Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.

Genome manipulation by guide-directed Argonaute cleavage.

Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses.

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for vs [nicotine] (δ-slope, 2.

Microbiome for Mars: surveying microbiome connections to healthcare with implications for long-duration human spaceflight, virtual workshop, July 13, 2020.

The inaugural "Microbiome for Mars" virtual workshop took place on July 13, 2020. This event assembled leaders in microbiome research and development to discuss their work and how it may relate to long-duration human space travel. The conference focused on surveying current microbiome research, future endeavors, and how this growing field could broadly impact human health and space exploration.

Attempts to develop an enzyme converting DHIV to KIV.

Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD).

Protein stability engineering insights revealed by domain-wide comprehensive mutagenesis.

The accurate prediction of protein stability upon sequence mutation is an important but unsolved challenge in protein engineering. Large mutational datasets are required to train computational predictors, but traditional methods for collecting stability data are either low-throughput or measure protein stability indirectly. Here, we develop an automated method to generate thermodynamic stability data for nearly every single mutant in a small 56-residue protein.

Particulate methane monooxygenase contains only mononuclear copper centers.

Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (Cu) and one ~2 nanometers away in the membrane-bound PmoC subunit (Cu).

Monomerization of far-red fluorescent proteins.

-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λ ∼ 600-700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations.

ProtaBank: A repository for protein design and engineering data.

We present ProtaBank, a repository for storing, querying, analyzing, and sharing protein design and engineering data in an actively maintained and updated database. ProtaBank provides a format to describe and compare all types of protein mutational data, spanning a wide range of properties and techniques. It features a user-friendly web interface and programming layer that streamlines data deposition and allows for batch input and queries.

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted.

Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.

Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers.

Computational design of co-assembling protein-DNA nanowires.

Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive.

Computational design and experimental verification of a symmetric protein homodimer.

Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein-protein interfaces can serve as a bottom-up method to further our understanding of protein interactions.

Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor.

The pursuit of circuits and metabolic pathways of increasing complexity and robustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription-translation (TX-TL) system for responsiveness to a new targeted effector.

Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants.

In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region.

Computational Design of the β-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization.

Computational design has been used with mixed success for the design of protein surfaces, with directed evolution heretofore providing better practical solutions than explicit design. Directed evolution, however, requires a tractable high-throughput screen because the random nature of mutation does not enrich for desired traits. Here we demonstrate the successful design of the β-sheet surface of a red fluorescent protein (RFP), enabling control over its oligomerization.

The structure of a far-red fluorescent protein, AQ143, shows evidence in support of reported red-shifting chromophore interactions.

Engineering fluorescent proteins (FPs) to emit light at longer wavelengths is a significant focus in the development of the next generation of fluorescent biomarkers, as far-red light penetrates tissue with minimal absorption, allowing better imaging inside of biological hosts. Structure-guided design and directed evolution have led to the discovery of red FPs with significant bathochromic shifts to their emission. Here, we present the crystal structure of one of the most bathochromically shifted FPs reported to date, AQ143, a nine-point mutant of aeCP597, a chromoprotein from Actinia equina.

Precision is essential for efficient catalysis in an evolved Kemp eliminase.

Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates.

Recovery of red fluorescent protein chromophore maturation deficiency through rational design.

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore's covalent structure.

GRID: a high-resolution protein structure refinement algorithm.

The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates.

Iterative approach to computational enzyme design.

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date.

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes.

Three semi-rational approaches, combinatorial site-saturation mutagenesis (CSSM) using a reduced amino acid set and two libraries based on C(orbit) and CRAM computational design algorithms targeting up to 10 active site residues, were used to engineer cytochrome P450 BM3 to demethylate dimethyl ether and hydroxylate propane and ethane. These small libraries (343-1028 variants) were all enriched with respect to the fraction functional and maximal activities compared with a random mutagenesis library and individual site-saturation libraries targeting the same residues. Despite high average amino acid substitution levels of 2.

A structural study of Hypocrea jecorina Cel5A.

Interest in generating lignocellulosic biofuels through enzymatic hydrolysis continues to rise as nonrenewable fossil fuels are depleted. The high cost of producing cellulases, hydrolytic enzymes that cleave cellulose into fermentable sugars, currently hinders economically viable biofuel production. Here, we report the crystal structure of a prevalent endoglucanase in the biofuels industry, Cel5A from the filamentous fungus Hypocrea jecorina.