Biology subjects

Goldsmith, M.

Publications and source records attributed to Goldsmith, M..

4 recordsLinked to original sources

bioRxiv2023

Stable mammalian serum albumins designed for bacterial expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40{degrees}C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/534334v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@71ef93org.highwire.dtl.DTLVardef@27a975org.highwire.dtl.DTLVardef@1e657fforg.highwire.dtl.DTLVardef@19b3de3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Computational design stabilized human and bovine serum albumins - Designs express solubly in E. coli and exhibit up to 40 {degrees}C increased thermostability - Some designs exhibit identical ligand binding properties - Crystal structure confirms design accuracy - Designs can be used in cell culture and in vitro applications

biochemistry↗

bioRxiv2022

An Effective mRNA-LNP Vaccine Against the Lethal Plague Bacterium

Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of mankind and is caused by the gram-negative bacterium Yersinia pestis. Currently, the disease is treated effectively with antibiotics. However, in the case of an outbreak caused by a multiple-antibiotic-resistant strain, alternative countermeasures are required. Despite the many efforts to develop a safe vaccine against the disease, there is still no vaccine approved for use in western countries. mRNA Lipid Nanoparticle (mRNA-LNP) vaccines have been demonstrated during the Covid-19 pandemic to be a versatile, clinically relevant, and rapidly manufactured vaccine platform. However, harnessing this platform for bacterial pathogens remains a formidable challenge. Here, we describe the design of several mRNA-LNP vaccine versions against Y. pestis, based on the F1 capsular antigen. We demonstrate that mRNA-LNP vaccines encoding the F1 antigen with either no signal sequences or conjugated to human Fc, provide substantial cellular and humoral responses. Most importantly, these vaccine candidates fully protect animals against Y. pestis infection. The results of this study suggest that mRNA-LNPs can be effective as anti-bacterial vaccines, and further developed to combat other bacterial pathogens, which are urgently needed, given the looming threat of antibiotic resistance. One-Sentence SummaryA novel mRNA-LNP vaccine against Y. pestis, the etiological agent of plague and the first documented mRNA-LNP vaccine to protect against a lethal bacterial pathogen infection.

microbiology↗

bioRxiv2022

Designed high-redox potential laccases exhibit high functional diversity

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for green-chemistry applications due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative directed evolution protocols have been applied to improve their expression, activity and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express: first, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step towards the generation of an effective lignin-degrading consortium of enzymes that can be secreted from yeast. More broadly, the stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

biochemistry↗

bioRxiv2020

Bringing BOS to light: Uncovering the key enzyme in the biosynthesis of the neurotoxin β-ODAP in Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, {beta}-N-oxalyl-L-,{beta}-diaminopropionic acid ({beta}-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of {beta}-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme {beta}-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

plant biology↗