Identifying Exchangeable Protons in a 1D NMR Spectrum by Spatially Selective Exchange-Editing.

The front cover artwork is provided by Markus Rotzinger at the University of Graz, Austria. The front cover picture illustrates the way the 1D exchange-editing NMR method makes exchanging protons visible by sign alteration. The depicted spectrum of D-glucose shows all exchanging signals inverted, thus allowing a fast qualitative determination, potentially in a single scan. Read the full text of the Research Article at 10.1002/cphc.202300713.

Identifying Exchangeable Protons in a 1D NMR Spectrum by Spatially Selective Exchange-Editing.

Signals undergoing chemical or conformational exchange in one-dimensional NMR spectra are often identified by deuterium exchange. In order to obtain quantitative information about the dynamic processes involved, one frequently used method is EXchange SpectroscopY (EXSY). To detect all exchange processes, the EXSY experiment requires the acquisition of time-consuming two-dimensional spectra. Here we report a faster alternative, an experiment which uses spatial encoding to extract similar information in a 1D exchange-edited experiment. Thereby, all protons are observed at once, but in different slices of the detection volume. The experiment can be carried out in a single scan to identify exchanging sites in a 1D spectrum by changes in signal intensity indicating exchange processes. If the exchanging partner, for example water is in molar excess the exchange-editing method easily identifies mobile protons by negative signals in the 1D 1H NMR spectrum.

Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Accelerative Solid-State Oxidation Behaviour of Amorphous and Partially Crystalline Venetoclax.

There is a growing focus on solid-state degradation, especially for its relevance in understanding interactions with excipients. Performing a solid-state degradation of Venetoclax (VEN), we delve into VEN's stability in different solid-state oxidative stress conditions, utilizing Peroxydone™ complex and urea peroxide (UHP). The investigation extends beyond traditional forced degradation scenarios, providing insights into VEN's behavior over 32 h, considering temperature and crystallinity conditions. Distinct behaviors emerge in the cases of Peroxydone™ complex and UHP. The partially crystalline (PC-VEN) form proves more stable with Peroxydone™, while the amorphous form (A-VEN) shows enhanced stability with UHP. N-oxide VEN, a significant degradation product, varies between these cases, reflecting the impact of different oxidative stress conditions. Peroxydone™ complex demonstrates higher reproducibility and stability, making it a promising option for screening impurities in solid-state oxidative stress scenarios. This research not only contributes to the understanding of VEN's stability in solid-state but also aids formulators in anticipating excipient incompatibilities owing to presence of reactive impurities (peroxides) and oxidation in the final dosage form.

Influence of PLGA End Groups on the Release Profile of Dexamethasone from Ocular Implants.

The present study deals with the development of dexamethasone (DM)-loaded implants using ester end-capped Resomer RG 502 poly(lactic acid-co-glycolic acid) (PLGA) (502), acid end-capped Resomer RG 502H PLGA (502H), and a 502H:502 mixture (3:1) via hot melt extrusion (HME). The prepared intravitreal implants (20 and 40% DM loaded in each PLGA) were thoroughly investigated to determine the effect of different end-capped PLGA and drug loading on the long-term release profile of DM. The implants were characterized for solid-state active pharmaceutical ingredient (APIs) using DSC and SWAXS, water uptake during stability study, the crystal size of API in the implant matrix using hot-stage polarized light microscopy, and in vitro release profile. The kinetics of PLGA release was thoroughly investigated using quantitative 1H NMR spectroscopy. The polymorph of DM crystal was found to remain unchanged after the extrusion and stability study. However, around 3 times reduction in API particle size was observed after the HME process. The morphology and content uniformity of the RT-stored samples were found to be comparable to the initial implant samples. Interestingly, the samples (mainly 502H) stored at 40 °C and 75% RH for 30 d demonstrated marked deformation and a change in content uniformity. The rate of DM release was higher in the case of 502H samples with a higher drug loading (40% w/w). Furthermore, a simple digital in vitro DM release profile derived for the formulation containing a 3:1 ratio of 502H and 502 was comparable with the experimental release profile of the respective polymer mixture formulation. The temporal development of pores and/or voids in the course of drug dissolution, evaluated using µCT, was found to be a precursor for the PLGA release. Overall, the release profile of DM was found to be dependent on the PLGA type (independent of subtle changes in the formulation mass and diameter). However, the extent of release was found to be dependent on DM loading. Thus, the present investigation led to a thorough understanding of the physicochemical properties of different end-capped PLGAs and the underlying formulation microstructure on the release profile of a crystalline water-insoluble drug, DM, from the PLGA-based implant.

Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR.

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.

The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines.

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags.

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

Phase Behavior of Drug-Lipid-Surfactant Ternary Systems toward Understanding the Annealing-Induced Change.

The present study systematically investigates the effect of annealing conditions and the Kolliphor P 407 content on the physicochemical and structural properties of Compritol (glyceryl behenate) and ternary systems prepared via melt cooling (Kolliphor P 407, Compritol, and a hydrophilic API) representing solid-lipid formulations. The physical properties of Compritol and the ternary systems with varying ratios of Compritol and Kolliphor P 407 were characterized using differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SWAXS) and infrared (IR) spectroscopy, and hot-stage microscopy (HSM), before and after annealing. The change in the chemical profiles of different Compritol components as a function of annealing was evaluated using 1H NMR spectroscopy. While no change in the polymorphic form of API and Kolliphor P 407 occurred during annealing, a systematic conversion of the α- to ß-form was observed in the case of Compritol. Furthermore, the polymorphic transformation of Compritol was found to be dependent on the Kolliphor P 407 content. As per the Flory-Huggins mixing theory, higher miscibility was observed in the case of monobehenin-Kolliphor P 407, monobehenin-dibehenin, and dibehenin-tribehenin binary mixtures. The miscibility of Kolliphor P 407 with monobehenin and 1,2-dibehenin was confirmed by 1H NMR analysis. The observed higher miscibility of Kolliphor P 407 with monobehenin and 1,2-dibehenin is proposed as the trigger for the physical separation from the 1,3-diglyceride and triglycerides during melt solidification of the formulations. The phase separation is postulated as the mechanism underlying the formation of a stable ß-polymorphic form (a native form of 1,3-diglyceride) of Compritol upon annealing. This finding is expected to have an important implication for developing stable solid-lipid-surfactant-based drug formulations.

Vibrio natriegens: An Alternative Expression System for the High-Yield Production of Isotopically Labeled Proteins.

Isotopic labeling of recombinant proteins is crucial for studying proteins by liquid state NMR spectroscopy. Nowadays, conventional E. coli-based expression systems like BL21 (DE3) are typically used to express recombinant proteins. Still, the production of isotopically labeled proteins is often costly and time-consuming, and yields are not sufficient enough for structural studies. Here, we present Vibrio natriegens (Vmax) as an alternative expression system in M9 minimal medium. Due to our optimized M9 minimal medium and conditions and the early time point of induction, we obtained a 2- to 4-fold higher protein yield for two test proteins, FKBP and EYFP, compared to E. coli BL21 (DE3). Production of proteins in V. natriegens in minimal medium is not only more cost-effective and convenient but also less time-consuming than in E. coli. Comparing 15N HSQC spectra of FKBP and EYFP expressed in Vmax and BL21 (DE3) revealed correct folding during expression.

A cation-π interaction in a transmembrane helix of vacuolar ATPase retains the proton-transporting arginine in a hydrophobic environment.

Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg735) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-π interaction in TM7 between Arg735 and two highly conserved nearby aromatic residues, Tyr733 and Trp737 We propose a mechanism by which the transient, membrane-embedded cation-π complex provides the necessary energy to keep the charged side chain of Arg735 within the hydrophobic membrane. Such cation-π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.

In Situ Observation of Photoswitching by NMR Spectroscopy: A Photochemical Analogue to the Exchange Spectroscopy Experiment.

We present 1D and 2D NMR experiments that provide in situ insights into photoinduced isomerizations. Irradiation during the mixing period of an exchange spectroscopy (EXSY) experiment leads to characteristic cross peaks in 2D spectra. The phototriggered exchange of magnetization occurring in photoswitchable (Z)- and (E)-isomers of three selected azo compounds provides information on the dynamic E/Z equilibria. We report the dependence of the diagonal-to-cross-peak ratio on concentration, light intensity, and mixing time. In analogy to exchange spectroscopy, this ratio mirrors the efficiency of light induced molecular transformations. Furthermore, we present a time-saving 1D version and a combined light/phase cycle scheme for enhanced detectability of photoinduced changes in the spectrum. This insight into light-induced structural changes is highly suited to study macromolecules, in which photoswitchable units trigger conformational changes.

Solvent-independent determination of heteroatom protonation states from NMR spectra by differential deuterium isotope shifts.

The NMR-spectroscopy based structure elucidation of organic molecules containing heteroatoms is often obstructed by the difficulties in determining the heteroatom protonation states. Here we describe a simple but broadly applicable approach for the determination of the protonation states of heteroatoms. Differential deuterium isotope shifts observed upon the addition of small amounts of H2O or D2O to any solvent can be used to determine the protonation states of heteroatoms.

Trimethylsilyl tag for probing protein-ligand interactions by NMR.

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.

High Resolution for Chemical Shifts and Scalar Coupling Constants: The 2D Real-Time J-Upscaled PSYCHE-DIAG.

The facile determination of chemical shift and scalar coupling constants in NMR spectra is often prevented by spectral overlap and limited resolution. Here, we present a high-resolution NMR experiment for the simultaneous detection of both resonance frequencies and coupling patterns even with small J-values. A PSYCHE-decoupled DIAG (Pure Shift Yielded by Chirp Excitation- DIAGonal) experiment, which resolves chemical shift in the indirect dimension of a 2D experiment is combined with real-time J-upscaling in order to visualize small coupling constants that would otherwise be hidden in the linewidth of a regular proton or DIAG spectrum.

Investigating Protein-Ligand Interactions by Solution Nuclear Magnetic Resonance Spectroscopy.

Protein-ligand interactions are of fundamental importance in almost all processes in living organisms. The ligands comprise small molecules, drugs or biological macromolecules and their interaction strength varies over several orders of magnitude. Solution NMR spectroscopy offers a large repertoire of techniques to study such complexes. Here, we give an overview of the different NMR approaches available. The information they provide ranges from the simple information about the presence of binding or epitope mapping to the complete 3 D structure of the complex. NMR spectroscopy is particularly useful for the study of weak interactions and for the screening of binding ligands with atomic resolution.

Extracting unresolved coupling constants from complex multiplets by a real-time J-upscaled SERF experiment.

The measurement of small homonuclear coupling constants is often prevented by either their small size and/or overlap with other signal splittings. Here, we present a real-time method to extract such couplings without interference from other splittings, with a resolution that is beyond conventional NMR spectra. In this real-time J-upscaled SERF experiment, homonuclear coupling is removed by slice-selective pure shift NMR, whereas scalar coupling to only one selected signal is reintroduced by selective refocusing. The remaining couplings are enhanced by real-time J-upscaling during interruptions of the FID data acquisition. The resulting spectrum is not only simplified by the restriction of the scalar coupling but also its resolution enhanced. This improved resolution results from a reduction of signal broadening due to magnetic field inhomogeneities from 2 different sources: slice-selective excitation and the spin-echo type J-upscaling element.

Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain.

Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.

Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding.

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

A 2-O-Methylriboside Unknown Outside the RNA World Contains Arsenic.

Lipid-soluble arsenic compounds, also called arsenolipids, are ubiquitous marine natural products of currently unknown origin and function. In our search for clues about the possible biological roles of these compounds, we investigated arsenic metabolism in the unicellular green alga Dunaliella tertiolecta, and discovered an arsenolipid fundamentally different from all those previously identified; namely, a phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside. The discovery is of particular interest because 2-O-methylribosides have, until now, only been found in RNA. We briefly discuss the significance of the new lipid in biosynthesis and arsenic biogeochemical cycling.