Efficient Design Strategy for Whole-Cell and Cell-Free Biosensors based on Engineered Riboswitches

Abstract

We have applied dual genetic selection to design a bacterial riboswitch with improved sensitivity by employing a high-affinity heterologous thiamine pyrophosphate (TPP) aptamer and modified selection conditions in Escherichia coli. The cells transformed with the engineered TPP riboswitches were characterized as whole-cell biosensors. The riboswitches were further studied in cell-free translation systems where they exhibited characteristics similar to those in vivo and a shorter response time for analysis. The flexibility of the riboswitch-based biosensors to accommodate different reporter genes was also demonstrated. Tuning of biosensor characteristics in vivo enables efficient development of aptamer-based whole-cell and cell-free biosensors.     

Riboswitches Based on Kissing Complexes for the Detection of Small Ligands

Biosensors derived from aptamers were designed for which folding into a hairpin shape is triggered by binding of the cognate ligand. These aptamers (termed aptaswitches) thus switch between folded and unfolded states in the presence and absence of the ligand, respectively. The apical loop of the folded aptaswitch is recognized by a second hairpin called the aptakiss through loop–loop or kissing interactions, whereas the aptakiss does not bind the unfolded aptaswitch. Therefore, the formation of a kissing complex signals the presence of the ligand. Aptaswitches were designed that enable the detection of GTP and adenosine in a specific and quantitative manner by surface plasmon resonance when using a grafted aptakiss or in solution by anisotropy measurement with a fluorescently labeled aptakiss. This approach is generic and can potentially be extended to the detection of any molecule for which hairpin aptamers have been identified, as long as the apical loop is not involved in ligand binding.     

Protein knots and fold complexity: some new twists

The current knowledge on topological knots in protein structure is reviewed, considering in turn, knots with three, four and five strand crossings. The latter is the most recent to be identified and has two distinct topological forms. The knot observed in the protein structure is the form that requires the least number of strand crossings to become un-knotted. The position of the chain termini must also correspond to a position that allows (un) knotting in one move. This is postulated as a general property of protein knots and other more complex knots with this property are proposed as the next most likely knots that might be found in a protein. It is also noted that the "Jelly-roll" fold found in some all-beta proteins would provide likely candidates. Alternative measures of knottedness and entanglement are reviewed, including the occurrence of slip-knots. These measures are related to the complexity of the protein fold and may provide useful filters for selecting predicted model structures.     

Protein fold recognition

Summary An important, yet seemingly unattainable, goal in structural molecular biology is to be able to predict the native three-dimensional structure of a protein entirely from its amino acid sequence. Prediction methods based on rigorous energy calculations have not yet been successful, and best results have been obtained from homology modelling and statistical secondary structure prediction. Homology modelling is limited to cases where significant sequence similarity is shared between a protein of known structure and the unknown. Secondary structure prediction methods are not only unreliable, but also do not offer any obvious route to the full tertiary structure. Recently, methods have been developed whereby entire protein folds are recognized from sequence, even where little or no sequence similarity is shared between the proteins under consideration. In this paper we review the current methods, including our own, and in particular offer a historical background to their development. In addition, we also discuss the future of these methods and outline the developments under investigation in our laboratory.     

Alpha/Beta-hydrolase fold enzymes: structures,functions and mechanisms

The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined. Relationships between active site anatomy, topology and conformational rearrangements in the protein molecule is discussed in the context of enzyme mechanism of action.     

Direct correlation analysis improves fold recognition

The extraction of correlated mutations through the method of direct information (DI) provides predicted contact residue pairs that can be used to constrain the three dimensional structures of proteins. We apply this method to a large set of decoy protein folds consisting of many thousand well-constructed models, only tens of which have the correct fold. We find that DI is able to greatly improve the ranking of the true (native) fold but others still remain high scoring that would be difficult to discard due to small shifts in the core beta sheets.     

Mutation-induced fold switching among lattice proteins

Recent experiments uncovered a mutational pathway between two proteins, along which a single mutation causes a switch in fold. Searching for such paths between real proteins remains, despite this achievement, a true challenge. Here, we analyze fold switching in the minimalistic hydrophobic/polar model on a square lattice. For this analysis, we generate a comprehensive sequence-structure database for chains of length ≤ 30, which exceeds previous work by five units. Single-mutation-induced fold switching turns out to be quite common in the model. The switches define a fold network, whose topology is roughly similar to what one would expect for a set of randomly connected nodes. In the combinatorially challenging search for fold switches between two proteins, a tempting strategy is to only consider paths containing the minimum number of mutations. Such a restricted search fails to correctly identify 40% of the single-mutation-linked fold pairs that we observe. The thermodynamic stability is correlated with mutational stability and is, on average, markedly reduced at the observed fold switches.     

O2 penetration and proton burial depth in proteins: applicability to fold family recognition

Paramagnetically induced relaxation effects of O2 and the nitroxide 4-hydroxy TEMPO were measured for the amide protons of perdeuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium pasteurianum. For both O2 and the impermeant nitroxide, the induced relaxation at the static solvent inaccessible amide sites is dominated by long-range interactions with the paramagnetic species in the bulk aqueous phase. The upper bound of O2 solubility in the internal matrix of the rubredoxins is one-tenth that of the bulk aqueous phase. Furthermore, the difference between the oxygen solubilities inside the two rubredoxins is at most 1% that of bulk water O2 solubility, suggesting that there are only modest differences in this measure of fluidity for the mesophile vs hyperthermophile protein interiors. Calculations based on the assumption of a paramagnet uniformly distributed on the protein exterior yield accurate predictions at nearly all amide sites for the minimum relaxation value observed from either the O2 or nitroxide data. Model calculations indicate that the readily obtained paramagnetically induced relaxation effects should prove effective in recognition of structural homology for proteins that are too widely diverged for sequence-based recognition.     

Oligoresorcinols fold into double helices in water

We report the first double helices with a controlled helicity in water based on oligoresorcinols as a new, simplest water-soluble structural motif. The molecular strands of the oligoresorcinols self-assemble into double helices with the aid of aromatic interactions in water as characterized by 1H NMR and absorption spectroscopies together with the X-ray crystallographic study of the pentamer. The double helix formation is sensitive to the chain length, solvent composition, and temperature. Moreover, a bias in the screw sense of the double helices was achieved by covalently attaching chiral substituents to both ends of the molecular strands.     

Folding of a miniprotein with mixed fold

Using the 28 residue betabetaalpha protein FSD-EY as a target system, we examine correction terms for the ECEPP/3 force field. We find an increased probability of formation of the native state at low temperatures resulting from a reduced propensity to form alpha helices and increased formation of beta sheets. Our analysis of the observed folding events suggests that the C-terminal helix of FSD-EY is much more stable than the N-terminal beta hairpin and forms first. The hydrophobic groups of the helix provide a template which promotes the formation of the beta hairpin that is never observed to form without the helix.     

Sequence-controlled oligomers fold into nanosolenoids and impart unusual optical properties

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR‐MS revealed the exact masses of these sequence‐controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring‐current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero‐chromophoric dimer supports a rotated, cofacial π‐stacking orientation separated by about 3.5 Å. Electron–phonon coupling is much stronger than excitonic coupling in these self‐folded PDI oligomers; thus, Franck–Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left‐handed helices or the 1,3‐eclipsed structures. Both theory and experiments validate the nano‐π‐solenoids and their novel photophysical properties.     

Engineering the quadruplex fold: nucleoside conformation determines both folding topology and molecularity in guanine quadruplexes

Nucleic acid quadruplexes, based on the guanine quartet, can arise from one or several strands, depending on the sequence. Those consisting of a single strand are usually folded in one of two principal topologies: antiparallel, in which all or half of the guanine stretches are antiparallel to each other, or parallel, in which all guanine stretches are parallel to each other. In the latter, all guanine nucleosides possess the anti conformation about the glycosidic bond, while in the former, half possess the anti conformation, and half possess the syn conformation. While antiparallel is the more common fold, examples of biologically important, parallel quadruplexes are becoming increasingly common. Thus, it is of interest to understand the forces that determine the quadruplex fold. Here, we examine the influence of individual nucleoside conformation on the overall folding topology by selective substitution of rG for dG. We can reverse the antiparallel fold of the thrombin binding aptamer (TBA) by this approach. Additionally, this substitution converts a unimolecular quadruplex into a bimolecular one. Similar reverse substitutions in the all-RNA analogue of TBA result in a parallel to antiparallel change in topology and alter the strand configuration from bimolecular to unimolecular. On the basis of the specific substitutions made, we conclude that the strong preference of guanine ribonucleosides for the anti conformation is the driving force for the change in topology. These results demonstrate how conformational properties of guanine nucleosides govern not only the quadruplex folding topology but also impact quadruplex molecularity and provide a means to control these properties.     

Estimations of fold surface densities using space-filling models

The model of a tight-fold adjacent reentry fold surface for polyethylene single crystals has been questioned, because it has been speculated that a tight-fold surface should have a density approximating that of the unit cell (～1.00 g/cm³). This would lead to an overall lamella density of close to unity. In contrast, the majority of measured values are in the range 0.96 to 0.97⁺ g/cm³. These lower values have been taken as evidence to disprove a tight-fold–surface model. The present calculations made on space filling models of tight folds indicates a fold surface density on the order of 0.75 g/cm³. This produces no inconsistency between a tight-fold model and accepted lamella density values. Further, calculated values of the weight fraction crystallinity of lamella, as a function of the number of carbons in the fold, limits this number to approximately 9 to 11 carbons per fold.