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Summary Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies., Graphical abstract, Highlights • Crystal structures unveil molecular basis of SEFL2.2 and CPM technologies • Next gen structure-guided design of CPMs to steer HC-HC pairing • Top CPMs show high pairing efficiency and optimal expression and stability • Balancing CPM charge distribution minimizes impact of sequence diversity, Biochemistry; Bioengineering; Biomolecular engineering; Structural biology

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Summary Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed., Graphical abstract, Highlights • Dual fluorescence approach was used for in situ observation of living cell processes • Radiation-induced effects of mitochondrial biogenesis and apoptosis were observed • Relationship between mitochondrial biogenesis and apoptosis was revisited • Assessing early mitochondrial biogenesis is critical for predicting later fate of cells, Biochemistry methods; Biomolecular engineering; Cell biology

Diseases are caused by mutations in DNA sequences. DNA tends to form double-helix structures but in more recent times, it has been found that it can also form non-canonical structures in response to changes in the environment around it. Japanese researchers have now unearthed some exciting findings about the chain reaction that results, potentially leading to diseases such as cancer. Associate Professor Hisae Tateishi-Karimata and Professor Naoki Sugimoto are based at the Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University in Japan, and are collaborating on important work to better understand the complex inner workings of cells in disease. An area of focus for this research is the elucidation of mechanisms of diseases caused by non-canonical structures of DNA and regulation for DNA function.

Regulatory mechanisms in biological systems are analogous to process control exerted in many chemical and biomolecular engineering processes. Ribonucleic acid (RNA) has been identified as a ubiquit...

The halogen bond (or X-bond) is a noncovalent interaction that is increasingly recognized as an important design tool for engineering protein-ligand interactions and controlling the structures of proteins and nucleic acids. In the past decade, there have been significant efforts to characterize the structure-energy relationships of this interaction in macromolecules. Progress in the computational modeling of X-bonds in biological molecules, however, has lagged behind these experimental studies, with most molecular mechanics/dynamics-based simulation methods not properly treating the properties of the X-bond. We had previously derived a force field for biological X-bonds (ffBXB) based on a set of potential energy functions that describe the anisotropic electrostatic and shape properties of halogens participating in X-bonds. Although fairly accurate for reproducing the energies within biomolecular systems, including X-bonds engineered into a DNA junction, the ffBXB with its seven variable parameters was considered to be too unwieldy for general applications. In the current study, we have generalized the ffBXB by reducing the number of variables to just one for each halogen type and show that this remaining electrostatic variable can be estimated for any new halogenated molecule through a standard restricted electrostatic potential calculation of atomic charges. In addition, we have generalized the ffBXB for both nucleic acids and proteins. As a proof of principle, we have parameterized this reduced and more general ffBXB against the AMBER force field. The resulting parameter set was shown to accurately recapitulate the quantum mechanical landscape and experimental interaction energies of X-bonds incorporated into DNA junction and T4 lysozyme model systems. Thus, this reduced and generalized ffBXB is more readily adaptable for incorporation into classical molecular mechanics/dynamics algorithms, including those commonly used to design inhibitors against therapeutic targets in medicinal chemistry and materials in biomolecular engineering.

Although natural or artificial modified pyrimidine nucleobases represent important molecules with valuable properties as constituents of DNA and RNA, no systematic analyses of the structural aspects of bromo derivatives of cytosine have appeared so far in the literature. In view of the biochemical and pharmaceutical relevance of these compounds, six different crystals containing proton-transfer derivatives of 5-bromocytosine are prepared and analyzed in the solid-state by single crystal X-ray diffraction. All six compounds are organic salts, with proton transfer occurring to the Nimino atom of the pyridine ring. Experimental results are then complemented with Hirshfeld surface analysis to quantitively evaluate the contribution of different intermolecular interactions in the crystal packing. Furthermore, theoretical calculations, based on different arrangements of molecules extracted from the crystal structure determinations, are carried out to analyze the formation mechanism of halogen bonds (XBs) in these compounds and provide insights into the nature and strength of the observed interactions. The results show that the supramolecular architectures of the six molecular salts involve extensive classical intermolecular hydrogen bonds. However, in all but one proton-transfer adducts, weak to moderate XBs are revealed by C–Br…O short contacts between the bromine atom in the fifth position, which acts as XB donor (electron acceptor). Moreover, the lone pair electrons of the oxygen atom of adjacent pyrimidine nucleobases and/or counterions or water molecules, which acts as XB acceptor (electron donor).

Classical hydrogen bonds have, for many decades, been the dominant non-covalent interaction in the toolbox that chemists and chemical engineers have used to design and control the structures of compounds and molecular assemblies as novel materials. Recently, a set of non-classical non-covalent (NC-NC) interactions have emerged that exploit the properties of the Group IV, V, VI, and VII elements of the periodic table (the tetrel, pnictogen, chalcogen, and halogen bonds, respectively). Our research group has been characterizing the prevalence, geometric constraints, and structure-function relationship specifically of the halogen bond in biological systems. We have been particularly interested in exploiting the biological halogen bonds (or BXBs) to control the structures, stabilities, and activities of biomolecules, including the DNA Holliday junction and enzymes. In this review, we first provide a set of criteria for how to determine whether BXBs or any other NC-NC interactions would have biological relevance. We then navigate the trail of studies that had led us from an initial, very biological question to our current point in the journey to establish BXBs as a tool for biomolecular engineering. Finally, we close with a perspective on future directions for this line of research.

Proteins are one of the most fundamental biomolecules present in living organisms. They are indispensable to the sustenance of these organisms owing to the multitude of functions that they perform. They serve as structural constituents of cells and are involved in cell signaling, transport of biological components, cell adhesion, eliciting immune responses, catalyzing chemical reactions, etc. In order to get better insights into these functional aspects, it is quintessential to understand not only the physicochemical features of proteins, but also their structural organization (starting from primary sequences and ending in quaternary structures), and the various interactions that stabilize the higher-order structures. Many biological functions in the cell are mediated when a protein interacts with one or two other proteins in a phenomenon known as protein–protein interaction (PPI). The chapter concludes by providing an overview of PPIs including their functional importance.

Advanced diagnostic techniques, such as PCR and ELISA, are widely used in well-equipped laboratories. However, they are expensive and are not accessible in resource-limited settings. With growing evidence that point-of-care testing (POCT) improves clinical outcomes, there exists a need for simple, fast, low cost, yet sensitive POCT technique. We present a single pot “molecular adaptor”, which we name DNA split proximity circuit (SPC), that can be used as an isothermal, enzyme-free and wash-free diagnostics platform. SPC consists of a pair of “plug-and-play” DNA initiators readily adaptable to various biomarkers ranging from nucleic acids to protein complexes, which then triggers a signal amplification readout based on Hybridization Chain Reaction (HCR). We first improved the kinetics and the sensitivity of HCR by refining HCR hairpin designs. Using biomarkers related to breast cancer as model systems, we applied the optimized HCR to our SPC system and demonstrated its capability to detect microRNA (miRNA) and cell surface receptors. SPC demonstrated good limit of detection in the femtomolar range within 30 min for all miRNA targets tested. It also showed high specificity and an ability to differentiate between different cell surface protein markers. Being isothermal and enzyme-free, our system is less sensitive to temperature and buffer conditions, making it highly robust, thus showing great potential for bedside use. With applications in biomarker analysis, cell visualization and disease diagnostics, herein we improved and established the potential of SPC as a powerful next-generation POCT assay.

Biosurfactants are surface-active molecules originated from renewable resources, which are produced by microbial fermentation or chemical/enzymatic catalysis. These molecules present important advantages as compared to petrochemical surfactants, given their resistance to extreme conditions, biodegradability, specificity, and environmental compatibility. Besides that, the high production costs hinder its commercialization. In this way, this article aimed to analyze microbial biosurfactants production, focusing on the optimization of metabolic pathways and production processes, to identify key aspects and provide alternatives to allow a cost-effective production at industrial scale. This was achieved by a broad analysis of biosurfactants properties, applications, and biosynthetic pathways (in terms of yield, cofactors, and energy), in addition to an assessment of production-associated costs. As a result of the present extensive data survey and analysis, key production aspects are disclosed. The metabolic pathway yield analysis demonstrated that production of biosurfactants can be significantly improved (highest theoretical yield was 0.47 gbiosurfactant /gsubstrate ) by the use of biomolecular engineering techniques to generate optimized synthetic pathways. With an alternative proposed pathway for surfactin, yield was improved and imbalance in cofactors and ATP was reduced. Analysis of productive costs indicated that to make rhamnolipids commercial production feasible, the main efforts should focus on lowering substrate costs as well as the identification of energy-efficient unit operations to lower electricity cost, since these parameters accounted for 19.36 and 78.22%, respectively, of the production costs. The data generated by this analysis highlight the need for multidisciplinary collaboration to make rhamnolipids economically feasible, including biomolecular engineering and process intensification.

Many cell behaviors are significantly affected by cell culture geometry, though it remains unclear which geometry from two- to three-dimensional (2D to 3D) culture is appropriate for probing a specific cell function and mimicking native microenvironments. Toward addressing this, we established a 2.5D culture geometry, enabling initial cell spreading while reducing polarization to bridge between 2D and 3D geometries, and examined the responses of wound healing cells, human pulmonary fibroblasts, within it. To achieve this, we used engineered biomimetic hydrogels formed by photopolymerization, creating robust layered hydrogels with spread fibroblasts at the interface. We found that fibroblast responses were similar between 2D and 2.5D culture and different from 3D culture, with some underlying differences in mechanotransduction. These studies established the 2.5D cell culture geometry in conjunction with biomimetic synthetic matrices as a useful tool for investigations of fibroblast activation with relevance to the study of other cell functions and types.

Summary Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs., Graphical Abstract, Highlights • Non-invasive, microscopic imaging or screening of protein-protein interactions • Intracellular assembly of sensor polypeptides with luminescent Tb(III) complexes • High dynamic range with time-gated detection of Tb(III)-to-GFP sensitized emission, Sensor; Molecular Spectroscopy Techniques; Molecular Interaction; Biomolecular Engineering

The rational design of a multi-responsive protein-based supramolecular system that can predictably respond to more than one stimulus remains an essential but highly challenging goal in biomolecular engineering. Herein, we report a novel chemical method for the construction of multi-responsive supramolecular nanoassemblies using custom-designed facially amphiphilic monodisperse protein-dendron bioconjugates. The macromolecular synthons contain a globular hydrophilic protein domain site-specifically conjugated to photo-responsive hydrophobic benzyl-ether dendrons of different generations through oligo(ethylene glycol) linkers of defined length. The size of the protein nanoassemblies can be systematically tuned by choosing an appropriate dendron or linker of defined length. Exposure of protein nanoassemblies to light results in partial rather than complete disassembly of the complex. The newly formed protein nanoparticle no longer responds to light but could be disassembled into constitutive monomers under acidic conditions or by further treatment with a small molecule. More interestingly, the distribution ratio of the assembled versus disassembled states of protein nanoassemblies after photochemical reaction does not depend on dendron generation, the nature of the linker functionality or the identity of the protein, but is heavily influenced by the linker length. In sum, this work discloses a new chemical method for the rational design of a monodisperse multi-responsive protein-based supramolecular system with exquisite control over the disassembly process.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.Topical HeadingBiomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4`-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

Nanostructures built from biomolecules such as proteins, DNA and RNA are attracting attention in many areas of biological and materials sciences. Such nanoscale engineering was pioneered with proteins, yet the use of DNA is rapidly gaining traction. What are the advantages of the different biopolymers and which is best suited for a given molecular structure, function or application? In this Review, we evaluate the different structural properties of proteins and nucleic acids, as well as possible designs and synthetic routes for functional nanostructures. By comparing protein engineering and DNA nanotechnology, we highlight molecular architectures that are relevant in biotechnology, biomedicine and synthetic biology research, and identify emerging areas for research such as hybrid materials composed of protein and DNA/RNA. Proteins, DNA and RNA can be used to build functional nanostructures. This Review compares protein and DNA/RNA in terms of biochemical properties and ease of engineering in three major areas of application: biomolecular recognition, biocatalysis and structural support.

International audience; ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular re- sponses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensi- tizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and nec- roptosis. We also decrypted a temporally shifted amplitude- and frequency-modu- lated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Molecular hydrogen is a promising currency in the future energy economy due to the uncertain availability of finite fossil fuel resources and environmental effects from their combustion. It also has important uses in the production of fertilizers and platform chemicals as well as in upgrading conventional fuels. Conventional methods for producing molecular hydrogen from natural gas produce carbon dioxide and use a finite resource as feedstock. However, these issues can be overcome by using light energy from the Sun combined with microorganisms and their molecular machinery capable of photosynthesis. In the presence of light, the proteins involved in photosynthesis coupled with appropriate catalysts in higher plants, algae, and cyanobacteria can produce molecular hydrogen, and optimization via genetic modifications and biomolecular engineering further improves production rates. In this review, we will discuss techniques that have been utilized to improve rates of hydrogen production in biological systems based on the protein machinery of photosynthesis coupled with appropriate catalysts. We will also suggest areas for improvement and future directions for work in the field.

Peptide nucleic acids (PNAs) are polyamide analogues of nucleic acids, very effective in terms of affinity and selectivity in DNA/RNA recognition. As other supramolecular entities, the PNA structure has been an interesting scaffold for the development of new molecules, aimed to DNA and RNA recognition, with improved or completely new properties. This review describes recent work, with the aim of describing how the design of these molecules has evolved in recent years, using increasingly effective tools, from simple crystal structure analysis to molecular dynamics and metadynamics. Modified PNA with additional modules appended, either on the backbone or on the nucleobase, are described. Polyfunctional molecules with both backbone and nucleobase modification are then considered. Finally, recent examples of architectures obtained by conjugation of PNAs to inorganic nanostructures as cargo systems for diagnostics and nano-biotechnology are presented.

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

Halogenation is widespread in Nature, as witnessed by the several thousands of naturally occurring halogenated compounds. This review seeks to provide an overview on the pathways and multiple roles of halogenation in Nature, as a source of inspiration to chemists. Remarkably, several halogenation pathways were recently found in humans, sparking an increasing interest into halogens as biomolecular recognition sites. A description of the molecular mechanisms underlining the role of halogenation in the biomolecular context is provided, highlighting how the strategic use of halogen-driven interactions can be exploited to tune the properties of both small compounds and macromolecules. The complexity, versatility and bio-orthogonality of halogen-driven interactions are indeed key figures of merit in promoting novel opportunities for halogenation in a range of applications, spanning from drug design to self-assembly and biomolecular engineering. Specifically, the most relevant and impactful recent contributions in the field of peptide and protein science are presented and critically discussed, with a particular emphasis on the existing opportunities to control their conformational and self-assembly properties towards novel therapeutics and functional materials.

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

Novel methods of molecular modeling help solving urgent problems in drug design, directed evolution of biocatalysts and biosensors, and a lot of other research fields. Implementation of such methods to organophosphorus hydrolase being perfect research object that hydrolyzes dangerous neurotoxic organophosphates could intensify development of antidote and protective preparations to treat poisoning. Structures of enzyme-polyelectrolyte complexes (EPCs) based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) with different biopolymers (various modifications of polyglutamic and polyaspartic acid, as well as hydroxyethyl starch and succinylated gelatin) were simulated at different pH using molecular docking. A number of EPCs with expected “positive” effect on maintaining the maximum level of His6-OPH activity, and some “negative” options were produced, and their catalytic performance was studied. The theoretical results were experimentally confirmed for four of the six “positive” options. EPCs obtained possessed up to 20–40% higher catalytic efficiency in hydrolysis reactions of Paraoxon and Parathion-methyl as compared with that of the native His6-OPH. The results obtained may be a good proof of concept for implementation of molecular docking to calculate model complexes of proteins with (bio)polymers of 6.4–105.5 kg/mol. Also, the approach used here could be interesting as alternative or addition to the directed modifications of enzymes to alter their catalytic characteristics.

Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering.

Control over the functionality of interfaces through biomolecular engineering is a central tool for nanoscale technology as well as many current applications of biology. In this work we designed fusion proteins that combined the surface adhesion and interfacial activity of a hydrophobin-protein together with the high affinity biotin-binding capability of an avidin-protein. We found that an overall architecture that was based on a circularly permuted version of avidin, dual-chain avidin, and hydrophobin gave a highly functional combination. The protein was produced in the filamentous fungus Trichoderma reesei and was efficiently purified using an aqueous two-phase partitioning procedure. The surface adhesive properties were widely different compared to wild-type avidin. Functional characterization showed that the protein assembled on hydrophobic surfaces as a thin layer even at very low concentrations and efficiently bound a biotinylated compound. The work shows how the challenge of creating a fusion protein with proteins that form multimers can be solved by structural design and how protein self-assembly can be used to efficiently functionalize interfaces.

Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts.

Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.

The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale self-assembled structures, antisense therapeutics, microarray diagnostics, and biosensors. Such applications frequently require DNA to be modified and conjugated to other macromolecules, including proteins, polymers, or fatty acids, in order to equip the system with properties required for a particular application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3' hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations. As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures.

The recent recognition that halogen bonding (XB) plays important roles in the recognition and assembly of biological molecules has led to new approaches in medicinal chemistry and biomolecular engineering. When designing XBs into strategies for rational drug design or into a biomolecule to affect its structure and function, we must consider the relationship between this interaction and the more ubiquitous hydrogen bond (HB). In this review, we explore these relationships by asking whether and how XBs can replace, compete against or behave independently of HBs in various biological systems. The complex relationships between the two interactions inform us of the challenges we face in fully utilizing XBs to control the affinity and recognition of inhibitors against their therapeutic targets, and to control the structure and function of proteins, nucleic acids and other biomolecular scaffolds.

International audience; Mycotoxin contamination of food and feed is now ubiquitous. Exposures to mycotoxin via contact or ingestion can potentially induce adverse health outcomes. Affordable mycotoxin-monitoring systems are highly desired but are limited by (a) the reliance on technically challenging and costly molecular recognition by immuno-capture technologies; and (b) the lack of predictive tools for directing the optimization of alternative molecular recognition modalities. Our group has been exploring the development of ochratoxin detection and monitoring systems using the peptide NFO4 as the molecular recognition receptor in fluorescence, electrochemical and multimodal biosensors. Using ochratoxin as the model mycotoxin, we share our perspective on addressing the technical challenges involved in biosensor fabrication, namely: (a) peptide receptor design; and (b) performance evaluation. Subsequently, the scope and utility of molecular modeling and simulation (MMS) approaches to address the above challenges are described. Informed and enabled by phage display, the subsequent application of MMS approaches can rationally guide subsequent biomolecular engineering of peptide receptors, including bioconjugation and bioimmobilization approaches to be used in the fabrication of peptide biosensors. MMS approaches thus have the potential to reduce biosensor development cost, extend product life cycle, and facilitate multi-analyte detection of mycotoxins, each of which positively contributes to the overall affordability of mycotoxin biosensor monitoring systems.

Natural products are complex small molecules produced by an array of organisms and which humans have put to diverse uses, including as pesticides, antibiotics, and anticancer agents. While a wide range has already been uncovered it is clear that there are many more potential biosynthetic pathways for which the products have never been studied. Using the tools of synthetic biology allows us to manipulate these pathways and produce the desired compounds. The genes involved can also be transferred into other organisms which can be more easily manipulated. Recent advances in understanding these complex systems have also informed the targeted manipulation of biosynthetic pathways to produce specific modifications of the final natural product. In the future it may be possible, rather than relying on chemical synthesis, to design and build artificial pathways to synthesize completely new compounds as desired.

Proteins are the key biomolecules in almost all the physiological and pathological processes that are occurring in the cell. Functionality of proteins is related to its conformation, which is ultimately dictated by their unique amino acid sequence. In the current chapter, a brief overview will be provided on the nature of amino acids, structural characteristics and functional versatility of proteins. A glimpse on various interactions present in stabilizing the protein fold and the methods for unraveling their atomic level structures are also discussed. Finally, we present the role of engineered synthetic proteins for the welfare of humanity with huge potential in research and industrial sectors including biotechnological and biomedical fields under the emerging concepts of biomolecular/protein engineering.

Erratum Erratum to ‘‘Pharmacophore modeling strategies for the development of novel nonsteroidal inhibitors of human aromatase (CYP19)” [Bioorg. Med. Chem. Lett. 20 (2010) 3050] Yagmur Muftuoglu , Gabriela Mustata b,* Departments of Biophysics and Chemical Biomolecular Engineering, Johns Hopkins University, Baltmore, MD 21218, United States Department of Computational Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, United States

A review of current processing practices in preparation of recombinant single chain antibody fragments is presented. Single chain antibody fragments which are superior to their Fab and IgG counterparts due to their higher affinity for target antigens while imposing minimal antigenicity in recipient hosts, have sparked breakthroughs in immunology and the medical field at large. The rapidly increasing market demand for pure single chain antibodies for research and therapeutic applications, necessitates viable manufacture routes that can produce large amounts of these antibodies efficiently and as cheaply as possible. Medium- to high-producing expression systems reported for recombinant single chain antibody production are reviewed, and their reported or potential success for efficient commercial-viable preparation of pure antibodies discussed. The effects of expression host system choice on product molecular constraints, ease of processing, and flowsheet design and scale-up are compared. It is concluded that there is no unique host system that can consistently yield high expression levels for a wide range of single chain antibodies; instead, product sequence and end application often dictate the optimum choice of expression host. Irrespective of host systems, adequate a priori design and engineering of the molecular construct supported by good biophysical understanding of the single chain fragment molecules, is a crucial pre-requisite for improved product stability and downstream recovery, which will favourably impact final product yield and functionality.

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.

Due to their structural and mechanical properties, 1D helical protein assemblies represent highly attractive design targets for biomolecular engineering and protein design. Here we present a designed, tetrameric protein building block, Zn(8)R(4), which assembles via Zn coordination interactions into a series of kinetically stable, crystalline, helical nanotubes whose widths can be controlled by solution conditions. X-ray crystallography and transmission electron microscopy (TEM) measurements indicate that all three classes of protein nanotubes are constructed through the same 2D arrangement of Zn(8)R(4) tetramers held together by Zn coordination. The mechanical properties of these nanotubes are correlated with their widths. All Zn(8)R(4) nanotubes are found to be highly flexible despite possessing crystalline order, owing to their small inter-building-block interaction surfaces that are mediated solely by metal coordination.

Understanding the microscopic structure and macroscopic properties of condensed matter from a molecular perspective is important for both traditional and modern chemical engineering. A cornerstone of such understanding is provided by statistical mechanics, which bridges the gap between molecular events and the structural and physiochemical properties of macro- and mesoscopic systems. With ever-increasing computer power, molecular simulations and ab initio quantum mechanics are promising to provide a nearly exact route to accomplishing the full potential of statistical mechanics. However, in light of their versatility for solving problems involving multiple length and timescales that are yet unreachable by direct simulations, phenomenological and semiempirical methods remain relevant for chemical engineering applications in the foreseeable future. Classical density functional theory offers a compromise: on the one hand, it is able to retain the theoretical rigor of statistical mechanics and, on the other hand, similar to a phenomenological method, it demands only modest computational cost for modeling the properties of uniform and inhomogeneous systems. Recent advances are summarized of classical density functional theory with emphasis on applications to quantitative modeling of the phase and interfacial behavior of condensed fluids and soft materials, including colloids, polymer solutions, nanocomposites, liquid crystals, and biological systems. Attention is also given to some potential applications of density functional theory to material fabrications and biomolecular engineering. © 2005 American Institute of Chemical Engineers AIChE J, 2006

With recent advances in biomolecular engineering, the bioremediation of persistent organic pollutants (POPs) using genetically modified microorganisms has become a rapidly growing area of research for environmental protection. Two main biomolecular approaches, rational design and directed evolution, have been developed to engineer enhanced microorganisms and enzymes for the biodegradation of POPs. This review describes the most recent developments and applications of these biomolecular tools for enhancing the capability of microorganisms to bioremediate three major classes of POPs – polycyclic aromatic hydrocabons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Most of the examples focused on the redesign of various features of the enzymes involved in the bioremediation of POPs, including the enzyme expression level, enzymatic activity and substrate specificity. Overall, the rapidly expanding potential of biomolecular engineering techniques has created the exciting potential of remediating some of the most recalcitrant and hazardous compounds in the environment.