Biology subjects

Parmeggiani, F.

Publications and source records attributed to Parmeggiani, F..

8 recordsLinked to original sources

bioRxiv2025

Structure-guided design of protein attachment points for functional augmentation of complex molecular machines

Protein-protein conjugation systems are a powerful way of creating fusion proteins and enable the dynamic combination of protein domains with diverse functionalities. However, the insertion of these systems into enzymes is often performed with little consideration of the structural impact they might have. This is particularly relevant when modifying complex molecular machines that transition between numerous conformational states. Here, we address this issue by developing SIMPLIFE, a computational workflow that supports the design of optimal insertion sites for conjugation tags based on the structure of the proteins involved and performs localised residue redesign where needed. We demonstrate how SIMPLIFE can be used to effectively augment the function of T7 RNA polymerase using the DogCatcher-DogTag system, enabling diverse and dynamically varying mutations within a targeted region of DNA. This work demonstrates the power of combining biophysical and machine learning based approaches for protein structure prediction to efficiently augment the function of molecular machines, accelerating our ability to combine complex biochemical functionalities in new ways.

synthetic biology↗

bioRxiv2025

A global survey of intramolecular isopeptide bonds

Many protein domains harbour covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large-scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template-based software Isopeptor. Our findings reveal an extensive phyletic distribution in surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA-like or CnaB-like, from a relatively small set of related Pfam families, including ten novel families that we predict to contain intramolecular isopeptide bonds. One CnaA-like domain of unknown function, DUF11 (renamed here to "CLIPPER") is broadly distributed in cell-surface proteins from Gram-positive bacteria, Gram-negative bacteria, and archaea, and is structurally and biophysically characterised in this work. Using X-ray crystallography, we resolve a CLIPPER domain from a Gram-negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond-containing protein domains in nature, and structurally resolve the previously cryptic CLIPPER domain.

bioinformatics↗

bioRxiv2025

CLIMBS: assessing Carbohydrate-Protein interactions through a graph neural network classifier using synthetic negative data

I.Carbohydrate-protein interactions are essential for biological processes, such as cellular signaling and metabolism, and represent a large pool of untapped targets for diagnostics and therapeutics. However, current design and prediction methods fail to accurately evaluate the affinity and specificity of proteins for carbohydrates such as glucose and galactose. Here, we describe a machine learning classifier, named CLIMBS, as a novel scoring method for protein-carbohydrate interactions and train it on crystal structures and synthetic data from unsuccessfully designed binders, to effectively assess if carbohydrate-protein complexes represent realistic, native like structures. Compared to other methods, CLIMBS has outstanding accuracy, excellent carbohydrate specificity, sub-second runtime per sample, minimal bias towards either negative or positive samples, and can be employed to improve selection of successful docking and design models of carbohydrate-protein complexes.

bioinformatics↗

bioRxiv2024

Scalable design of repeat protein structural dynamics via probabilistic coarse-grained models

Computational protein design has emerged as a powerful tool for creating proteins with novel functionalities. However, most existing methods ignore structural dynamics even though they are known to play a central role in many protein functions. Furthermore, methods like molecular dynamics that are able to simulate protein movements are computationally demanding and do not scale for the design of even moderately sized proteins. Here, we develop a probabilistic coarse-grained model to overcome these limitations and support the design of the structural dynamics of modular repeat proteins. Our model allows us to rapidly calculate the probability distribution of structural conformations of large modular proteins, enabling efficient screening of design candidates based on features of their dynamics. We demonstrate this capability by exploring the design landscape of 4-6 module repeat proteins. We assess the flexibility, curvature and multi-state potential of over 65,000 protein variants and identify the roles that particular modules play in controlling these features. Although our focus here is on protein design, the methods developed are easily generalised to any modular structure (e.g., DNA origami), offering a means to incorporate dynamics into diverse biological design workflows.

synthetic biology↗

bioRxiv2023

Silk Road Revealed: Mechanism of silk fibre formation in Bombyx mori

The transition of silk fibroin from liquid to solid is fundamental to silk-fibre production and key to the superior materials properties of native silks. Here we discover that the fibroin heavy chain from the silkworm moth Bombyx mori folds into a novel {beta}-solenoid structure, where the N-terminal domain (NTD) promotes higher-order oligomerization driven by pH reduction. These findings elucidate the complex rheological behaviour of silk and the liquid crystalline textures within the silk gland. We also find that NTD undergoes hydrolysis during standard regeneration, explaining differences between native and regenerated silk feedstocks. Overall, this study establishes a fibroin heavy chain fold, which could be relevant for other similar proteins, and explains mechanistically its liquid-to-solid transition, driven by pH reduction and stress. One sentence summaryThis study redefines the molecular structure of fibroin heavy chain and its role in the transition from solution to fibre.

biochemistry↗

bioRxiv2022

Precisely patterned nanofibers made from extendable protein multiplexes

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha helical coiled coils have such symmetric extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here, we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibers can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibers can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.

bioengineering↗

bioRxiv2021

Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

synthetic biology↗

bioRxiv2020

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a promiscuous glycosyltransferase

The {beta}-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of {beta}-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

biochemistry↗