Biology subjects

Waudby, C. A.

Publications and source records attributed to Waudby, C. A..

10 recordsLinked to original sources

bioRxiv2026

Solution structure, dynamics and fragment binding of unbound MERS-CoV nsp10

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) poses a significant public health threat, with a fatality rate of 37% and no approved therapeutics. Non-structural protein 10 (nsp10) is an essential cofactor that activates both the nsp14 3'-5' exoribonuclease (ExoN) activity required for RNA proofreading and the nsp16 2'-O-methyltransferase (2'-O-MTase) implicated in viral RNA cap formation. Despite its functional importance, the unbound structure and dynamics of MERS-CoV nsp10 in solution remain uncharacterised. Here we report a near-complete NMR backbone and sidechain assignment and characterise the solution structure and dynamics of the protein by NMR. In contrast to the folded 1 helix observed in crystal structures of coronavirus nsp10, we find that this region is intrinsically disordered in solution, with residues 10-22 undetectable under standard conditions and further evidenced by pH titration, temperature-dependent NMR, and CLEANEX-PM experiments. Analysis of NOE contacts confirmed that the core adopts the conserved coronavirus nsp10 fold, consistent with the AlphaFold-predicted structure. 15N backbone relaxation measurements indicated a rigid, well-ordered core with only localised flexibility, despite a relatively low proportion of secondary structure elements, and CPMG and CEST experiments detected no conformational exchange on the s-ms timescale. Building on this structural and dynamic characterisation, we explored the ligandability of nsp10 by 19F NMR fragment screening. Screening of a 463-compound library identified 23 initial hits (4.97% hit rate), of which 20 were confirmed by 15N SOFAST-HMQC and eight gave quantifiable affinities by MST (Kd 0.5-6.9 mM), the remainder being too weak for reliable determination. Chemical shift perturbations clustered near functional surfaces of the folded core, indicating that MERS-CoV nsp10 is ligandable and providing chemical starting points for antiviral development.

biochemistry↗

bioRxiv2025

Long-range electrostatic forces govern how proteins fold on the ribosome

Protein biosynthesis and folding are tightly intertwined processes regulated by the ribosome and auxiliary factors. Nascent proteins can begin to fold on their parent ribosome but formation of the native state is often inhibited well beyond the emergence of the necessary residues from the exit tunnel. The dominant forces driving this apparent destabilisation have remained unclear. We investigate this here, combining NMR experiments and atomistic simulations of a folded nascent chain on and off the ribosome. While its native structure and internal dynamics remain unaltered, co-translational folding is disfavoured due to intermolecular electrostatic repulsion between the negatively charged ribosome surface and nascent protein. Partially folded intermediates are less destabilised, resulting in their high populations. Specifically, we show that the polypeptides net charge is the dominant factor determining nascent folding thermodynamics, with smaller contributions from charge distribution. Consequently, positively charged proteins can fold on the ribosome without populating stable intermediates. These findings reconcile conflicting observations of previous studies and establish the general physical principles underpinning de novo protein folding.

biophysics↗

bioRxiv2024

Amyloid forming human lysozyme intermediates are stabilised by non-native amide-π interactions

Mutational variants of human lysozyme cause a rare but fatal hereditary form of systemic amyloidosis by populating an intermediate state that self-assembles into amyloid fibrils. Despite its significance in lysozyme amyloidosis, the intermediate state has been recalcitrant to detailed structural investigation as it is only transiently and sparsely populated. Here, we investigated the intermediate state of a mutational variant of human lysozyme (I59T) using CEST and CPMG RD NMR at low pH. 15N CEST profiles probed the thermal unfolding of the native state into the denatured ensemble and revealed an additional state distinct from the two major states. Global fitting of 15N CEST and CPMG data provided kinetic and thermodynamic parameters for the exchange between all three states, characterising the intermediate state populated at 0.6%. 1H CEST data also confirmed the presence of the intermediate state displaying unusually high or low 1HN chemical shifts. To further investigate the structural details of the intermediate state we used molecular dynamics (MD) simulations, which recapitulated the experimentally observed folding pathway and free energy landscape. A high-energy intermediate state with a locally disordered {beta}-domain and C-helix was observed, revealing non-native hydrogen bonding and amide-{pi} interactions. These interactions account for the anomalous 1H chemical shifts and likely stabilise the transient intermediate state structure. Together, our NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.

biophysics↗

bioRxiv2023

Structural Insights into Regulation of Insulin Expression Involving i-Motif DNA Structures in the Insulin-Linked Polymorphic Region.

The insulin linked polymorphic region (ILPR) is a variable number of tandem repeats (VNTR) region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of VNTR repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the ILPR form different DNA secondary structures and insulin expression is dependent on formation of i-motif and G-quadruplex structures. The first crystal structure and dynamics of an intramolecular i-motif also reveal sequences within the loop regions forming additional stabilising interactions, which are critical to formation of the stable i-motif structures that modulate insulin expression. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to alter insulin gene expression.

biophysics↗

bioRxiv2021

An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

biochemistry↗

bioRxiv2021

Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

Transverse nuclear spin relaxation can be a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two four-spin transitions in selectively protonated methyl groups within perdeuterated biomacromolecules, alongside control experiments for measurement of 1H and 13C chemical shift anisotropies. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and determines the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. The analysis can be combined with CPMG relaxation dispersion measurements to provide improved precision, particularly in the determination of 1H chemical shift differences.

biophysics↗

bioRxiv2020

Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

NMR measurements of cross-correlated nuclear spin relaxation provide powerful probes of polypeptide dynamics and rotational diffusion, free from contributions due to chemical exchange or interactions with external spins. Here, we report on the development of a sensitivity-optimized pulse sequence for the measurement of cross-correlated relaxation in methyl spin systems by analysis of the differential relaxation of transitions within the 13C multiplet. We describe the application of optimal design theory to implement a real-time on-the-fly adaptive sampling scheme that maximizes the accuracy of the measured rate constants. The increase in sensitivity obtained using this approach enables, for the first time, quantitative measurements of rotational diffusion within folded states of translationally-arrested ribosome-nascent chain complexes of the FLN5 filamin domain, and can be used to place strong limits on interactions between the domain and the ribosome surface.

biophysics↗

bioRxiv2019

Catalysis by a rigid enzyme

Many enzymes are dynamic entities, sampling conformational states that are relevant for catalytic activity. Crystal structures of catalytic intermediates suggest, however, that not all enzymes require structural changes for activity. The single-domain enzyme xylanase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose. We demonstrate that BCX in solution undergoes minimal structural changes during catalysis. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow, enzyme induced substrate distortion. Therefore, we propose a model in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates catalysis.\n\nOne Sentence SummaryThe rigid matrix of BCX uses substrate flexibility in Michaelis complex formation.

biochemistry↗

bioRxiv2019

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

NMR spectroscopy provides a powerful approach for the characterisation of chemical exchange and molecular interactions by analysis of series of experiments acquired over the course of a titration measurement. The appearance of NMR resonances undergoing chemical exchange depends on the frequency difference relative to the rate of exchange, and in the case of one-dimensional experiments chemical exchange regimes are well established and well known. However, two-dimensional experiments present additional complexity, as at least one additional frequency difference must be considered. Here we provide a systematic classification of chemical exchange regimes in two-dimensional NMR spectra. We highlight important differences between exchange in HSQC and HMQC experiments, that on a practical level result in more severe exchange broadening in HMQC spectra, but show that complementary alternatives to the HMQC are available in the form of HZQC and HDQC experiments. We present the longitudinal relaxation optimised SOFAST-H(Z/D)QC experiment for the simultaneous acquisition of sensitivity-enhanced HZQC and HDQC spectra, and the longitudinal and transverse relaxation optimised BEST-ZQ-TROSY for analysis of large molecular weight systems. We describe the application of these experiments to the characterisation of the interaction between the Hsp90 N-terminal domain and a small molecule ligand, and show that the independent analysis of HSQC, HMQC, HZQC and HDQC experiments provides improved confidence in the fitted dissociation constant and dissociation rate. Joint analysis of such data may provide improved sensitivity to detect and analyse more complex multi-state interaction mechanisms such as induced fit or conformational selection.

biophysics↗

bioRxiv2017

Systematic mapping of the free energy landscapes of a growing immunoglobulin domain identifies a kinetic intermediate associated with co-translational proline isomerization

Co-translational folding is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the wasteful or hazardous formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of co-translational folding pathways. Here we contrast observations in translationally-arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome. By using this approach we identify and characterize two folding intermediates, including a partially folded intermediate associated with the isomerization of a conserved proline residue, which, together with measurements of folding kinetics, raises the prospect that neighboring unfolded domains might accumulate during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation, and show that catalysis of folding by peptidyl-prolyl isomerases is essential to eliminate this hazard.

biophysics↗