Biology subjects

Baldwin, A. J.

Publications and source records attributed to Baldwin, A. J..

10 recordsLinked to original sources

bioRxiv2025

Isostere 18F-protein post-translational editing enables dynamic tracking of neurodegeneration biomarkers

The neurofilament light chain protein (NfL) is a suggested general marker for neuronal loss. Its release from brain parenchyma into cerebral spinal fluid, and presumed detection in blood has seen it established as a first blood-based marker of disease activity and drug efficacy in multiple sclerosis (MS) and in the presymptomatic diagnosis and assessment of disease course for other neurodegenerative disorders.1 However, the lack of characterisation of its behaviour in circulation, largely due to its antibody-dependent measurement, have hampered the biological interpretation of these measurements, especially after acute injury such as in MS relapse or head trauma.2 Here, we describe a strategy for exploiting positron emission tomography (PET) imaging using isosteric protein mimics following the installation of a fluorine-18 label that is benign enough to provide sensitive, real-time information on the dynamics and trafficking of NfL protein. This circumvents the limits of current methods that integrate 18F into proteins through the bio-conjugation of bulky, unnatural groups, which we show perturb NfLs assembly and functional properties from those in the natural state. We use a visible-light-driven reaction to access radioactive isostere proteins that are unperturbed and so closely resemble their native form. In this way, generation of [18F]fluoroalkyl radicals that can be rapidly reacted at pre-defined sites on proteins creates mimics of proteinogenic side chains bearing near-zero-size labels to probe proteins in functionally true form. These prosthetic-free, protein radiotracers can be generated in excellent radiochemical yield (up to 67%) via a semi-automated protocol in just 15 mins. High associated molar activities (precursor up to 102 GBq mol-1) allowed high sensitivity dynamic observations in blood, brain and cerebrospinal fluid, enabling even the first unambiguous observations of spinal flow kinetics using proteins. These dynamics, including the high rate of spinal flow (on the order of mm per min) and drainage of NfL from CSF into sacral lymph nodes, now provides evidence that the slow fall rate of antibody-detected markers that is observed after acute neural insults is not due to a long half-life, but rather reflects sustained neuronal loss. This discovery will now help to better correlate clinical and radiological features of disease with NfL blood levels. Our methodology now demonstrates the broad potential of a near-zero-size labelling method for the functional study of proteins in whole organisms without interfering with their biological activity and native assembly.

synthetic biology↗

bioRxiv2024

Seedless: On-the-fly pulse calculation for NMR experiments

All NMR experiments require sequences of RF pulses to manipulate nuclear spins. Signal is lost due to non-uniform excitation of nuclear spins resonating at different energies (chemical shifts) and inhomogeneity in the RF actually generated by hardware over the sample volume. Here, we present Seedless, a tool to calculate NMR pulses that compensate for these effects to enhance control of magnetisation and boost signal. The calculations take only a few seconds using an optimised GRadient Ascent Pulse Engineering (GRAPE) implementation, allowing pulses to be generated on-the-fly, optimised for individual samples and spectrometers. Each calculated pulse requires bands of chemical shift to be identified, over which one of 4 transforms will be performed, selected from a set that covers all commonly used applications: a universal rotation (e.g. 90{degrees} about the x axis), including as a special case an identity operation (return spins in the same state as they started), state-to-state (e.g. Z->Y), an XYcite (Z->XY plane), or a novel type, a suppression, that leaves spins minimally perturbed at all times during the pulse. Using imaging experiments we demonstrate our pulses effectively increase the size of the coil volume and effectively increase signal-to-noise in all experiments. We illustrate the approach by devising ultra-broadband pulses (300 ppm excitation pulse for 19F 1D spectra), a 15N HSQC with 58% increased S/N (950 MHz spectrometer + cryoprobe), triple resonance biomolecular NMR experiments such as HNCACO with 55% increased S/N (600 MHz spectrometer + RT probe), and a highly efficient pulse sequence for water suppression. The 8 optimised pulse sequences presented required 54 bespoke pulses all calculated on-the-fly. Seedless provides a means to enhance sensitivity in all pulse sequences in a manner that can be tailored to all samples/hardware being used.

biophysics↗

bioRxiv2023

Haptenic adducts of beta-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Many classes of small-molecule drugs form protein adducts in vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although {beta}-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones--the MIL series--predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other {beta}-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level. Highlights- PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions. - PenG-protein adduction in vitro or in vivo is sufficient to elicit penicillin-specific IgG responses. - Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin. - The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.

immunology↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Molecular determinants of dynamic protein-protein interactions in the functional cycle of the membrane protein DsbD

Molecular recognition is of central importance in biology. The molecular determinants shaping recognition of one protein domain by another are incompletely understood, especially in the context of the complex function of molecular machines. Here, we combine NMR experiments and molecular dynamics simulations to elucidate the determinants of recognition of the C-terminal (cDsbD) domain of the transmembrane reductant conductor DsbD by its cognate partner, the N-terminal domain of the protein (nDsbD). As part of the natural cycle of this oxidoreductase, which effectively transfers electrons from the cytoplasm to the periplasm of Gram-negative bacteria, cDsbD and nDsbD toggle between oxidised and reduced states, something that modulates the affinity of the domains for each other and prevents otherwise unproductive reactions. We find that the redox state of cDsbD determines the dissociation rate of cDsbD-nDsbD complexes. Molecular dynamics simulations demonstrate how the redox-state of the active site determines the stability of inter-domain hydrogen bonds and thus the dissociation rate. AlphaFold modelling and atomistic molecular dynamics simulations of full-length DsbD in a realistic bacterial membrane again highlights the close proximity of the periplasmic domains and the importance of tuning the strength of the interactions of the periplasmic domains to enable electron transfer to cognate periplasmic partners such as CcmG. Our AlphaFold models are consistent with in vivo functional assays of DsbD mutants, which together help to reveal for the first-time a putative binding site for thioredoxin on the cytoplasmic side of DsbD.

biophysics↗

bioRxiv2020

Repulsive electrostatic interactions modulate dense and dilute phase properties of biomolecular condensates

Liquid-like membraneless organelles form via multiple, weak interactions between biomolecules. The resulting condensed states constitute novel solvent environments inside eukaryotic cells that partition biomolecules and may favour particular biochemical reactions. Here we demonstrate that, in addition to attractive interactions, repulsive electrostatic interactions modulate condensate properties. We find that net charge modulates the formation, morphology and solvent properties of model Ddx4 condensates in cells and in vitro and that a net negative charge is conserved across germ cell-specific Ddx4 orthologues. This conserved net charge provides a sensitivity to multivalent cations that is not observed in somatic paralogues. The disfavouring effect of a net negative charge in Ddx4 orthologues appears to be offset by increased charge patterning, indicating that fine tuning of both attractive and repulsive interactions can create responsive solvent environments inside biomolecular condensates.

cell biology↗

bioRxiv2019

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

Neurodegenerave diseases such as amyotrophic lateral sclerosis (ALS) are oten associated with mutaons in proteins that are associated with stress granules. Stress granules are condensates formed by liquid-liquid phase separaon which, when aberrant, can lead to altered condensaon behaviours and disease phenotypes. Here, we identified lipoamide, a small molecule which specifically prevents cytoplasmic condensaon of stress granule proteins. Thermal proteome profiling showed that lipoamide preferentially stabilises intrinsically disordered domain-containing proteins. These include SRSF1 and SFPQ, stress granule proteins necessary for lipoamide activity. The redox state of SFPQ correlates with its condensate-dissolving behaviour, in concordance with the importance of the dithiolane ring for lipoamide activity. In animals, lipoamide ameliorates aging-associated aggregaon of a stress granule reporter, improves neuronal morphology, and recovers motor defects caused by expression of ALS-associated FUS and TDP-43 mutants. In conclusion, lipoamide is a well-tolerated small molecule modulator of stress granule condensaon and dissection of its molecular mechanism identified a cellular pathway for redox regulaon of stress granule formaon.

cell biology↗

bioRxiv2018

Atomic Level Characterisation of Millisecond-Time Scale Protein Motions through a Combined Molecular Simulations and NMR Approach

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these excited conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1{rho} solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.

biophysics↗

bioRxiv2018

Local unfolding of the HSP27 monomer regulates chaperone activity

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved -crystallin domain, and provide the first structural characterization of a small heat-shock protein monomer. While HSP27 assembles into oligomers, we show that the transiently populated monomers released upon reduction are highly active chaperones in vitro, but are kinetically unstable and susceptible to uncontrolled aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance spectroscopy, we reveal that the pair of {beta}-strands that mediate dimerization become partially disordered in the monomer. Strikingly, we note that numerous HSP27 mutations associated with inherited neuropathies cluster to this unstructured region. The high degree of sequence conservation in the -crystallin domain amongst mammalian sHSPs suggests that partially unfolded monomers may be a general, functional feature of these molecular chaperones.

biophysics↗

bioRxiv2018

αB-crystallin inhibits amyloidogenesis by disassembling aggregation nuclei

Amyloid formation is implicated in a range of neurodegenerative conditions including Alzheimers and Parkinsons diseases. The small heat-shock protein B-crystallin (BC) is associated with both, and directly inhibits amyloid formation in vitro and its toxicity in cells. Studying the mechanism of aggregation inhibition is challenging owing to sample heterogeneity and the dynamic nature of the process. Here, by means of NMR spectroscopy and chemical kinetics, we establish the mechanism by which the protein -lactalbumin aggregates and forms amyloid, and how this is inhibited by BC. In particular, we characterise the lifetime of the unstable aggregation nucleus, and determine that this species is specifically destabilised by BC. This mechanism allows the chaperone to delay the onset of aggregation, although it is overwhelmed on longer timescales. The methodology we present provides a mechanistic understanding of how BC reduces the toxicity of amyloids, and is widely applicable to other complex mixtures.

biophysics↗