2020-04, Braun, Theresa S., Widder, Pia, Osswald, Uwe, Groß, Lina, Williams, Lara, Schmidt, Moritz J., Helmle, Irina, Summerer, Daniel, Drescher, Malte

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.

2016, Roser, Patrick, Schmidt, Moritz J., Drescher, Malte, Summerer, Daniel

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.

2015, Schmidt, Moritz J., Fedoseev, Artem, Bücker, Dennis, Borbas, Julia, Peter, Christine, Drescher, Malte, Summerer, Daniel

The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

2014, Kubik, Grzegorz, Schmidt, Moritz J., Penner, Johanna E., Summerer, Daniel

Gene expression is extensively regulated by specific patterns of genomic 5-methylcytosine (mC), but the ability to directly detect this modification at user-defined genomic loci is limited. One reason is the lack of molecules that discriminate between mC and cytosine (C) and at the same time provide inherent, programmable sequence-selectivity. Programmable transcription-activator-like effectors (TALEs) have been observed to exhibit mC-sensitivity in vivo, but to only a limited extent in vitro. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome.

2019-02-07, Kugele, Anandi, Braun, Theresa S., Widder, Pia, Williams, Lara, Schmidt, Moritz J., Summerer, Daniel, Drescher, Malte

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.

2016, Lee, Yan-Jiun, Schmidt, Moritz J., Tharp, Jeffery M., Weber, Annemarie, Koenig, Amber L., Zheng, Hong, Gao, Jianmin, Waters, Marcey L., Summerer, Daniel, Liu, Wenshe R.

Fluorophenylalanines bearing 2-5 fluorine atoms at the phenyl ring have been genetically encoded by amber codon. Replacement of F59, a phenylalanine residue that is directly involved in interactions with trimethylated K9 of histone H3, in the Mpp8 chromodomain recombinantly with fluorophenylalanines significantly impairs the binding to a K9-trimethylated H3 peptide.

2014-01-29, Schmidt, Moritz J., Borbas, Julia, Drescher, Malte, Summerer, Daniel

We report the genetic encoding of a noncanonical, spin-labeled amino acid in Escherichia coli. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chemical labeling step usually required for protein electron paramagnetic resonance (EPR) studies. The amino acid can be introduced at multiple, user-defined sites of a protein and is stable in E. coli even for prolonged expression times. It can report intramolecular distance distributions in proteins by double-electron electron resonance measurements. Moreover, the signal of spin-labeled protein can be selectively detected in cells. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.

2018, Schmidt, Moritz J., Summerer, Daniel

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.

2015, Schmidt, Moritz J., Fedoseev, Artem, Summerer, Daniel, Drescher, Malte

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and interactions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL-EPR studies and enables the investigation of native, endogenous proteins with minimal structural perturbation, and without the need to create unique reactive sites for chemical labeling. We report detailed experimental procedures for the efficient synthesis of SLK-1, the expression and purification of SLK-1-containing proteins under conditions that ensure maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR distance measurements in proteins by double electron-electron resonance.

2014, Schmidt, Moritz J., Summerer, Daniel, Drescher, Malte

Elektronenspinresonanz- (ESR-) Spektroskopie kombiniert mit ortsspezifischer Spinmarkierung ist bestens geeignet, um Struktur, Dynamik und Wechselwirkungen von Proteinen zu untersuchen. Besonders interessant sind Abstandsmessungen zwischen zwei identischen, verhältnismäßig kleinen Spinmarkern, beispielsweise Nitroxiden. Diese Abstandsmessungen beruhen auf der Dipol-Dipol-Wechselwirkung zwischen den beiden Spinmarkern, die mit 1/r3 abfällt. Präzise Messungen von Abstandsverteilungen sind über einen großen Bereich, etwa 1 bis 10 Nanometer möglich. Für die Messung der Dipol-Dipol-Wechselwirkung verwendet man meist gepulste ESR-Methoden. Für die traditionelle ortsspezifische Spinmarkierung eines Proteins wird über Mutagenese gezielt ein Cystein in die Aminosäurensequenz eingebaut. Mit spezifischen Spinmarkern, wie z.B. MTSL (1-oxyl-2,2,5,5-tetra-methyl-pyrroline-3-methyl-methanethiosulfonate), wird über eine Disulfidbindung eine Modifikation des Cysteins in vitro herbeigeführt. Dieser Ansatz benötigt also chemische Markierungsschritte, zugängliche Aminosäuren auf der Proteinoberfläche und die Entfernung der nativen Cysteine.