Roman J. Lichtenecker

Selective Isotope Labelling of Leucine Residues by Using α-Ketoacid Precursor Compounds

You can have one without the other: A new metabolic precursor compound can selectively introduce (13)C and (2)H patterns at leucine residues in proteins in cell-based expression systems without interfering with valine metabolism. The protocol provides selectively labelled macromolecules well suited for probing structure and dynamics in high-molecular-weight proteins by NMR spectroscopy.

α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression

Abstract13C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of 15N-chiral amino acid synthesis using organic chemistry.

Novel Approaches in Selective Tryptophan Isotope Labeling by Using Escherichia coli Overexpression Media

NMR‐based investigations of large protein complexes require optimized isotopic labeling schemes. We report new methods to introduce stable isotopes into tryptophan residues; these are fine‐tuned to the requirements of the particular protein NMR experiment. Selective backbone labeling was performed by using a new α‐ketoacid precursor as an additive in cell‐based overexpression media. Additionally, we developed synthetic routes to certain isotopologues of indole with 13C–1H spin systems surrounded by 12C and 2H. The corresponding proteins, overexpressed in the presence of these precursor compounds, can be effectively analyzed for conformational changes in tryptophan residues in response to external stimuli, such as interaction with other proteins or small molecules.

Iterative Antimicrobial Candidate Selection from Informed D-/L-Peptide Dimer Libraries

Growing resistance to antibiotics, as well as newly emerging pathogens, stimulate the investigation of antimicrobial peptides (AMPs) as therapeutic agents. Here, we report a new library design concept based on a stochastic distribution of natural AMP amino acid sequences onto half‐length synthetic peptides. For these compounds, a non‐natural motif of alternating D‐ and L‐backbone stereochemistry of the peptide chain predisposed for β‐helix formation was explored. Synthetic D‐/L‐peptides with permuted half‐length sequences were delineated from a full‐length starter sequence and covalently recombined to create two‐dimensional compound arrays for antibacterial screening. Using the natural AMP magainin as a seed sequence, we identified and iteratively optimized hit compounds showing high antimicrobial activity against Gram‐positive and Gram‐negative bacteria with low hemolytic activity. Cryo‐electron microscopy characterized the membrane‐associated mechanism of action of the new D‐/L‐peptide antibiotics.

Dynamic Combinatorial Enrichment of Polyconformational D‐/L‐Peptide Dimers

D-/L-peptides such as gramicidin A (gA) adopt unique dimeric β-helical structures of different topologies. To overcome their conformational promiscuity and enrich individual components, a dynamic combinatorial approach assisted by thiol tags was developed. This method led to identification of the preferential formation of antiparallel dimers under a broad range of conditions, which was independent of peptide side-chain polarity. Exclusive formation of an antiparallel cyclic dimer was achieved in the presence of cesium ions.

Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever‐expanding owing to progress in experimental techniques, hardware development, and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodological gap, we present a novel metabolic precursor for cell‐based protein overexpression to assemble 13C/2H isotope patterns in the peptide backbone, as well as in side chain positions of a mechanistically distinguished histidine residue.

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when 13Cβ and 13C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using 13Cα connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs.

Late metabolic precursors for selective aromatic residue labeling

In recent years, we developed a toolbox of heavy isotope containing compounds, which serve as metabolic amino acid precursors in the E. coli-based overexpression of aromatic residue labeled proteins. Our labeling techniques show excellent results both in terms of selectivity and isotope incorporation levels. They are additionally distinguished by low sample production costs and meet the economic demands to further implement protein NMR spectroscopy as a routinely used method in drug development processes. Different isotopologues allow for the assembly of optimized protein samples, which fulfill the requirements of various NMR experiments to elucidate protein structures, analyze conformational dynamics, or probe interaction surfaces. In the present article, we want to summarize the precursors we developed so far and give examples of their special value in the probing of protein–ligand interaction.

Bioactivity of topologically confined gramicidin A dimers

The d-/l-peptide gramicidin A (gA) is well known as a pivotal ion channel model and shows a broad spectrum of bioactivities such as antibiosis, antimalarial activity, as well as hemolysis. We applied inter-chain disulfide bonds to constrain the conformational freedom of gA into parallel and antiparallel dimeric topologies. Albeit the constructs were not found to be monoconformational, CD- and IR-spectroscopic studies suggested that this strategy indeed restricted the conformational space of the d-/l-peptide construct, and that β-helical secondary structures prevail. Correlative testing of gA dimers in antimicrobial, antimalarial, and ion conduction assays suggested that the tail-to-tail antiparallel single stranded β6.3 helix dominantly mediates the bioactivity of gA. Other conformers are unlikely to contribute to these activities. From these investigations, only weakly ion conducting gA dimers were identified that retained nM antimalarial activity.