Split Variants Research Articles

Boronic Acid Assisted Self-Assembly of Functional RNAs.

Boronate esters formed by reaction of an oligonucleotide carrying a 5'-boronic acid moiety with the 3'-terminal cis-diol of another have been shown previously to assist assembly of fragmented DNAzymes. Here we demonstrate that boronate esters replacing the natural phosphodiester linkage at selected sites of two functional RNAs, the hairpin ribozyme and the Mango aptamer, allow assembly of functional structures. The hairpin ribozyme, a small naturally occurring RNA that supports the reversible cleavage of appropriate RNA substrates, is very sensitive to fragmentation. Splitting the ribozyme at four different sites led to a significant decrease or even loss of cleavage and ligation activity. Ribozymes assembled from fragments capable of boronate ester formation showed restoration of cleavage activity in some but not all cases, dependent on the split site. Ligation proved to be more challenging, no supportive effect of the boronate ester was observed. Split variants of the Mango aptamer also showed a dramatic loss of functionality, which however, was restored when 5'-boronic acid modified fragments were used for assembly. These studies show for the first time that boronate esters as internucleoside linkages can act as surrogates of natural phosphodiesters in functional RNA molecules.

A split ribozyme that links detection of a native RNA to orthogonal protein outputs

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing.

Fluorescent labels and biosensors play central roles in biological and medical research. Targeted to specific biomolecules or cells, they allow noninvasive imaging of the machinery that govern cells and organisms in real time. Recently, chemogenetic reporters made of organic dyes specifically anchored to genetic tags have challenged the paradigm of fully genetically encoded fluorescent proteins. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded tags, these chemogenetic reporters open new exciting prospects for studying cell biochemistry and biology. In this Account, we present the growing toolbox of fluorescence-activating and absorption-shifting tags (FASTs), small monomeric proteins of 14 kDa (125 amino acids residues) that can be used as markers to monitor gene expression and protein localization in live cells and organisms. Engineered by directed protein evolution from the photoactive yellow protein (PYP) from the bacterium Halorhodospira halophila, prototypical FAST binds and stabilizes the fluorescent state of live-cell compatible hydroxybenzylidene rhodanine chromophores. This class of chromophores are normally dark when free in solution or in cells because they dissipate light energy through nonradiative processes. The protein cavity of FAST allows the stabilization of the deprotonated state of the chromophore and blocks the chromophore into a planar conformation, which leads to highly fluorescent protein-chromophore assemblies. The use of such fluorogenic dyes (also called fluorogens) enables the imaging of FAST fusion proteins in cells with high contrast without the need to remove unbound ligands through separate washing steps. Fluorogens with various spectral properties exist nowadays allowing investigators to adjust the spectral properties of FAST to their experimental conditions. Molecular engineering allowed furthermore to generate membrane-impermeant fluorogens for the selective labeling of cell-surface proteins. Over the years, we generated a collection of FAST variants with expanded spectral properties or fluorogen selectivity using a concerted strategy involving molecular engineering and directed protein evolution. Moreover, protein engineering allowed us to adapt FASTs for the design of fluorescent biosensors. Circular permutation enabled the generation of FAST variants with increased conformational flexibility for the design of biosensors in which fluorogen binding is conditioned to the recognition of a given analyte. Bisection of FASTs into two complementary fragments allowed us furthermore to create split variants with reversible complementation that allow the detection and imaging of dynamic protein-protein interactions. We provide, here, a general overview of the current state of development of these different systems and their applications for advanced live cell imaging and biosensing and discuss potential future directions.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein.

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein‐splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non‐redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class‐3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high‐resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors.

T7 RNA polymerase (RNAP) is a powerful protein scaffold for the construction of synthetic biology tools and biosensors. However, both T7 RNAP and its split variants are intolerant to C-terminal modifications or fusions, thus placing a key limitation on their engineering and deployment. Here, we use rapid continuous-evolution approaches to evolve both full-length and split T7 RNAP variants that tolerate modified C termini and fusions to entire other proteins. Moreover, we show that the evolved split C-terminal RNAP variants can function as small-molecule biosensors, even in the context of large C-terminal fusions. This work provides a panel of modified RNAP variants with robust activity and tolerance to C-terminal fusions, and provides insights into the biophysical requirements of the C-terminal carboxylic acid functional group of T7 RNAP.

Enhanced catalytic properties of novel (αbγ)2 heterohexameric Rhodobacter capsulatus xanthine dehydrogenase by separate expression of the redox domains in Escherichia coli

Post-translational proteolysis is usually necessary for the commercial production of xanthine dehydrogenases (XDHs), such as bovine (αβγ)2 XDH, to increase its catalytic activity. The proteolysis approach suffers from low controllability and inefficient purification. To obviate these disadvantages, we have developed a method that translates active Rhodobacter capsulatus (αβγ)2 XDH by directly expressing the iron-sulfur domain, the flavin adenine dinucleotide domain and the sulfurated molybdenum domain as three separate proteins in Escherichia coli. Two (αβγ)2 XDH variants, Split166 and Split178, which were designed by splitting the small subunit of R. capsulatus CGMCC 1.3366 (αβ)2 XDH at the N- and C-terminal ends of the L167-A178 peptide linking the iron–sulfur clusters and flavin adenine dinucleotide domains, respectively, were expressed in E. coli. Compared to the wild type, both split variants increased the thermostability by 11°C and Split178 enhanced the turnover number and catalytic efficiency by 1.15-fold and 1.66-fold, while Split166 decreased these parameters by 3.2-fold and 5-fold, respectively. This study is the first successful trial to express an active split (αβγ)2 XDH directly by manipulating genes encoding redox domains, and the enhanced properties of the expressed (αβγ)2 XDH using the in vivo splitting strategy may be a promising technique for the commercial production of XDHs.

Bimolecular fluorescence complementation based on the red fluorescent protein FusionRed

The aim of this work is to construct split variants of the red fluorescent protein FusionRed, each of which consists of two separate polypeptides, nonfluorescent parts of FusionRed, that can form functional fluorescent proteins upon reassociation. At the first stage, various circularly permuted FusionRed variants have been created (in circular permutants the protein polypeptide chain is divided into two parts, which change places so that the C-terminal part is followed by the N-terminal part). Two variants with the highest rate of chromophore maturation (fluorescence development) have been selected out of 23 tested permutation points. These proteins called cpFR76-73 and cpFR189-188 (the first number indicates the last amino acid residue of the N-terminal part; the second number, the first residue of the C-terminal part) are spectrally similar to parental FusionRed but possess lower fluorescence quantum yields. Split variants corresponding to these two circular permutants have been tested in mammalian cells. For reassembly of the fluorescent protein fragments, heterodimerizing leucine zippers have been used. It has been shown that split variant FR189-188 matures at 37°C and possesses fluorescence brightness similar to that of FusionRed. Consequently, FR189-188 is potentially suitable for a wide range of applications, for example, the study of protein–protein interactions or visualization of cell populations, in which two target gene promoters are simultaneously active.

Combining Fragmentation of an Amino Acyl tRNA Synthetase with Fusion to Interacting Proteins to Increase Control Over Protein Metabolic Labeling

Protein synthesis and degradation rates change during development and disease onset, but it remains difficult to measure these changes in multicellular organisms because we lack sufficient spatial and temporal control over proteomic measurements. One method of resolving this issue is to use a modified methionyl tRNA synthetase (MetRS) to regulate noncanonical amino acid tagging of newly synthesized protein with azidonorleucine (Anl). Noncanonical amino acids with azide groups (e.g., Anl) are particularly useful for proteomics because bioorthogonal azides allow for selective “click” chemistry for imaging, enrichment, and sequencing. Anl labeling has been previously accomplished using E. coli MetRS (EcMetRS) harboring NLL (L13N/Y260L/H301L) active site mutations that direct substrate specificity from methionine to Anl. To expand control of this genetically encoded tool for metabolic protein labeling, we built libraries of fragmented EcMetRS that encode all possible fragmented EcMetRS as fusions to different pairs of interacting proteins. Selection of these libraries for active MetRS revealed a large number of backbone cleavage sites that can be tolerated without disrupting protein function. Some of these split variants showed dependence on the protein-protein interaction while others showed activity in the absence of an interaction. Ongoing efforts are examining the activities of these split EcMetRS in the presence of NLL mutations, which are required for Anl labeling. These new split NLL-EcMetRS are expected to enable post-translation regulation of metabolic protein labeling with Anl, which will be useful for increasing control over proteomics in complex cellular settings by allowing for regulation of NLL-EcMetRS through a combination of transcriptional and post-translational control. This work was funded by the Houston Area Molecular Biophysics Training Program and the National Science Foundation.

Increasing Spatiotemporal Control over Azidonorleucine Protein Labeling within Cells to Enable Higher Resolution Proteomics

As many signaling pathways are interconnected in complex ways, it is difficult to predict the protein production changes accompanying most changes of cellular state, such as during development or disease onset. One method of resolving this issue is to use a modified methionyl tRNA synthetase (MetRS) for bioorthogonal noncanonical amino acid tagging of newly synthesized protein with azidonorleucine (Anl). Use of noncanonical amino acids with azide groups (e.g., Anl) are particularly useful because azides are uncommon within cells, thus allowing for selective “click” chemistry for imaging and sequencing. Anl labeling can be accomplished using E. coli MetRS (EcMetRS) harboring NLL (L13N/Y260L/H301L) active site mutations that direct substrate specificity from Met to Anl. Using a conditional promoter, EcMetRS activity can be spatiotemporally controlled. To expand spatiotemporal control, we built split two‐fragment EcMetRS libraries with various protein‐protein interactions and linkers with or without Tobacco Etch Virus (TEV) specific protease cleavage sites. The libraries were subject to selection and revealed diverse functional split sites across the protein sequence. Some of these split variants showed dependence on the protein‐protein interaction and on presence/absence of TEV. Overall, these findings suggest that incorporation of the NLL mutations in these new EcMetRS variants will allow for novel regulation of metabolic labeling for proteomics through a combination of transcriptional and post‐translational control. This work was funded by the Houston Area Molecular Biophysics Training Program, the Howard Hughes Medical Institute Med into Grad Program, and the Welch Foundation.

Functional analysis of the second extracellular loop of rhodopsin by characterizing split variants

The second extracellular loop (ECL2) of the family 1 G protein-coupled receptors (GPCR) is known to function in ligand recognition and to show structural diversity in a variety of GPCRs. In rhodopsin, ECL2 forms a rigid structure including a beta-sheet and interacts with the transmembrane region via a disulfide bond, hydrogen bonds, and hydrophobic interactions. It forms the chromophore-binding pocket with transmembrane helices and directly interacts with the 11-cis-retinal chromophore. To clarify the functional role of the rigid ECL2 in bovine rhodopsin, we designed split rhodopsins in which the polypeptide chain of rhodopsin was cleaved at the C-terminal end and/or N-terminal end of ECL2 by genetic engineering. A reconstitution study of pigment from two peptide fragments and 11-cis-retinal showed that fixation of the N-terminus of ECL2 to the transmembrane region is essential for folding into the native rhodopsin structure. Split rhodopsin was resistant to hydroxylamine and activated transducin upon light absorption similarly to wild-type rhodopsin, but was readily disassembled by photobleaching. Analyses of the photobleaching processes of split rhodopsins and rhodopsin mutant lacking disulfide bond showed that the rigid structure of ECL2 is required for facilitating the formation of the active state. These results suggest that ECL2 'mechanically' drives the conformational change of rhodopsin.

Nitrous oxide emissions from cattle-impacted pasture soil amended with nitrate and glucose

There is little information concerning N2O fluxes in the pasture soil that has received large amounts of nutrients, such as urine and dung, for several years. The aims of this study were to (1) experimentally quantify the relationship between mineral N input and N2O emissions from denitrification, (2) describe the time course of N2O fluxes resulting in N inputs, and (3) find whether there exists an upper limit of the amount of nitrogen escaping the soil in the form of N2O. The study site was a grassland used as a cattle overwintering area. It was amended with KNO3 and glucose corresponding to 10–1,500 kg N and C per hectare, covering the range of nutrient inputs occurring in real field conditions. Using manual permanent chambers, N2O fluxes from the soil were monitored for several days after the amendments. The peak N2O emissions were up to 94 mg N2O–N m−2 h−1, 5–8 h after amendment. No upper limit of N2O emissions was detected as the emissions were directly related to the dose of nutrients in the whole range of amendments used, but the fluxes reflected the soil and environmental conditions, too. Thus, in three different experiments performed during the season, the total cumulative losses of N2O–N ranged from 0.2 to 5.6% of the applied 500kg \(\operatorname{N} \operatorname{O} _{3} ^{ - } - \operatorname{N} \) ha−1. Splitting of high nutrient doses lowered the rate of N2O fluxes after the first amendment, but the effect of splitting on the total amount of N2O–N released from the soil was insignificant, as the initial lower values of emissions in the split variants were compensated for by a longer duration of gas fluxes. The results suggest that the cattle-impacted soil has the potential to metabolize large inputs of mineral nitrogen over short periods (∼days). Also, the emission factors for \( \operatorname{N} \operatorname{O} _{3} ^{ - } \, - \operatorname{N} \) did not exceed values reported in literature.

Expression of CD44 variant proteins in adenocarcinoma of Barrett's esophagus and its relation to prognosis.

None of the commonly used staging criteria accurately determine the prognosis of a patient with adenocarcinoma of Barrett's esophagus. The authors therefore assessed the expression pattern and prognostic impact of CD44 standard and CD44 isoforms CD44v4, v5,v6,v7, and v10 in adenocarcinoma of Barrett's esophagus. Specimens from 41 patients with adenocarcinoma of Barrett's esophagus who underwent esophageal resection were embedded in paraffin and studied immunohistochemically to determine the expression of CD44 splice variants. Histomorphologic parameters and survival time were not known at the time of the investigation. Correlations between favorable clinical or histomorphologic parameters and CD44s or any of the split variants could not be established. Down-regulation of CD44s and the split variant v10 was significantly correlated with pT classification. Furthermore, down-regulation of CD44v10 and up-regulation of CD44v7 were significantly correlated with ploidy. There was a significant correlation between CD44s and split variants in tumorous and nontumorous tissue from the same patient. Down-regulation of CD44s and CD44v4 had a significant influence on prognosis in that it was associated with shortened life expectancy. Multivariate analysis revealed that the expression of CD44v4 was an independent factor in prognosis. The results obtained for this small patient sample suggest that CD44v4 is a new independent prognostic parameter for adenocarcinoma of Barrett's esophagus that can be determined preoperatively by biopsy. It may therefore be helpful in planning therapy by allowing the identification of patients who may benefit from esophageal resection as well as those who are at high risk for morbidity and mortality even when the tumor is otherwise resectable. Further studies of larger patient samples are required to validate the results of the current study.