Engineering Protein-Peptide Interfaces via Combinatorial Mutagenesis and Mass Photometric Screening.

The SpyTag-SpyCatcher system, developed by the Howarth lab, is based on splitting the CnaB2 domain from Streptococcus pyogenes into two parts: a 13-amino-acid SpyTag and a 116-amino-acid SpyCatcher. Upon incubation, they spontaneously form a covalent isopeptide bond between Asp7 (SpyTag) and Lys31 (SpyCatcher). This study explores whether the interaction specificity can be modulated by altering hydrophobic residues within the SpyCatcher binding pocket and corresponding SpyTag positions, potentially to create orthogonal SpyTag-SpyCatcher pairs.

A New Highly Specific and Soluble Protease for Precise Removal of N‑Terminal Purification Tags.

Biotherapeutics production has been significantly enhanced by affinity purification. After purification, however, it is often necessary to remove the affinity purification tag. Thus, we aim for a protease suitable for such a task with properties that include high production yields, good solubility and stability, high cleavage specificity, sufficiently fast turnover, and tolerance of the amino acid identity at the P' position (the C-terminus of the recognition site).

Cell-Free Protein Synthesis as a Method to Rapidly Screen Machine Learning-Generated Protease Variants.

Machine learning (ML) tools have revolutionized protein structure prediction, engineering, and design, but the best ML tool is only as good as the training data it learns from. To obtain high-quality structural or functional data, protein purification is typically required, which is both time and resource consuming, especially at the scale required to train ML tools. Here, we showcase cell-free protein synthesis as a straightforward and fast tool for screening and scoring the activity of protein variants in ML workflows.

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT.

Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells.

Applications of cell free protein synthesis in protein design.

In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs.

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins.

Modulating the Viscoelastic Properties of Covalently Crosslinked Protein Hydrogels.

Protein engineering allows for the programming of specific building blocks to form functional and novel materials with customisable physical properties suitable for tailored engineering applications. We have successfully designed and programmed engineered proteins to form covalent molecular networks with defined physical characteristics. Our hydrogel design incorporates the SpyTag (ST) peptide and SpyCatcher (SC) protein that spontaneously form covalent crosslinks upon mixing.

Uncovering diffusive states of the yeast membrane protein, Pma1, and how labeling method can change diffusive behavior.

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2.

Chemically cross-linked hydrogels from repetitive protein arrays.

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype.

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach.

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells.

Core packing of well-defined X-ray and NMR structures is the same.

Numerous studies have investigated the differences and similarities between protein structures determined by solution NMR spectroscopy and those determined by X-ray crystallography. A fundamental question is whether any observed differences are due to differing methodologies or to differences in the behavior of proteins in solution versus in the crystalline state. Here, we compare the properties of the hydrophobic cores of high-resolution protein crystal structures and those in NMR structures, determined using increasing numbers and types of restraints.

Self-Assembling Protein Surfaces for Capture of Cell-Free-Synthesized Proteins.

We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using "Catcher/Tag" technology.

PAINT using proteins: A new brush for super-resolution artists.

PAINT (points accumulation for imaging in nanoscale topography) refers to methods that achieve the sparse temporal labeling required for super-resolution imaging by using transient interactions between a biomolecule of interest and a fluorophore. There have been a variety of different implementations of this method since it was first described in 2006. Recent papers illustrate how transient peptide-protein interactions, rather than small molecule binding or DNA oligonucleotide duplex formation, can be employed to perform PAINT-based single molecule localization microscopy (SMLM).

LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions.

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein.

Rational Design and Self-Assembly of Coiled-Coil Linked SasG Protein Fibrils.

Protein engineering is an attractive approach for the self-assembly of nanometer-scale architectures for a range of potential nanotechnologies. Using the versatile chemistry provided by protein folding and assembly, coupled with amino acid side-chain functionality, allows for the construction of precise molecular "protein origami" hierarchical patterned structures for a range of nanoapplications such as stand-alone enzymatic pathways and molecular machines. The surface protein SasG is a rigid, rod-like structure shown to have high mechanical strength due to "clamp-like" intradomain features and a stabilizing interface between the G5 and E domains, making it an excellent building block for molecular self-assembly.

Void distributions reveal structural link between jammed packings and protein cores.

Dense packing of hydrophobic residues in the cores of globular proteins determines their stability. Recently, we have shown that protein cores possess packing fraction ϕ≈0.56, which is the same as dense, random packing of amino-acid-shaped particles.

The past, present and future of protein-based materials.

Protein-based materials are finding new uses and applications after millennia of impacting the daily life of humans. Some of the earliest uses of protein-based materials are still evident in silk and wool textiles and leather goods. Today, even as silks, wools and leathers are still be used in traditional ways, these proteins are now seen as promising materials for biomaterials, vehicles of drug delivery and components of high-tech fabrics.

A threonine zipper that mediates protein-protein interactions: Structure and prediction.

We present the structure of an engineered protein-protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close-packing of the Thr residues, with only one intermonomer hydrogen bond (H-bond) between two of the Thr residues. This Thr-rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues.

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA.

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces, leading to the formation of flattened sessile water drops.

Intensification: A Resource for Amplifying Population-Genetic Signals with Protein Repeats.

Large-scale genome sequencing holds great promise for the interpretation of protein structures through the discovery of many, rare functional variants in the human population. However, because protein-coding regions are under high selective constraints, these variants occur at low frequencies, such that there is often insufficient statistics for downstream calculations. To address this problem, we develop the Intensification approach, which uses the modular structure of repeat protein domains to amplify signals of selection from population genetics and traditional interspecies conservation.

Protein-Based Hydrogels for Tissue Engineering.

The tunable mechanical and structural properties of protein-based hydrogels make them excellent scaffolds for tissue engineering and repair. Moreover, using protein-based components provides the option to insert sequences associated with promoting both cellular adhesion to the substrate and overall cell growth. Protein-based hydrogel components are appealing for their structural designability, specific biological functionality, and stimuli-responsiveness.