We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)—the product of SAM-dependent methyl group transfer reactions—and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational ‘OFF’-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific C labeled RNA building blocks - a 5-C-uridine and a 2,8-C-adenosine building block - are used to introduce isolated C-H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-C-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-C-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. TheC-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.