Biology subjects

Williamson, M. P.

Publications and source records attributed to Williamson, M. P..

8 recordsLinked to original sources

bioRxiv2026

Universal nucleic acid preservation in biological fluids with boron clusters

Nucleic acid preservation remains a critical bottleneck for diagnostics and therapeutics, with small molecule inhibitors such as EDTA showing limited spectrum against diverse nucleases, while protein-based alternatives requiring costly cold-chain storage. Here, we report dodecaborate cluster [B12H12]2- as a novel class of pan-nuclease inhibitor with a fundamentally different mechanism--preventing protein-nucleic acid interactions rather than targeting cofactor dependencies. [B12H12]2- inhibited all six DNases and ten RNases tested, making it the broadest spectrum nuclease inhibitor known. Remarkably, its inhibition is reversible via {gamma}-cyclodextrin complexation. We demonstrate that [B12H12]2- preserved a physiological range of DNA for 14 days and that of RNA for 3-7 days at room temperature in human plasma and urine, achieving up to 323,972-fold better RNA retention than controls. A newly developed blood collection tube using [B12H12]2- enables whole-blood circulating cell-free RNA sequencing after 7 days of room-temperature storage, with preserved transcript integrity. Mechanistic studies suggest [B12H12]2- binds to nuclease active sites and creates electrostatic barriers that prevent substrate binding. This chemically stable, indefinitely shelf-stable inhibitor enables cold-chain-free biological sample transport, potentially transforming accessibility of nucleic acid-based diagnostics worldwide. Graphical abstract

bioengineering↗

bioRxiv2024

The Enterococcal Polysaccharide Antigen: from structure to biosynthesis and function

L-Rhamnose-containing polysaccharides are produced by Streptococci and Enterococci. They define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The Enterococcal Polysaccharide Antigen produced by the opportunistic pathogen Enterococcus faecalis plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer for E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, we elucidate the structure of the intact EPA produced by E. faecalis OG1RF. We report the structure of the linkage unit, revealing an unprecedented complexity of the rhamnose backbone and decorations. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA for the rational design of therapeutic strategies against a group of important pathogens.

microbiology↗

bioRxiv2022

Improved methodology for protein NMR structure calculation using hydrogen bond restraints and ANSURR validation: the SH2 domain of SH2B1

Protein structures calculated using NMR data are less accurate and less well defined than they could be. Here we use the program ANSURR to show that this deficiency is at least in part due to a lack of hydrogen bond restraints. We then describe a protocol to introduce hydrogen bond restraints into the structure calculation of the SH2 domain from SH2B1 in a systematic and transparent way, and show that the structures generated are more accurate and better defined as a result. We also show that ANSURR can be used as a guide to know when the structure calculation is good enough to stop.

biophysics↗

bioRxiv2022

The accuracy of protein structures in solution determined by AlphaFold and NMR

In the recent CASP (Critical Assessment of Structure Prediction) competition, AlphaFold2 performed outstandingly. Its worst predictions were for NMR structures, which has two alternative explanations: either the NMR structures were poor, implying that AlphaFold may be more accurate than NMR; or there is a genuine difference between crystal and solution structures. Here, we use the program ANSURR, which measures the accuracy of solution structures, and show that one of the NMR structures was indeed poor. We then compare AlphaFold predictions to NMR structures, and show that AlphaFold tends to be more accurate than NMR ensembles, in particular correctly more rigid in loops. There are however some cases where the NMR ensembles are more accurate. These tend to be dynamic structures where AlphaFold had low confidence. We suggest that AlphaFold could be used as the model for NMR structure refinements, and that AlphaFold structures validated by ANSURR require no further refinement.

biophysics↗

bioRxiv2021

The accuracy of NMR protein structures in the Protein Data Bank

We recently described a method, ANSURR, for measuring the accuracy of NMR protein structures. It is based on comparing residue-specific measures of rigidity from backbone chemical shifts via the random coil index, and from structures. Here, we report the use of ANSURR to analyse NMR ensembles within the Protein Data Bank (PDB). NMR structures cover a wide range of accuracy, which improved over time until about 2005, since when accuracy has not improved. Most structures have accurate secondary structure, but are too floppy, particularly in loops. There is a need for more experimental restraints in loops. The best current accuracy measures are Ramachandran distribution and number of NOE restraints per residue. The precision of structure ensembles correlates with accuracy, as does the number of hydrogen bond restraints per residue. If a structure contains additional components (such as additional polypeptide chains or ligands), then their inclusion improves accuracy. Analysis of over 7000 PDB NMR ensembles is available via our website ansurr.com.

biophysics↗

bioRxiv2020

31P NMR Spectroscopy Demonstrates Large Amounts of Phosphohistidine in Mammalian Cells

Protein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH [≤] 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 mol/g and 68 mol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The 31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells.

biochemistry↗

bioRxiv2020

A method for validating the accuracy of NMR protein structures

We present a method, Accuracy of NMR Structures using Random Coil Index and Rigidity (ANSURR), that measures the accuracy of NMR protein structures. It provides a residue-by-residue comparison of two measures of local rigidity: the Random Coil Index [RCI] (a measure of the extent to which backbone chemical shifts adopt random coil values); and local rigidity predicted by mathematical rigidity theory using the computational method Floppy Inclusion and Rigid Substructure Topology [FIRST], calculated from an NMR structural model. We compare RCI and FIRST using a correlation score (which assesses the location of secondary structure), and an RMSD score (which measures overall rigidity, and mainly assesses hydrogen bond correctness). We test the performance of ANSURR using: (a) structures refined in explicit solvent, which have much better RMSD score than unrefined structures, though similar correlation; (b) decoy structures generated for 89 NMR structures. The experimental NMR structures are usually better, though helical and sheet structures behave differently; (c) conventional predictors of structural accuracy such as number of restraints per residue, restraint violations, energy of structure, RMSD of the ensemble (precision of the calculation), Ramachandran distribution, and clashscore. Comparisons of NMR to crystal structures show that secondary structure is equally accurate in both, but crystal structures tend to be too rigid in loops, whereas NMR structures tend to be too floppy overall.

biophysics↗

bioRxiv2018

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and sensitivity to effector molecules of innate immunity

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

microbiology↗