Biology subjects

Vongsouthi, V.

Publications and source records attributed to Vongsouthi, V..

3 recordsLinked to original sources

bioRxiv2024

Ancestral reconstruction of polyethylene terephthalate degrading cutinases reveals a rugged and unexplored sequence-fitness landscape

The enzymatic degradation of polyethylene terephthalate (PET) is a promising method of advanced plastic recycling. Traditional protein engineering methods often fall short in exploring protein sequence space for optimal enzymes due to structural and rational design limitations. Our study addresses this by using multiplexed ancestral sequence reconstruction (mASR) to explore the evolutionary sequence space of PET-degrading cutinases. With a dataset of 397 cutinase sequences, we created a diverse library of ancestral sequences. Experimental characterization of 48 ancestral sequences revealed a wide range of PETase activities, highlighting the value of mASR in uncovering functional variants when compared to traditional ASR. Our results showed that PETase activity in cutinases evolved through diverse pathways involving distal mutations to the active site, and is readily accessible within this family. Additionally, our analysis of the PETase fitness landscape using one-hot encoding (OHE) and local ancestral sequence embedding (LASE) highlighted the effectiveness of LASE in capturing sequence features relevant to activity. This work emphasizes the utility of mASR as a protein engineering tool for identifying enhanced PET-degrading enzymes, and the advantages of the LASE embedding scheme in mapping the PETase fitness landscape.

biochemistry↗

bioRxiv2023

A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus

The biological significance of a small supernumerary marker chromosome that results in dosage alterations to chromosome 9p24.1, including triplication of the GLDC gene encoding glycine decarboxylase, in two patients with psychosis is unclear. In an allelic series of copy number variant mouse models, we identify that triplication of Gldc reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) but not in CA1, suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, and further demonstrate that GLDC negatively regulates long-term synaptic plasticity at specific hippocampal synapses, possibly contributing to the development of neuropsychiatric disorders.

neuroscience↗

bioRxiv2020

A computationally designed fluorescent biosensor for D-serine

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability and conformational change to accomplish diverse functions in small molecule transport, sensing and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here we have engineered a D-alanine-specific SBP into a fluorescent biosensor with specificity for the signaling molecule D-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 {+/-} 0.5 M), specificity (40-fold increase vs. glycine), thermostability (Tm = 79 {degrees}C) and dynamic range (~14%). This sensor allowed measurement of physiologically relevant changes in D-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity and thermostability, and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

biochemistry↗