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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]

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N‐terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context‐specifically by modulating interactions with the surface of the folded C‐terminal domain. These observations revise the long‐standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder‐to‐order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi‐step process. [ABSTRACT FROM AUTHOR]

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]

5‐Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl‐CpG‐binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain‐dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light‐control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD‐DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin‐associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Their approach offers otherwise inaccessible kinetic insights into the domain‐specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states. [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]

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, protondetected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atomspecific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution. [ABSTRACT FROM AUTHOR]

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly "rigid protein" that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines. [ABSTRACT FROM AUTHOR]

Round and round The intrinsic dynamics of small‐molecule ligands while bound to their protein targets is of interest for optimization of the entropic contributions to binding affinity. In their Communication (e202313947), Rasmus Linser et al. show that NMR proton relaxation dispersion is able to capture an aromatic ring flip within a high‐affinity pharmacological inhibitor on the µs timescale...By Julia Kotschy; Benedikt Söldner; Himanshu Singh; Suresh K. Vasa and Rasmus LinserReported by Author; Author; Author; Author; Author [Extracted from the article]

Round and round The intrinsic dynamics of small‐molecule ligands while bound to their protein targets is of interest for optimization of the entropic contributions to binding affinity. In their Communication (e202313947), Rasmus Linser et al. show that NMR proton relaxation dispersion is able to capture an aromatic ring flip within a high‐affinity pharmacological inhibitor on the µs timescale...By Julia Kotschy; Benedikt Söldner; Himanshu Singh; Suresh K. Vasa and Rasmus LinserReported by Author; Author; Author; Author; Author [Extracted from the article]

TROSY type techniques in triple resonance experiments which enable the assignment of resonances in loop regions of a microcrystalline protein could be used to overcome the impediment during the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. The proposed TROSY approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

Analyses of solution [super 15]N relaxation data and solid-state [super 1][H.sup.N]-[super 15]N dipolar couplings from a small globular protein, [alpha]-spectrin SH3 domain, have produced similar pattern of order parameters, suggesting that there is no ns-[micro]s dynamics in the structured portion of the backbone. The results obtained by the molecular dynamics (MD) simulations of [alpha]-spectrin SH3 in a hydrated crystalline environment and in solution is consistent with the protein dynamics.

Heteronuclear correlation experiments using perdeuterated proteins have helped in understanding the hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. A biexponential decay behavior is observed for those hydroxyl groups that are involved in side chain-side chain C-O-H-O[double bond]C hydrogen bonds and correlations are observed between amide proton and carbonyl atoms.

The presence of paramagnetic compounds in the bulk solvent has induced a site-specific relaxation in addition to local dynamics, which is dependent on the surface accessibility of the respective amide proton in the protein. The relaxation is mediated by hydroxyl groups, which transfer relaxation by their ability to exchange with the quickly relaxing bulk solvent, and relaxation is transferred by water molecules that diffuse into the protein structure and produce an efficient difference Paramagnetic Relaxation Enhancement (PRE) in flexible regions of the protein.

A modified version of the centerband-only detection of exchange (CODEX) NMR experiment is used for the site-resolved study of millisecond to second (ms to s) motions in a protein in the solid (microcrystalline) state. The NMR study of the internal dynamics of proteins in the solid state on the ms to s time scale with site-specific spectral resolution has provided both time-scale and geometry information about molecular motions.

Fast-magic-angle-spinning solid-state NMR is a developing technique for determination of protein structure and dynamics. Proton–proton correlations usually lead to rough distance restraints, a serious hurdle towards high-resolution structures. Analogous to the “eNOE” concept in solution, an integrative approach for more accurate restraints enables improved structural accuracy with minimal analytical effort. [ABSTRACT FROM AUTHOR]

Solid‐state NMR spectroscopy has recently enabled structural biology with small amounts of non‐deuterated proteins, largely alleviating the classical sample production demands. Still, despite the benefits for sample preparation, successful and comprehensive characterization of complex spin systems in the few cases of higher‐molecular‐weight proteins has thus far relied on traditional 13C‐detected methodology or sample deuteration. Herein we show for a 29 kDa carbonic anhydrase:acetazolamide complex that different aspects of solid‐state NMR assessment of a complex spin system can be successfully accessed using a non‐deuterated, 500 μg sample in combination with adequate spectroscopic tools. The shown access to protein structure, protein dynamics, as well as biochemical parameters in amino acid sidechains, such as histidine protonation states, will be transferable to proteins that are not expressible in E. coli. The faster, the better? Fast magic angle spinning (MAS) makes solid‐state NMR characterization of complex proteins possible even in the absence of deuteration. It is shown for a 29 kDa complex of hCAII:acetazolamide that structure, dynamics, as well as side‐chain proton characteristics become accessible despite a large facilitation of sample production. [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.)