Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins.

With the rapid acceleration in the design and development of new biotherapeutics, ensuring consistent quality and understanding degradation pathways remain paramount, requiring an array of analytical methods including mass spectrometry. The incorporation of non-canonical amino acids, such as for synthetic selenoproteins, creates additional challenges. A comprehensive strategy to characterize selenoproteins should serve dual purposes of providing sequence confirmation and mapping of selenocysteine bridge locations and the identification of unanticipated side products. In the present study, a combined approach exploiting the benefits of both top-down and bottom-up mass spectrometry was developed. Both electron-transfer/higher-energy collision dissociation and 213 nm ultraviolet photodissociation were utilized to provide complementary information, allowing high quality characterization, localization of diselenide bridges for complex proteins, and the identification of previously unreported selenoprotein dimers.

Using fungible biosensors to evolve improved alkaloid biosyntheses.

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC50) < 30 µM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine. High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, a precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids.

DLPacker: Deep learning for prediction of amino acid side chain conformations in proteins.

Prediction of side chain conformations of amino acids in proteins (also termed "packing") is an important and challenging part of protein structure prediction with many interesting applications in protein design. A variety of methods for packing have been developed but more accurate ones are still needed. Machine learning (ML) methods have recently become a powerful tool for solving various problems in diverse areas of science, including structural biology. In this study, we evaluate the potential of deep neural networks (DNNs) for prediction of amino acid side chain conformations. We formulate the problem as image-to-image transformation and train a U-net style DNN to solve the problem. We show that our method outperforms other physics-based methods by a significant margin: reconstruction RMSDs for most amino acids are about 20% smaller compared to SCWRL4 and Rosetta Packer with RMSDs for bulky hydrophobic amino acids Phe, Tyr, and Trp being up to 50% smaller.

Directed Evolution of an Improved Aminoacyl-tRNA Synthetase for Incorporation of L-3,4-Dihydroxyphenylalanine (L-DOPA).

The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L-DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA-Fe chromophore.

Discovery of Novel Gain-of-Function Mutations Guided by Structure-Based Deep Learning.

Despite the promise of deep learning accelerated protein engineering, examples of such improved proteins are scarce. Here we report that a 3D convolutional neural network trained to associate amino acids with neighboring chemical microenvironments can guide identification of novel gain-of-function mutations that are not predicted by energetics-based approaches. Amalgamation of these mutations improved protein function in vivo across three diverse proteins by at least 5-fold. Furthermore, this model provides a means to interrogate the chemical space within protein microenvironments and identify specific chemical interactions that contribute to the gain-of-function phenotypes resulting from individual mutations.

The Role of tRNA in Establishing New Genetic Codes.

One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/L-shaped molecule must implement the first genetic code, via base pairing and wobble interactions, but is also largely responsible for the specificity of the second genetic code, the pairings between amino acids, tRNA synthetases, and tRNAs. Despite the overarching similarities between tRNAs, they must nonetheless be specifically recognized by cognate tRNA synthetases and largely rejected by noncognate synthetases. Conversely, despite the differences between tRNAs that allow such discrimination, they must be uniformly accepted by the ribosome, in part via the machinations of the translation elongation factors, which work with a diverse coterie of tRNA-amino acid conjugates to balance binding and loading. While it is easy to ascribe both discrimination and acceptance to the individual proteins (synthetases and EF-Tu/eEF-1) that recognize tRNAs, there is a large body of evidence that suggests that the sequences, structures, and dynamics of tRNAs are instrumental in the choices these proteins make.

Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains.

Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered ß-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.

Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates.

Pathogenic Escherichia coli, one of the primary causes of urinary tract infections, has shown significant resistance to the most popular antibiotic, trimethoprim (TMP), which inhibits dihydrofolate reductase (DHFR). The resistance is modulated by single point mutations of DHFR. The impact of two clinically relevant mutations, P21L and W30R, on the activity of DHFR was evaluated via measurement of Michaelis-Menten and inhibitory kinetics, and structural characterization was undertaken by native mass spectrometry with ultraviolet photodissociation (UVPD). Compared to WT-DHFR, both P21L and W30R mutants produced less stable complexes with TMP in the presence of co-factor NADPH as evidenced by the relative abundances of complexes observed in ESI mass spectra. Moreover, based on variations in the fragmentation patterns obtained by UVPD mass spectrometry of binary and ternary DHFR complexes, notable structural changes were localized to the substrate binding pocket for W30R and to the M20 loop region as well as the C-terminal portion containing the essential G-H functional loop for the P21L mutant. The results suggest that the mutations confer resistance through distinctive mechanisms. A novel propargyl-linked antifolate compound 1038 was shown to be a reasonably effective inhibitor of the P21L mutant.

Addicting diverse bacteria to a noncanonical amino acid.

Engineered orthogonal translation systems have greatly enabled the expansion of the genetic code using noncanonical amino acids (NCAAs). However, the impact of NCAAs on organismal evolution remains unclear, in part because it is difficult to force the adoption of new genetic codes in organisms. By reengineering TEM-1 ß-lactamase to be dependent on a NCAA, we maintained bacterial NCAA dependence for hundreds of generations without escape.

Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry.

The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.

Evolving tRNA(Sec) for efficient canonical incorporation of selenocysteine.

Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNA(Sec) needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNA(Sec) that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNA(Sec) allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.

Engineered rRNA enhances the efficiency of selenocysteine incorporation during translation.

We developed a new genetic selection approach to screen for mutations that can alter the efficiency of selenocysteine incorporation. We identified mutations in 16S rRNA that increase or decrease the efficiency of selenocysteine incorporation in Escherichia coli without influencing the efficiency or fidelity of canonical translation. Engineered ribosomes with improved selenocysteine incorporation provide valuable tools for synthetic biology and biotechnology.

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors.

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.