Person: Summerer, Daniel
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Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads
5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.
Double Nitroxide Labeling by Copper-Catalyzed Azide–Alkyne Cycloadditions with Noncanonical Amino Acids for Electron Paramagnetic Resonance Spectroscopy
Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling (SDSL) is an important tool to obtain long-range distance restraints for protein structural research. We here study a variety of azide- and alkyne-bearing noncanonical amino acids (ncAA) in terms of protein single- and double-incorporation efficiency via nonsense suppression, metabolic stability, yields of nitroxide labeling via copper-catalyzed [3 + 2] azide–alkyne cycloadditions (CuAAC), and spectroscopic properties in continuous-wave and double electron–electron resonance measurements. We identify para-ethynyl-l-phenylalanine and para-propargyloxy-l-phenylalanine as suitable ncAA for CuAAC-based SDSL that will complement current SDSL approaches, particularly in cases in which essential cysteines of a target protein prevent the use of sulfhydryl-reactive spin labels.
Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids
The directed evolution of orthogonal aminoacyl-tRNA synthetases (aaRS) for the genetic encoding of noncanonical amino acids (ncAA) has paved the way for the site-specific incorporation of >170 functionally diverse ncAAs into proteins in a large number of organisms [1, 2]. Here, we describe the directed evolution of orthogonal pyrrolysyl-tRNA synthetase (PylRS) mutants with new amino acid selectivities from libraries using a two-step selection protocol based on chloramphenicol and barnase reporter systems. Although this protocol focuses on the evolution of PylRS variants, this procedure can be universally employed to evolve orthogonal aaRS.
Site-directed spin labeling of proteins for distance measurements in vitro and in cells
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells.
Isoindoline-Based Nitroxides as Bioresistant Spin Labels for Protein Labeling via Cysteines and Alkyne Bearing Noncanonical Amino Acids
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool in protein structural research. Nitroxides are highly suitable spin labeling reagents, but suffer from limited stability in particular in cellular environment. Here, we present the synthesis of a maleimid- and an azide-modified tetraethyl-shielded isoindoline-based nitroxide (M- and Az-TEIO) for labeling of cysteines or the noncanonical amino acid para -ethynyl-l-phenylalanine ( p ENF). We demonstrate high stability of TEIO site-specifically attached to the protein thioredoxin (TRX) towards reduction in prokaryotic and eukaryotic environments, and conduct double electron-electron resonance (DEER) measurements. We further generate a rotamer library for the new residue p ENF-Az-TEIO that affords a distance distribution that is in agreement with the measured distribution.
Site-directed spin labelling of proteins by Suzuki–Miyaura coupling via a genetically encoded aryliodide amino acid
We report site-directed protein spin labelling via Suzuki–Miyaura coupling of a nitroxide boronic acid label with the genetically encoded amino acid 4-iodo-L-phenylalanine. The resulting spin label bears a rigid biphenyl linkage with lower flexibility than spin label R1. It is suitable to obtain defined electron paramagnetic resonance distance distributions and to report protein–membrane interactions and conformational transitions of α-synuclein.
Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity
We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases.
Combining site-directed spin labeling in vivo and in-cell EPR distance determination
Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.
Expanding the Genetic Code for Site-Directed Spin-Labeling
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL⁻EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL⁻EPR studies.
TALEored Epigenetics : A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis
Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development and disease. Besides 5-methylcytosine (mC), the oxidized mC derivatives 5 hydroxymethyl-, 5-formyl- and 5-carboxylcytosine (hmC, fC and caC) have recently been discovered. These are intermediates of an active demethylation pathway, but may also represent new epigenetic marks with individual biological roles. This increase in chemical complexity of DNA-encoded information has created a pressing need for new approaches that allow to read and edit this information. Transcription-activator-like effectors (TALEs) are DNA-binding domains with programmable sequence selectivity that offer the direct reading of epigenetic cytosine-modifications but also enable guiding enzymatic editing domains to genomic loci of choice. Here we review recent advances in employing TALEs for these applications.