Biology subjects

Ling, W. L.

Publications and source records attributed to Ling, W. L..

3 recordsLinked to original sources

bioRxiv2023

Phase separation and molecular ordering of the prion-like domain of the thermosensory protein EARLY FLOWERING 3

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalisation of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

biophysics↗

bioRxiv2021

More than meets the Kappa for Antibody Superantigen Protein L (PpL)

Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six V{kappa} and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-V{kappa}2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

immunology↗

bioRxiv2020

Nonclassical nucleation of protein mesocrystals via oriented attachment

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles which in turn become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not been observed for proteins. Here we employ cryoEM in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. The OA mechanism yields crystal morphologies that are not attainable through conventional crystallization routes. Based on these insights we revisit a system of protein crystallization that has long been classified as non-classical, but our data is in direct conflict with that conclusion supporting a classical mechanism that implicates OA. These observations raise further questions about past conclusions for other proteins and illustrate the importance of maturation stages after primary nucleation has taken place.

biochemistry↗