Chemoproteogenomic stratification of the missense variant cysteinome.

Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation.

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.

A suite of designed protein cages using machine learning and protein fragment-based protocols.

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, but their creation remains challenging. Here, we apply computational approaches to design a suite of tetrahedrally symmetric, self-assembling protein cages. For the generation of docked conformations, we emphasize a protein fragment-based approach, while for sequence design of the de novo interface, a comparison of knowledge-based and machine learning protocols highlights the power and increased experimental success achieved using ProteinMPNN.

Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library.

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides.

A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols.

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while for interface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN.

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å.

Design of Beta-2 Microglobulin Adsorbent Protein Nanoparticles.

Beta-2 microglobulin (B2M) is an immune system protein that is found on the surface of all nucleated human cells. B2M is naturally shed from cell surfaces into the plasma, followed by renal excretion. In patients with impaired renal function, B2M will accumulate in organs and tissues leading to significantly reduced life expectancy and quality of life.

Dynamic acylome reveals metabolite driven modifications in .

is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes.

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways.

Ethanol is a widely available carbon compound that can be increasingly produced with a net negative carbon balance. Carbon-negative ethanol might therefore provide a feedstock for building a wider range of sustainable chemicals. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochemicals, and how we can use the energy stored in ethanol to generate ATP, another key molecule important for powering biochemical pathways.

Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1.

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.

A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis.

Structural Perturbations of Form II RuBisCO Mutant Enzymes That Affect CO Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from , a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here.

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface.

Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response.

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin-antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues.

Structures of EccB1 and EccD1 from the core complex of the mycobacterial ESX-1 type VII secretion system.

Background: The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system.

Engineering an allosteric transcription factor to respond to new ligands.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery.

Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity.

Structure of the toxic core of α-synuclein from invisible crystals.

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy.

A Suite of Engineered GFP Molecules for Oligomeric Scaffolding.

Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition.

Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii.

Prior studies have indicated that MJ1099 from Methanocaldococcus jannaschii has roles in the biosynthesis of tetrahydromethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 Å resolution using anomalous scattering methods.

Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host.

Comparative proteomics identifies the cell-associated lethality of M. tuberculosis RelBE-like toxin-antitoxin complexes.

The Mycobacterium tuberculosis (Mtb) genome encodes approximately 90 toxin-antitoxin protein complexes, including three RelBE family members, which are believed to play a major role in bacterial fitness and pathogenicity. We have determined the crystal structures of Mtb RelBE-2 and RelBE-3, and the structures reveal homologous heterotetramers. Our structures suggest RelE-2, and by extension the closely related RelE-1, use a different catalytic mechanism than RelE-3, because our analysis of the RelE-2 structure predicts additional amino acid residues that are likely to be functionally significant and are missing from analogous positions in the RelE-3 structure.