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Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the "photon budget" require better and brighter fluorophores, causing a renewed interest in synthetic dyes. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical fluorophores to modern biological imaging.

The intrinsic and extrinsic factors that contribute to stem and neuronal precursor cell maintenance and/or differentiation remain poorly understood. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. We have taken a loss-of-function approach, by developing a library of bacterial lyases and sulfatases to specifically remodel the ECM and test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation.

Genetically encoded FRET probes enable us to visualize a variety of signaling events such as protein phosphorylation and G-protein activation in living cells. This unit focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and incorporated into a single molecule, i.e., a unimolecular probe. Advantages of these probes lie in their easy loading into cells, simple acquisition of FRET images, and clear evaluation of data. We have developed FRET probes for Ras-superfamily GTPases, designated Ras and interacting protein chimeric unit (Raichu) probes. We hereby describe strategies to develop Raichu-type FRET probes, procedures for their characterization, and acquisition and processing of images. Although improvements upon FRET probes are still based on trial-and-error, we provide practical tips for their optimization and briefly discuss the theory and applications of unimolecular FRET probes.

Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2 degrees structure, 3 degrees structure, and 4 degrees structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes. Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2 degrees structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3 degrees structure, 4 degrees structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.

The number of known protein structures is growing exponentially (Berman et al., 2000), but the structural mapping of essential domain-domain and protein-protein interaction surfaces has advanced more slowly. It is particularly difficult to analyze the interaction surfaces of membrane proteins on a structural level, both because membrane proteins are less accessible to high-resolution structural analysis and because the membrane environment is often required for native complex formation. The Protein-Interactions-by-Cysteine-Modification (PICM) method is a generalizable, in vitro chemical scanning approach that can be applied to many protein complexes, in both membrane-bound and soluble systems. The method begins by engineering Cys residues on the surface of a protein of known structure, then a bulky probe is coupled to each Cys residue. Next, the effects of both Cys substitution and bulky probe attachment are measured on the assembly and the activity of the target complex. Bulky probe coupling at an essential docking site disrupts complex assembly and/or activity, while coupling outside the site typically has little or no effect. PICM has been successfully applied to the core signaling complex of the E. coli and S. typhimurium chemotaxis pathway, where it has mapped out essential docking surfaces on transmembrane chemoreceptor (Tar) and histidine kinase (CheA) components (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The approach shares similarities with other important scanning methods like alanine and tryptophan scanning (Cunningham and Wells, 1989; Sharp et al., 1995a), but has two unique features: (1) functional effects are determined for both small volume (Cys) and large volume (bulky probe) side chain substitutions in the same experiment, and (2) nonperturbing positions are identified at which Cys residues and bulky probes can be introduced for subsequent biochemical and biophysical studies, without significant effects on complex assembly or activity.

Today different database systems for molecular structures (genes and proteins) and metabolic pathways are available. All these systems are characterized by the static data representation. For progress in biotechnology the dynamic representation of this data is important. The metabolism can be characterized as a complex biochemical network. Different models for the quantitative simulation of biochemical networks are discussed, but no useful formalization is available. This paper shows that the theory of Petrinets is useful for the quantitative modeling of biochemical networks.

Chemical peptide synthesis is well-established but has drawbacks, notably the need to protect side-chain functional groups during reactions. Proteolytic enzymes may be used 'in reverse' to catalyse peptide synthesis without side-chain protection and avoiding the danger of racemization. Enzymic syntheses may use either an equilibrium or a kinetic strategy. Equilibrium synthesis is simply the reversal of hydrolysis, using any type of protease. Kinetic synthesis involves time-dependent acyl transfer using activated substrates and a serine or cysteine protease. Enzymic synthesis often takes place in partially aqueous or non-aqueous reaction media; the medium can often desirably influence the protease's properties. It is possible to tailor a protease to improve its usefulness in peptide synthesis; one can alter the enzyme's properties by means such as chemical modification, PEG-coupling or protein engineering.

Intracellular lipid binding proteins (iLBPs) play crucial roles in lipid transport and cellular metabolism across the animal kingdom. Recently, a fat‐to‐neuron axis was described in Caenorhabditis elegans, in which lysosomal activity in the fat liberates polyunsaturated fatty acids (PUFAs) that signal to neurons and extend lifespan with durable fecundity. In this study, we investigate the structure and binding mechanisms of a lifespan‐extending lipid chaperone, lipid binding protein‐3 (LBP‐3), which shuttles dihomo‐γ‐linolenic (DGLA) acid from intestinal fat to neurons. We present the first high‐resolution crystal structure of LBP‐3, which reveals a classic iLBP fold with an unexpected and unique homodimeric arrangement via interstrand interactions that is incompatible with ligand binding. We identify key ionic interactions that mediate DGLA binding within the lipid binding pocket. Molecular dynamics simulations further elucidate LBP‐3's preferential binding to DGLA due to its rotational freedom and access to favorable binding conformations compared to other 20‐carbon PUFAs. We also propose that LBP‐3 dimerization may be a unique regulatory mechanism for lipid chaperones. [ABSTRACT FROM AUTHOR]

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging unsolved problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign tasks, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 19 new homo-oligomers and structurally recombined them to make 24 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system have identical or nearly identical backbones, and hence are ideal building blocks for generating and functionalizing larger symmetric and pseudosymmetric assemblies. Hetero-oligomeric proteins offer many advantages for bioengineering efforts but are difficult to make from scratch. Here, authors present a simple method for creating pseudosymmetric hetero-oligomers from input symmetrical proteins. [ABSTRACT FROM AUTHOR]

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes. Microbial keratinases can convert keratin wastes into valuable compounds for mankind. Since the market for keratinases cannot be satisfied by production from wild hosts, it is obligatory to develop hosts with high keratinase yields. The intention of this review is to evaluate microbial keratinase advancement through protein engineering, breeding techniques, and fermentation optimization. The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods. Moreover, modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield. Intending to accelerate the production of modified keratinase, this review attempts to highlight the optimization of expression elements, such as promoter engineering, UTR, signal peptide, and codon optimization. Moreover, the approaches of host engineering including strengthening precursor supply, membrane surface engineering, and optimization of secretion pathways were also explained here. Furthermore, it is also essential to optimize the medium composition and fermentation condition for high keratinase yield. This review also addressed the present advancements, difficulties, and tendencies in the field of microbial keratinase production, along with its potential.

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5' guanosine is very low thus limiting their utility as a transcriptome engineering toolset. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.

Neuroglobin is a hemoprotein expressed in several nervous system cell lineages with yet unknown physiological functions. Neuroglobin presents a very similar structure to that of the related globins hemoglobin and myoglobin, but shows an hexacoordinate heme as compared to the pentacoordinated heme of myoglobin and hemoglobin. While several reactions of neuroglobin have been characterized in vitro, the relative importance of most of those reactions in vivo is yet undefined. Neuroglobin, like other heme proteins, can reduce nitrite to nitric oxide, providing a possible route to generate nitric oxide in vivo in low oxygen conditions. The reaction kinetics are highly dependent on the nature of the distal residue, and replacement of the distal histidine His64(E7) can increase the reaction rate constants by several orders of magnitude. However, mutation of other distal pocket positions such as Phe28(B10) or Val68(E11) has more limited impact on the rates. Computational analysis using myoglobin as template, guided by the structure of dedicated nitrite reductases like cytochrome cd1 nitrite reductase, has pointed out that combined mutations of the residues B10 and CD1 could increase the nitrite reductase activity of myoglobin, by mimicking the environment of the distal heme pocket in cytochrome cd1 nitrite reductase. As neuroglobin shows high sequence and structural homology with myoglobin, we hypothesized that such mutations (F28H and F42Y in neuroglobin) could also modify the nitrite reductase activity of neuroglobin. Here we study the effect of these mutations. Unfortunately, we do not observe in any case an increase in the nitrite reduction rates. Our results provide some further indications of nitrite reductase regulation in neuroglobin and highlight the minor but critical differences between the structure of penta- and hexacoordinate globins.

Ubiquitin and its relatives are major players in many biological pathways, and a variety of experimental tools based on biological chemistry or protein engineering is available for their manipulation. One popular approach is the use of linear fusions between the modifier and a protein of interest. Such artificial constructs can facilitate the understanding of the role of ubiquitin in biological processes and can be exploited to control protein stability, interactions and degradation. Here we summarize the basic design considerations and discuss the advantages as well as limitations associated with their use. Finally, we will refer to several published case studies highlighting the principles of how they provide insight into pathways ranging from membrane protein trafficking to the control of epigenetic modifications. [Display omitted] • Flexible tethering facilitates the use of ubiquitin-like proteins in linear fusions. • Linear fusions are widely used to study the functions of ubiquitin-like modifiers. • Linear ubiquitin fusions can be used to control protein stability and degradation. [ABSTRACT FROM AUTHOR]

Global warming is caused by human activity, such as the burning of fossil fuels, which produces high levels of greenhouse gasses. As a consequence, climate change impacts all organisms and the greater ecosystem through changing conditions from weather patterns to the temperature, pH and salt concentrations found in waterways and soil. These environmental changes fundamentally alter many parameters of the living world, from the kinetics of chemical reactions and cellular signaling pathways to the accumulation of unforeseen chemicals in the environment, the appearance and dispersal of new diseases, and the availability of traditional foods. Some organisms adapt to extremes, while others cannot. This article asks five questions that prompt us to consider the foundational knowledge that biochemistry can bring to the table as we meet the challenge of climate change. We approach climate change from the molecular point of view, identifying how cells and organisms – from microbes to plants and animals – respond to changing environmental conditions. To embrace the concept of “one health” for all life on the planet, we argue that we must leverage biochemistry, cell biology, molecular biophysics and genetics to fully understand the impact of climate change on the living world and to bring positive change.

Direct reductive amination of ketones by reductive aminases (RedAms) is a promising method for the synthesis of primary, secondary and tertiary amines. In this work, five naturally occurring RedAms possessing reductive amination activity were identified using a structure-guided genome mining (SGGM) approach, which was based on highly conserved substrate binding and catalysis motif alignment. This RedAm toolbox facilitated the reductive amination of cyclohexanone and 4-fluoropropiophenone with various amine nucleophiles. In a preparative biotransformation (100 ​mg) with low stoichiometric ammonia donor and low catalyst loading, and three types of N-alkylcyclohexylamine were efficiently synthesized with a space-time yield up to 64.2 ​g ​L−1 ​d−1, demonstrating the potential of the RedAm enzymes as a biocatalyst toolbox for the synthesis of primary and secondary amines. Additionally, through protein engineering, the W205F variant of KfRedAm from Kribbella flavida was obtained with a 2-fold increased catalytic efficiency (kcat/Km), and the general applicability of the variant was verified, providing useful guidance for further evolution of RedAms within this family.

Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein-Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the 'linker', from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle.

Keratinases belong to a class of proteases that are able to degrade keratins into amino acids. Microbial keratinases play important roles in turning keratin-containing wastes into value-added products by participating in the degradation of keratin. Keratin is found in human and animal hard tissues, and its complicated structures make it resistant to degradation by common proteases. Although breaking disulfide bonds are involved in keratin degradation, keratinase is responsible for the cleavage of peptides, making it attractive in pharmaceutical and feather industries. Keratinase can serve as an important tool to convert keratin-rich wastes such as feathers from poultry industry into diverse products applicable to many fields. Despite of some progress made in isolating keratinase-producing microorganisms, structural studies of keratinases, and biochemical characterization of these enzymes, effort is still required to expand the biotechnological application of keratinase in diverse fields by identifying more keratinases, understanding the mechanism of action and constructing more active enzymes through molecular biology and protein engineering. Herein, this review covers structures, applications, biochemistry of microbial keratinases, and strategies to improve its efficiency in keratin degradation. [ABSTRACT FROM AUTHOR]

Methanotrophic bacteria represent a potential route to methane utilization and mitigation of methane emissions. In the first step of their metabolic pathway, aerobic methanotrophs use methane monooxygenases (MMOs) to activate methane, oxidizing it to methanol. There are two types of MMOs: a particulate, membrane-bound enzyme (pMMO) and a soluble, cytoplasmic enzyme (sMMO). The two MMOs are completely unrelated, with different architectures, metal cofactors, and mechanisms. The more prevalent of the two, pMMO, is copper-dependent, but the identity of its copper active site remains unclear. By contrast, sMMO uses a diiron active site, the catalytic cycle of which is well understood. Here we review the current state of knowledge for both MMOs, with an emphasis on recent developments and emerging hypotheses. In addition, we discuss obstacles to developing expression systems, which are needed to address outstanding questions and to facilitate future protein engineering efforts. [ABSTRACT FROM AUTHOR]

With the recent progress in biotechnology, a wide variety of novel enzymes with unique physicochemical properties and diverse applications has been introduced, and new application list continues to extend in the future. Enzymes obtained from microorganisms, including bacteria, fungi, yeast are widely applied in numerous food formulations for intensifying their texture and taste. Owing to several desirable characteristics such as easy, cost-efficient and stable production, microbial-derived enzymes are preferred source in contrast to animals or plants. Enzymatic processes have a considerable impact in controlling the characteristics such as (1) physiochemical properties, (2) rheological functionalities, (3) facile process as compared to the chemical-based processing, (4) no or minimal consumption of harsh chemicals, (5) overall cost-effective ratio, (6) sensory and flavor qualities, and (7) intensifying the stability, shelf life and overall quality of the product, etc. in the food industry. Also, enzyme-catalyzed processing has also been designed for new food applications such as extraction of bioactive compounds, nutrient-rich and texture improved foods production, and eliminating food safety hazards. Herein, we reviewed recent applications of food-processing enzymes and highlighted promising technologies to diversify their application range in food industries. Immobilization technology enabled biocatalysts to be used cost-effectively due to reusability with negligible or no activity loss. Integrated progress in novel enzyme discovery, and recombinant DNA technology, as well as protein engineering and bioprocess engineering strategies, are believed to rapidly propagate biocatalysis at industrial-scale food processing or green and sustainable chemical manufacturing. [ABSTRACT FROM AUTHOR]