Anne Claire Conibear

Dysfunction of the microtubule‐associated protein Tau is a central feature of Alzheimer's disease and related tauopathies, yet how site‐specific lysine modifications modulate the functional and pathological states of Tau remains poorly understood. Here, we combine protein semi‐synthesis with segmental isotope labelling and NMR spectroscopy of full‐length Tau, to dissect how lysine acetylation and carboxymethylation within the microtubule‐binding region of Tau regulate its interactions with tubulin, microtubules, and amyloid assembly. Site‐specific modification at lysine 294 delays Tau‐mediated tubulin polymerization and fibril formation, whereas acetylation at lysine 311 exerts more moderate effects, but alters fibril morphology. NMR spectroscopy of segmentally isotope‐labelled Tau variants indicates that single lysine acetylation does not measurably weaken Tau binding to pre‐formed microtubules; however, bivalent acetylation reduces microtubule binding, possibly by cumulative charge neutralization. Together, these results indicate that lysine acetylation redistributes Tau between functional states in a site‐ and valency‐dependent manner. Our study helps to establish a mechanistic framework linking combinatorial lysine modifications to Tau dysfunction and highlights the utility of region‐resolved structural approaches to decipher posttranslational modification–dependent equilibria in intrinsically disordered proteins.

Dysfunction of the microtubule‐associated protein Tau is a central feature of Alzheimer's disease and related tauopathies, yet how site‐specific lysine modifications modulate the functional and pathological states of Tau remains poorly understood. Here, we combine protein semi‐synthesis with segmental isotope labelling and NMR spectroscopy of full‐length Tau, to dissect how lysine acetylation and carboxymethylation within the microtubule‐binding region of Tau regulate its interactions with tubulin, microtubules, and amyloid assembly. Site‐specific modification at lysine 294 delays Tau‐mediated tubulin polymerization and fibril formation, whereas acetylation at lysine 311 exerts more moderate effects, but alters fibril morphology. NMR spectroscopy of segmentally isotope‐labelled Tau variants indicates that single lysine acetylation does not measurably weaken Tau binding to pre‐formed microtubules; however, bivalent acetylation reduces microtubule binding, possibly by cumulative charge neutralization. Together, these results indicate that lysine acetylation redistributes Tau between functional states in a site‐ and valency‐dependent manner. Our study helps to establish a mechanistic framework linking combinatorial lysine modifications to Tau dysfunction and highlights the utility of region‐resolved structural approaches to decipher posttranslational modification–dependent equilibria in intrinsically disordered proteins.

Chemical protein synthesis is a powerful tool that combines solid‐phase peptide synthesis with protein ligations to join synthetic protein segments together. This technology allows for the incorporation of site‐specific native and artificial modifications required for chemical biology research and therapeutic applications. Native chemical ligation (NCL), in which protein segments are joined together through a native amide bond, is the most widely used protein ligation strategy and has been employed in the synthesis and semi‐synthesis of many modified proteins. Whilst recent advances have extended the range of chemical strategies and ligation junctions available, the selection and optimization of an efficient route to a target protein can be challenging and time‐consuming. For most target proteins, several different approaches are possible, and their efficiency and sequence compatibility, as well as the protein properties and purpose of the target protein, must be evaluated. Here, several approaches to the synthesis of a target case study protein, the histone H2A.B variant are demonstrated, illustrating some of the potential challenges and troubleshooting options. This work provides a demonstration of the leading tools for NCL, to guide new users in the selection and optimization of appropriate strategies for targeting a diverse range of protein sequences and properties.

Conotoxins are disulfide-rich peptides isolated from the venoms of marine cone snails. These natural products have inspired the development of several drug candidates and novel therapeutic leads. In addition to disulfide bonds, many conotoxins are highly modified with posttranslational modifications (PTMs) such as proline hydroxylation, C-terminal amidation and glycosylation, among others. These modifications can alter the charge, size and hydrophobicity of the conotoxin, influencing its interaction with target receptors and modulating its potency and selectivity. PTMs can also affect the folding kinetics and conformational stability of the peptide, which further affects its biological activity. Although conotoxins undergo a variety of PTMs, the functions of many of these modifications remain unclear. Here, we explored the structural and functional implications of PTMs in two representative conotoxins, PIIIA and TIIIA of the µ-pharmacological family. We synthesised a series of PIIIA and TIIIA peptides bearing native hydroxyproline and C-terminal amidation PTMs, along with their unmodified counterparts. Solid phase peptide synthesis and non-selective disulfide bond formation provided access to pure forms of the eight possible variants for in vitro comparison of their oxidative folding. Structural studies using nuclear magnetic resonance (NMR) spectroscopy, alongside electrophysiological and serum stability assays, were conducted to characterise the functional roles of the PTMs in these conotoxins. Our results suggest that, whereas C-terminal amidation has a crucial role in folding and structural integrity, proline hydroxylation significantly influences the in vitro oxidative folding, stability and biological activity of these conotoxin peptides.

Ultrahigh-throughput methods such as flow cytometry are ideal tools for the directed evolution of enzymes by enabling the screening of up to 10⁹ variants per day. In this study, we developed an assay based on fluorescence-activated cell sorting (FACS) for the detection and engineering of amidase activity in whole cells. The assay establishes a stable genotype–phenotype linkage by coupling coumarin-based hydrolysis products to intracellular glutathione via a recombinantly coexpressed glutathione S-transferase. To demonstrate the applicability of the FACS-based assay, we engineered an amidase from Sphingomonas alpina (SaAmd) by screening combinatorial libraries with multiple amino acid positions randomized simultaneously. SaAmd variants containing proximal double mutations exhibited not only almost 5-fold improved activity against structurally different amide substrates but also coevolved promiscuous carbamate- and ester-hydrolyzing activities, which exceeded the wildtype activity up to 6-fold. Importantly, triple variants featuring distal mutations in three highly flexible loop regions, displayed up to 16-fold enhanced specific activities toward small molecules containing highly stable N-aryl amide and carbamate bonds. These motifs are commonly used as protecting groups for amines in organic synthesis but can also be found in environmental contaminants like pesticides and plastic waste. Therefore, the developed FACS-assisted assay has great potential to accelerate the engineering of amidases for versatile biotechnological applications.

The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the ‘chaperone code’), and its interactions with co‐chaperones and client proteins, still remains to be elucidated. The C‐terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co‐chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site‐selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C‐terminal phosphorylations do not significantly alter the anti‐aggregation activity for these clients. The optimization of expressed protein selenoester ligation (EPSL) to carry out several steps in one pot provides an efficient route to access site‐specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site‐specific PTMs to decipher the chaperone code.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three‐dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein‐protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome‐binding domain of an intrinsically disordered protein – HMGN1 – using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over‐predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction.

Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.

The early‐career researchers showcased in this ChemBioTalents special collection, and many others who have established their independent scientific careers over the last three years, have experienced a unique set of circumstances. The Covid‐19 pandemic necessitated new forms of communication and interpersonal interactions: From online interviews and virtual networking to relocating and establishing labs during a pandemic, we faced many challenges, but also unexpected opportunities. In this perspective, we reflect on this unique and formative time through personal anecdotes and viewpoints, trying to capture diverse experiences from the Chemical Biology community and beyond. We have tried to get a broad and varied set of perspectives, however, the selection is biased towards researchers who were able to start their independent careers.¹