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Stimuli-responsive flexible metal-organic frameworks (MOFs) remain at the forefront of porous materials research due to their enormous potential for various technological applications. Here, we introduce the concept of frustrated flexibility in MOFs, which arises from an incompatibility of intra-framework dispersion forces with the geometrical constraints of the inorganic building units. Controlled by appropriate linker functionalization with dispersion energy donating alkoxy groups, this approach results in a series of MOFs exhibiting a new type of guest- and temperature-responsive structural flexibility characterized by reversible loss and recovery of crystalline order under full retention of framework connectivity and topology. The stimuli-dependent phase change of the frustrated MOFs involves non-correlated deformations of their inorganic building unit, as probed by a combination of global and local structure techniques together with computer simulations. Frustrated flexibility may be a common phenomenon in MOF structures, which are commonly regarded as rigid, and thus may be of crucial importance for the performance of these materials in various applications. Responsiveness in metal-organic frameworks involving amorphous phases remains poorly understood. Here, the authors demonstrate MOFs that reversibly switch between well-defined crystalline and structurally degenerate amorphous states mediated by competing intra-framework forces.

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 |glum| 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. Melt‐quenching of enantiopure BINAP yields transparent chiral molecular glasses that retain axial chirality and display blue‐shifted luminescence together with strongly enhanced circularly polarized emission with dissymmetry factors |glum| of ≈10−2. These materials unite optical clarity, strong emission, and high chiroptical activity, highlighting vitrification as a powerful strategy for creating solid‐state chiral photonic materials.

1H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.

Site-specific heterogeneity of solid protein samples can be exploited as valuable information to answer biological questions ranging from thermodynamic properties determining fibril formation to protein folding and conformational stability upon stress. In particular, for proteins of increasing molecular weight, however, site-resolved assessment without residue-specific labeling is challenging using established methodology, which tends to rely on carbon-detected 2D correlations. Here we develop purely chemical-shift-based approaches for assessment of relative conformational heterogeneity that allows identification of each residue via four chemical-shift dimensions. High dimensionality diminishes the probability of peak overlap in the presence of multiple, heterogeneously broadened resonances. Utilizing backbone dihedral-angle reconstruction from individual contributions to the peak shape either via suitably adapted prediction routines or direct association with a relational database, the methods may in future studies afford assessment of site-specific heterogeneity of proteins without site-specific labeling.

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, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific 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 β-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the β-subunit from solution.

Given that solid-state NMR is being used for protein samples of increasing molecular weight and complexity, higher-dimensionality methods are likely to be more and more indispensable for unambiguous chemical shift assignments in the near future. In addition, solid-state NMR spectral properties are increasingly comparable with solution NMR, allowing adaptation of more sophisticated solution NMR strategies for the solid state in addition to the conventional methodology. Assessing first principles, here we demonstrate the application of automated projection spectroscopy for a micro-crystalline protein in the solid state.

Non-uniform sampling has been successfully used for solution and solid-state NMR of homogeneous samples. In the solid state, protein samples are often dominated by inhomogeneous contributions to the homogeneous line widths. In spite of different technical strategies for peak reconstruction by different methods, we validate that NUS can generally be used also for such situations where spectra are made up of complex peak shapes rather than Lorentian lines. Using the RMSD between subsampled and reconstructed data and those spectra obtained with uniform sampling for a sample comprising a wide conformational distribution, we quantitatively evaluate the identity of inhomogeneous peak patterns. The evaluation comprises Iterative Soft Thresholding (hmsIST implementation) as a method explicitly not assuming Lorentian lineshapes, as well as Sparse Multidimensional Iterative Lineshape Enhanced (SMILE) algorithm and Signal Separation Algorithm (SSA) reconstruction, which do work on the basis of Lorentian lineshape models, with different sampling densities. Even though individual peculiarities are apparent, all methods turn out principally viable to reconstruct the heterogeneously broadened peak shapes.

The atomic-level understanding of protein function and enzyme catalysis requires site-specific information on chemical properties such as protonation and hybridization states and chemical exchange equilibria. This information is encoded in NMR chemical shifts, which serve as important complementary information to structural data from other experimental techniques or structure prediction algorithms. This study demonstrates that comprehensive chemical-shift assignments are achievable for large and highly complex proteins, offering insights into chemical structure and dynamics. The access to the active-site chemistry in the 144-kDa (72-kDa asymmetric unit) enzyme tryptophan synthase demonstrated here extends the elucidation of chemical properties to a member of an important class of enzymes of interest in pharmacology and biotechnology. 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, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific 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 β-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the β-subunit from solution.

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.Competing Interest StatementThe authors have declared no competing interest.

Solid-state NMR has emerged as a potent technique in structural biology, suitable for the study of fibrillar, micro-crystalline, and membrane proteins. Recent developments in fast-magic-angle-spinning and proton-detected methods have enabled detailed insights into structure and dynamics, but molecular-weight limitations for the asymmetric part of target proteins have remained at ~30-40 kDa. Here we employ solid-state NMR for atom-specific characterization of the 72 kDa (asymmetric unit) microcrystalline protein tryptophan synthase, an important target in pharmacology and biotechnology, chemical-shift assignments of which we obtain via higher-dimensionality, 4D and 5D solid-state NMR experiments. The assignments for the first time provide comprehensive data for assessment of side chain chemical properties involved in the catalytic turnover, and, in conjunction with first-principles calculations, precise determination of thermodynamic and kinetic parameters is demonstrated for the essential acid-base catalytic residue βK87. The insights provided by this study expand by nearly a factor of two the size limitations widely accepted for NMR today, demonstrating the applicability of solid-state NMR to systems that have been thought to be out of reach due to their complexity. Competing Interest Statement The authors have declared no competing interest.