ZUSAMMENFASSUNG Proteinkinasen sind wichtige pharmazeutische Targets, doch die Entwicklung selektiver Modulatoren bleibt eine Herausforderung. Auf der Suche nach allosterischen Stellen in der Serin/Threonin‐Kinase p38α existiert eine „Lipidtasche” im C‐Lappen, die ein Potenzial für Bindung kleiner Moleküle aufweist. Die pharmakologische Signifikanz der ursprünglich identifizierten Liganden mit geringer Affinität ist jedoch nicht offensichtlich und wirft die übergeordnete Frage auf, ob eine Art Kommunikation zwischen dieser Tasche und den funktionellen Stellen des Enzyms existiert. Hier verwenden wir NMR‐Spektroskopie, um eine effektive Konnektivität dieser Stellen trotz ihrer räumlichen Distanz aufzuzeigen. Die Daten zeigen eine klare Interdependenz der Proteindynamik zwischen den verschiedenen Strukturelementen durch dynamische Allosterie, was die generelle Möglichkeit der pharmakologischen Entwicklung geeigneter Lipidtaschenliganden nahelegt, um die enzymatische Funktionalität im Krankheitskontext allosterisch zu adressieren.

Relaxation dispersion techniques allow access to μs time-scale protein motion both in solution and in solid-state NMR. For solids under fast magic-angle spinning, R1ρ relaxation dispersion for spin-lock fields around any rotary-resonance conditions (NERRD) adds highly versatile observables absent in solution that can report on site-specific μs time-scale fluctuations of anisotropic interactions. Proton NERRD, as opposed to 15N or 13C relaxation dispersion, can report on changes within the regional proton dipolar-coupling network around a given site. It is associated with distinct technical opportunities and, in principle, avoids the necessity for heteronuclear isotope labeling. Here we show that one of the current limitations─the presence of strong spin diffusion effects during proton R1ρ measurements, which can cause a deindividualization of the site-specific NERRD profiles and hence a blurring of their motional information─can effectively be ameliorated by off-resonance spin locks. Even though the fundamental limitations of proton relaxation compared to relaxation of more isolated nuclei cannot fully be overcome by technical innovations, the reduction of proton-proton crosstalk facilitates qualitative access to μs time-scale motion exclusively through protons in biology and materials.

The cytosine (C) modifications 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are central regulatory elements of mammalian genomes. Both marks occur in double-stranded DNA in either strand-symmetric or -asymmetric fashion, but it is still poorly understood how this symmetry information is selectively read out by the nuclear proteome as the basis of potential symmetry-dependent regulation. We report enrichment/proteomics studies with promoter probes being strand-symmetrically or asymmetrically modified with C, mC, and hmC, enabling comparison of their reader profiles in the same sequence, tissue, and experimental contexts. We identify a high number of tissue-specific readers for hmC-modified sequences that fall into distinct, probe-specific sub-groups, including members of important transcription factor classes and chromatin regulators. Among them, we discover the master regulators MYC and MAX that play central roles in cell (de)differentiation and cancer progression to read hmC in a sequence-dependent manner. We also find RFX5, a transcription factor involved in primary MHC class II deficiency, to discriminate between hmC symmetries in CpG dyads. Our findings provide further support for the hypothesis that hmC symmetry information can provide distinct regulatory outputs and provide a resource for studying the molecular mechanisms triggered by symmetric and asymmetric hmC modifications in chromatin regulation during development and disease.

Kinases are major drug targets especially in cancer therapy. However, the high degree of conservation of their active sites hinders the development of selective inhibitors, motivating a deeper understanding of kinase conformational ensembles and allosteric communication pathways. Here, we use dynamical network analysis to identify key residues involved in a dynamic allostery between the N- and C-lobes that connects the major functional units of the MAP kinase p38α. By combining NMR spectroscopy, activity assays, and in silico analysis of wildtype protein and mutants in the presence or absence of an active-site inhibitor, we experimentally validate the obtained architecture with respect to global protein motion and long-range allosteric modulation. Notably, the identified network highlights communication pathways across several functional sites, prominently involving the allosteric site, the activation loop, and even the lipid-binding domain with its embedded cryptic pocket in the C-lobe. These findings provide mechanistic insight into p38α allostery and suggest viable opportunities for the rational design of allosteric modulators of MAP kinases. Here, the authors apply dynamic network analysis, alongside a set of experimental validations, to probe correlated motions within p38α kinase. The data corroborate communication between its N- and C-lobes, including in particular the lipid pocket.

Protein kinases represent major pharmaceutical targets, but the development of selective modulators remains challenging. In search of allosteric sites in the serine/threonine kinase p38α, a "lipid pocket" in the C-lobe has been found to bear prospects for the binding of small molecules. A pharmacological potential of those low-affinity binders found initially has not become obvious, however, raising the overarching question whether any sort of communication between this pocket and the enzyme's functional sites exists. Here, we use NMR spectroscopy to reveal an effective connectivity of these sites in spite of their spatial distance. The data reveal a clear interdependency of protein dynamics between the different structural elements through dynamic allostery, together suggesting a pharmacological avenue for the development of suitable lipid pocket binders to allosterically alter enzymatic functionality in a disease context.

Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme that is a member of the nucleotidyltransferase (NTase) family and functions as a DNA sensor. The protein is comprised of a catalytic NTase core domain and an unstructured hypervariable N-terminal domain (NTD) that was reported to increase protein activity by providing an additional DNA-binding surface. We report nearly complete 1H, 15N, and 13C backbone chemical-shift assignments of mouse cGAS NTD (residues 5-146), obtained with a set of 3D and 4D solution NMR experiments. Analysis of the chemical-shift values confirms that the NTD is intrinsically disordered. These resonance assignments can provide the basis for further studies such as activation by DNA and protein-protein interactions.

Owing to fast magic-angle spinning, solid-state nuclear magnetic resonance has evolved as a versatile method to decipher protein structure, dynamics, and chemical properties. While higher-dimensional approaches such as 4D and 5D correlation spectra are generally capable of exceeding the established molecular-weight limitations by ameliorating signal overlap, their intrinsic implications for sensitivity and measurement times required have severely limited these important prospects in practice. Here we show that the extensive use of dedicated transverse-mixing optimal control pulses (TROPs) in high-dimensional experiments to enable concomitant transfer of complex signals along complex magnetization transfer pathways can reduce the necessary measurement time by an order of magnitude. Owing to the multiplicative benefits of the enhancement for successive indirect chemical-shift dimensions, combined with non-linear benefits upon spectral reconstruction, the combination of non-uniformly sampled, higher-dimensional approaches with an extensive use of TROPs hence presents itself as transformative for the conventional limitations of solid-state NMR.

Metal-organic frameworks (MOFs) are versatile materials with tunable properties and broad applications. Here, we report the first cadmium‐based zeolitic imidazolate framework (ZIF) glass, prepared by melt-quenching sub-micrometer-sized Cd(im)2 (im– = imidazolate) particles obtained via mechanochemical synthesis. This approach lowers the melting temperature from 461 °C (for larger solution-synthesized microcrystals) to 455 °C, mitigating thermal decomposition during melting. Crystalline Cd(im)2 adopts a two-fold interpenetrated diamondoid (dia-c) topology, assembled from tetrahedral Cd2+ centers and im– linkers. Rapid cooling yields a monolithic glass with a glass transition temperature (Tg) of 175 °C. Structural analysis confirms that short-range connectivity within individual networks is maintained, whereas interactions between the interpenetrated networks are disrupted. Upon reheating, partial recrystallization produces a single-component glass-ceramic with enhanced mechanical properties, an unprecedented behavior in melt-quenched ZIF glasses. Investigations of thermal parameters (cooling rates) and partial linker substitution reveal strategies for tuning the phase behavior of both glass and glass-ceramic. These findings extend ZIF glass systems to second-row transition metal ions and underscore mechanochemical synthesis as a tool for tailoring the thermal properties of MOFs. This dual‐phase functionality, combining glassy and crystalline domains of identical composition within a single material, offers potential for applications in thermal energy storage, phase change memory, and optics.

Chiral organic glasses combine unique optical properties with the processing advantages of amorphous solids. Here, melt‐quenching as a strategy for preparing optically active glasses from enantiopure BINAP (2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl), a pivotal ligand in asymmetric catalysis and for luminescent metal complexes is demonstrated. Thermal characterization reveals that only R ‐BINAP and S ‐BINAP, not rac ‐BINAP, form molecular glasses with glass transition temperatures near 100 °C. Pair distribution function analysis and circular dichroism confirm the retention of local structure and homochirality despite the loss of long‐range order. Remarkably, the glassy state has a beneficial influence on the molecular optoelectronic properties relative to the crystalline state, resulting in an increase of the radiative rate constant by ≈30%, attributed to more favourable Franck‐Condon factors. In addition, a highly unusual simultaneous enhancement of circularly polarized luminescence (CPL) by nearly an order of magnitude is observed, achieving dissymmetry factors | g lum | approaching 10 −2 that are competitive with the top‐performing purely organic molecular chiral emitters reported to date. These findings establish melt‐quenched chiral molecular glasses as promising platforms for advanced optoelectronic and photonic materials, combining exceptional chiroptical properties, strong luminescence, and processability without the constraints of crystallinity.

Bacterial signaling cascades have recently become of great relevance in the context of bacterial antibiotics resistance. Cyclic diadenylate monophosphate (c-di-AMP) is a key bacterial secondary messenger involved in growth, biofilm formation, virulence gene expression and others. The activation mechanisms of c-di-AMP receptors like the trimeric PII-like proteins upon messenger binding have, however, remained elusive due the pivotal role of highly flexible protein regions. Here, using solution NMR spectroscopy to elucidate the interplay between the ordered and disordered structural elements of the apo and messenger-bound forms of the 44 kDa homotrimeric PII-like signal transduction protein A (PstA), we reveal a sensitive modulation of the conformational ensemble of those extended loops thought to bind the downstream interaction partners by messenger association at the receptor core. The orchestration of the spatial properties of the loops, despite their retained internal dynamics, reveals the importance of allosteric effects even for disordered structural elements, whose steerable ensemble properties have long escaped the classical structural-biology understanding.

PEG detergents are important tools in the biophysical characterization of membrane protein whose utility is often limited by their intrinsic denaturing properties. This work addresses the question of whether changing the linker between PEG headgroup and nonpolar tail can modulate the denaturing properties of these detergents. To address this question, herein, we introduce the modular architecture of PEG550 detergents and explore its utility for protein purification from membranes and detergent exchange. Our results indicate that PEG550 detergents cannot efficiently solubilize proteins from lysed bacterial membranes. Varying the linker cannot eliminate the denaturing properties that PEG550 detergents can have on a protein during extraction and affinity purification. Interestingly, we find that PEG550 detergents can preserve the secondary structure and activity of the model membrane protein vitamin B12 transporter as good as the reference detergent n-dodecyl-β-D-maltoside following detergent exchange via drop dilution. Our findings clarify that denaturing properties of PEG550 detergents depend on both their chemical structure and the detergent exchange method with which proteins and detergents are brought together. Our drop dilution conditions are representative of those frequently employed in the biophysical characterization of membrane proteins. We anticipate PEG550 detergents will deliver a starting point for the optimization of sample properties in the biophysical characterization of membrane proteins.

Metal-organic frameworks (MOFs) are versatile materials with tunable properties and broad applications. Here, we report the first cadmium-based zeolitic imidazolate framework (ZIF) glass, prepared by melt-quenching sub-micrometer-sized Cd(im)2 particles (im- = imidazolate) obtained via mechanochemical synthesis. This route increases defect density and reduces crystallite domain size, lowering the melting temperature from 461 °C (for larger solution-synthesized microcrystals) to 455 °C, thereby mitigating thermal decomposition during melting. Crystalline Cd(im)2 adopts a two-fold interpenetrated diamondoid (dia-c) topology, assembled from tetrahedral Cd2+ centers and im- linkers. Rapid cooling of the Cd(im)2 melt yields a monolithic glass with a glass transition temperature (Tg) of 175 °C. Structural analysis confirms that short-range connectivity within individual networks is maintained, whereas interactions between the interpenetrated networks are disrupted in the glass. Upon reheating, partial recrystallization produces a single-component glass-ceramic with enhanced mechanical properties, an unprecedented behavior in melt-quenched ZIF glasses. Investigations of thermal parameters (cooling rates) and partial linker substitution reveal strategies for tuning the phase behavior of both glass and glass-ceramic. These findings extend ZIF glass systems to second-row transition metal ions and underscore mechanochemical synthesis as a tool for tailoring the thermal properties of MOFs. This dual-phase functionality, combining glassy and crystalline domains of identical composition within a single material, offers potential for applications in thermal energy storage, phase change memory, and optics.

Bakterielle Signalkaskaden sind im Kontext bakterieller Antibiotikaresistenzen von zunehmender Bedeutung geworden. Zyklisches Diadenosinmonophosphat (c‐di‐AMP) ist ein wichtiger bakterieller sekundärer Botenstoff, der unter anderem in Wachstum, Bildung von Biofilmen, Virulenz und Genexpression involviert ist. Die Aktivierungsmechanismen von c‐di‐AMP‐Rezeptoren, wie trimeren P II ‐ähnlichen Proteinen, infolge von Botenstoffbindung sind jedoch wegen der zentralen Rolle von hoch‐flexiblen Proteinregionen bisher schwer fassbar geblieben. Mithilfe von Flüssig‐NMR‐Spektroskopie zur Aufklärung des Zusammenspiels geordneter und ungeordneter Strukturelemente von apo‐ und botenstoffgebundenem, 44 kDa schwerem, homotrimerem P II ‐ähnlichem Signaltransduktionsprotein A (PstA) decken wir eine empfindliche Modulierung des Konformationsensembles jener ausgedehnten flexiblen Loops auf, von denen die Bindung an nachgeschaltete Interaktionspartner infolge von Botenstoffbindung im Rezeptorinneren angenommen wird. Die Orchestrierung räumlicher Eigenschaften der Loops trotz Aufrechterhaltung ihrer internen Dynamik zeigt die Bedeutung allosterischer Effekte selbst für ungeordnete Strukturelemente, deren steuerbare Ensembleeigenschaften auf der Grundlage der klassischen Strukturbiologie lange unerkannt geblieben sind.

With perdeuteration, a current standard for solid-state NMR spectroscopy, large proteins suffer from incomplete amide-proton back-exchange. Using a 72 kDa micro-crystalline protein, we show that deuteration exclusively via deuterated amino acids, largely suppressing sidechain protonation, provides spectral resolution comparable to perdeuterated preparations at intermediate spinning frequencies without proton back-exchange obstacles.

Halophilic organisms have adapted to multi-molar salt concentrations, their cytoplasmic proteins functioning despite stronger attraction between hydrophobic groups. These proteins, of interest in biotechnology because of decreasing fresh-water resources, have excess acidic amino acids. It has been suggested that conformational fluctuations - critical for protein function - decrease in the presence of a stronger hydrophobic effect, and that an acidic proteome would counteract this decrease. However, our understanding of the salt- and acidic amino acid dependency of enzymatic activity is limited. Here, using solution NMR relaxation and molecular dynamics simulations for in total 14 proteins, we show that salt concentration has a limited and moreover non-monotonic impact on protein dynamics. The results speak against the conformational-fluctuations model, instead indicating that maintaining protein dynamics to ensure protein function is not an evolutionary driving force behind the acidic proteome of halophilic proteins.

Motional properties of proteins govern recognition, catalysis, and regulation. The dynamics of tightly interacting residues can form intra molecular dynamic networks, dependencies fine-tuned by evolution to optimize a plethora of functional aspects. The constructive interaction of residues from different proteins to assemble inter molecular dynamic networks is a similarly likely case but has escaped thorough experimental assessment due to interfering association/dissociation dynamics. Here, we use fast-MAS solid-state 15 N R 1ρ NMR relaxation dispersion aided by molecular-dynamics simulations to mechanistically assess the hierarchy of individual μs timescale motions arising from a crystal-crystal contact, in the absence of translational motion. In contrast to the monomer, where particular mutations entail isolated perturbations, specific intermolecular interactions couple the motional properties between distant residues in the same protein. The mechanistic insights obtained from this conceptual work may improve our understanding on how intramolecular allostery can be tuned by intermolecular interactions via assembly of dynamic networks from previously isolated elements.

AbstractDie experimentelle Erfassung möglicher interner Dynamik eines Liganden ohne Isotopenmarkierung im proteingebundenen Zustand ist grundsätzlich schwierig. Hohe Temperaturfaktoren aus Kristallstrukturen können statische Heterogenität nicht von Bewegung unterscheiden, zudem können für symmetrische Bewegungen selbst bei niedrigsten B‐Faktoren schnelle Bewegungen vorhanden sein. Hier zeigen wir die sowohl in Lösung als auch im Kristall experimentell beobachtete interne μs‐Dynamik eines nicht‐isotopenmarkierten Liganden, während dieser mit hoher Affinität an das Enzym humane Carboanhydrase II (hCAII) gebunden ist. Der sprunghafte Wechsel zwischen den Rotameren der Benzolgruppe des Liganden verursacht sowohl in Lösungs‐ als auch Festkörper NMR mit schneller Rotation um den magischen Winkel 1H R1ρ Relaxationsdispersion, für die mittels Molecular Dynamics (MD)‐Simulationen mechanistische Einblicke erhalten werden. Die experimentelle Erfassung von konformationellem Austausch in gebundenen Liganden in Lösung und im Festkörper könnte einem besseren Verständnis von Wirt‐Gast‐Interaktionen in Biologie und supramolekularer und medizinischer Chemie dienen.

Rho‐GTPasen, räumliche Hauptregulatoren einer Vielzahl zellulärer Prozesse, werden durch Komplexbildung mit Guaninnukleotid‐Dissoziationsinhibitoren (RhoGDIs) reguliert. Man nimmt bisher an, dass diese einen unstrukturierten N‐Terminus aufweisen, der den Nukleotidaustausch ihres Klienten bei der Bindung/Faltung hemmt. Mithilfe von NMR‐Analysen, Molekulardynamiksimulationen und biochemischen Tests konnten wir stattdessen relevante strukturelle Eigenschaften des Terminus nachweisen, die sowohl in Anwesenheit als auch in Abwesenheit des Klienten transient existieren, allerdings kontextspezifisch variiert werden. Diese Beobachtungen revidieren das seit Langem bestehende Bild zum Mechanismus der Membranextraktion der GTPasen. Der komplexe und hochselektive Extraktionsprozess der RhoGTPasen wird nicht durch das gleichzeitige Falten und Binden eines sterisch blockierenden Peptides, sondern durch ein dynamisches Ensemble mit vordefinierten transienten strukturellen Eigenschaften orchestriert, die durch die spezifische Umgebung entlang des mehrstufigen Prozesses noch moduliert werden können.

By combining the porosity of crystalline metal-organic frameworks (MOFs) with the unique processability of the liquid state, melt-quenched MOF glasses offer exciting opportunities for molecular separation. However, progress in this field is limited by two factors. Firstly, only very few MOFs melt at elevated temperature and transform into stable glasses upon cooling of the corresponding MOF liquid. Secondly, the MOF glasses obtained thus far feature only very small porosities and extremely small pore sizes. Here we demonstrate solvent-assisted linker exchange (SALE) as a versatile method to prepare highly porous melt-quenched MOF glasses from the canonical ZIF-8. Two additional organic linkers are incorporated into the non-meltable ZIF-8, yielding high-entropy, linker-exchanged ZIF-8 derivatives undergoing crystal-to-liquid-to-glass phase transitions by thermal treatment. The ZIF-8 glasses demonstrate unprecedented porosities of about 25%, adsorb large amounts of technologically relevant C3 and C4 hydrocarbons, and feature high kinetic sorption selectivities for the separation of propylene from propane.

With perdeuteration, solid-state NMR spectroscopy of large proteins suffers from incomplete amide-proton back-exchange. Using a 72 kDa micro-crystalline protein, we show that deuteration exclusively via deuterated amino acids, well-established in solution to suppress sidechain protonation without proton back-exchange obstacles, provides spectral resolution comparable to perdeuterated preparations at intermediate spinning frequencies.