestion, it is important to be able to detect PFAS in drinking water as well as in agricultural water. Here we present an approach to designing a fluorescence-based biosensor for the rapid detection of PFAS based on human liver fatty acid binding protein (hLFABP). Introduction of solvatochromic fluorophores within the ligand binding pocket (L50) allowed for intrinsic detection of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) via blue-shifts in fluorescence emission spectra. Initially, a single tryptophan mutation (L50W) was found to be able to detect PFOA with a limit of detection (LOD) of 2.8 ppm. We improved the sensitivity of the biosensor by exchanging tryptophan for the thiol reactive fluorophore, acrylodan. The acrylodan conjugated C69S/F50C hLFABP variant is capable of detecting PFOA, PFOS, and PFHxS in PBS with LODs of 112 ppb, 345 ppb, and 1.09 ppm, respectively. The protein-based sensor is also capable of detecting these contaminants at similar ranges in spiked environmental water samples, including samples containing an interfering anionic surfactant sodium dodecyl sulfate. Overall, this study demonstrates engineered hLFABP is a useful platform for detection of PFAS in environmental water samples and highlights its ease of use and versatility in field applications.