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Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Motional properties of proteins govern recognition, catalysis, and regulation. The dynamics of tightly interacting residues can form intramolecular dynamic networks, dependencies fine‐tuned by evolution to optimize a plethora of functional aspects. The constructive interaction of residues from different proteins to assemble intermolecular 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 15N R1ρ 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. [ABSTRACT FROM AUTHOR]

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40–140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20–30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5‐methylation (mC), a total of four different 5‐modifications can theoretically co‐exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5‐carboxylcytosine (caC) dyads based on the methyl‐CpG‐binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC‐dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The possible internal dynamics of non‐isotope‐labeled small‐molecule ligands inside a target protein is inherently difficult to capture. Whereas high crystallographic temperature factors can denote either static disorder or motion, even moieties with very low B‐factors can be subject to vivid motion between symmetry‐related sites. Here we report the experimental identification of internal μs timescale dynamics of a high‐affinity, natural‐abundance ligand tightly bound to the enzyme human carbonic anhydrase II (hCAII) even within a crystalline lattice. The rotamer jumps of the ligand's benzene group manifest themselves both, in solution and fast magic‐angle spinning solid‐state NMR 1H R1ρ relaxation dispersion, for which we obtain further mechanistic insights from molecular‐dynamics (MD) simulations. The experimental confirmation of rotameric jumps in bound ligands within proteins in solution or the crystalline state may improve understanding of host‐guest interactions in biology and supra‐molecular chemistry and may facilitate medicinal chemistry for future drug campaigns. [ABSTRACT FROM AUTHOR]

The article in Angewandte Chemie discusses the importance of dynamic networks in allosteric regulation of protein function. Researchers used NMR spectroscopy and simulations to demonstrate how intermolecular association can create these networks from previously unrelated segments. The study highlights the potential for understanding and utilizing allosteric modulators in pharmacological and biotechnological applications. Other topics covered in the article include photodynamic therapy, electrocatalysis, photopharmacology, and photocatalysis, showcasing advancements in these areas of research. [Extracted from the article]

The article in the journal "Angewandte Chemie International Edition" discusses the importance of dynamic networks in allosteric regulation of protein function. Researchers used NMR spectroscopy and simulations to demonstrate how intermolecular association can create networks from previously unrelated motion segments. The study highlights the potential for understanding and utilizing allosteric modulators in pharmacological and biotechnological applications. Other topics covered in the article include photodynamic therapy, electrocatalysis, photopharmacology, and photocatalysis, showcasing advancements in these areas of research. [Extracted from the article]