Biology subjects

Mills, J. H.

Publications and source records attributed to Mills, J. H..

3 recordsLinked to original sources

bioRxiv2026

Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation

The pathogenic conversion of the cellular prion protein (PrPC) into the {beta}-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and non-infectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.

biochemistry↗

bioRxiv2024

A massively parallel in vivo assay of TdT mutants yields variants with altered nucleotide insertion biases

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdTs de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdTs intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro, revealing concordance between each variants in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

synthetic biology↗

bioRxiv2021

Structural origins of altered spectroscopic properties upon ligand binding in proteins containing a fluorescent non-canonical amino acid.

Fluorescent non-canonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red or blue shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups--contributed either by the protein or substrate--that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

biochemistry↗