RESUMO
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 ß-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.
RESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Humanos , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Conformação ProteicaRESUMO
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 temperatures and transform into stable glasses upon cooling the corresponding MOF liquid. Secondly, the MOF glasses obtained thus far feature only very small porosities and very 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 specific pore volumes of about 0.2 cm3g-1, adsorb large amounts of technologically relevant C3 and C4 hydrocarbons, and feature high kinetic sorption selectivities for the separation of propylene from propane.
RESUMO
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 (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-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.
RESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Soluções , Eletrólitos/química , Proteínas/química , Proteínas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Ressonância Magnética Nuclear BiomolecularRESUMO
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.
Assuntos
Aminoácidos , Prótons , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectroscopia de Ressonância MagnéticaRESUMO
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.
Assuntos
Citosina , Citosina/análogos & derivados , Metilação de DNA , Animais , Ilhas de CpG , Citosina/química , Epigênese Genética , Mamíferos/metabolismoRESUMO
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.
Assuntos
Cromatina , Proteínas de Ligação a DNA , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina , Heterocromatina , Metilação de DNA , Fatores de Transcrição/genética , DNA/metabolismo , Mamíferos/metabolismoRESUMO
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 anhydraseâ 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 1 H 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.
Assuntos
Proteínas , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínio Catalítico , Ligantes , Proteínas/química , Espectroscopia de Ressonância MagnéticaRESUMO
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.
Assuntos
Amidas , Proteínas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectroscopia de Ressonância Magnética/métodos , Amidas/química , Automação , PrótonsRESUMO
5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.
Assuntos
5-Metilcitosina , DNA , Epigênese Genética , Animais , Ilhas de CpG/genética , DNA/química , Metilação de DNA , Epigenômica , Mamíferos/metabolismo , Conformação MolecularRESUMO
Understanding macromolecular function, interactions, and stability hinges on detailed assessment of conformational ensembles. For solid proteins, accurate elucidation of the spatial aspects of dynamics at physiological temperatures is limited by the qualitative character or low abundance of solid-state nuclear magnetic resonance internuclear distance information. Here, we demonstrate access to abundant proton-proton internuclear distances for integrated structural biology and chemistry with unprecedented accuracy. Apart from highest-resolution single-state structures, the exact distances enable molecular dynamics (MD) ensemble simulations orchestrated by a dense network of experimental interproton distance boundaries gathered in the context of their physical lattices. This direct embedding of experimental ensemble distances into MD will provide access to representative, atomic-level spatial details of conformational dynamics in supramolecular assemblies, crystalline and lipid-embedded proteins, and beyond.
Assuntos
Proteínas , Prótons , Proteínas/química , Simulação de Dinâmica Molecular , Espectroscopia de Ressonância Magnética , Conformação MolecularRESUMO
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.
Assuntos
Proteínas , Prótons , Ressonância Magnética Nuclear Biomolecular/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Dobramento de ProteínaRESUMO
5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , DNA/isolamento & purificação , Proteína 2 de Ligação a Metil-CpG/química , Fragmentos de Peptídeos/química , DNA/química , Metilação de DNA , Evolução Molecular Direcionada , Células HEK293 , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Fragmentos de Peptídeos/genética , Domínios ProteicosRESUMO
Owing to fast-magic-angle-spinning technology, proton-detected solid-state NMR has been facilitating the analysis of insoluble, crystalline, sedimented, and membrane proteins. However, potential applications have been largely restricted by limited access to side-chain resonances. The recent availability of spinning frequencies exceeding 100 kHz in principle now allows direct probing of all protons without the need for partial deuteration. This potentiates both the number of accessible target proteins and possibilities to exploit side-chain protons as reporters on distances and interactions. Their low dispersion, however, has severely compromised their chemical-shift assignment, which is a prerequisite for their use in downstream applications. Herein, we show that unambiguous correlations are obtained from 5D methodology by which the side-chain resonances are directly connected with the backbone. When further concatenated with simultaneous 4D intra-side-chain correlations, this yields comprehensive assignments in the side chains and hence allows a high density of distance restraints for high-resolution structure calculation from minimal amounts of protein.
RESUMO
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 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.
Assuntos
Cristalografia por Raios X , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Triptofano Sintase/química , Cristalografia por Raios X/métodos , Peso Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Multimerização ProteicaRESUMO
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.
RESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Preparações Farmacêuticas/química , Proteínas/química , Sítios de Ligação , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Proteins depend on defined molecular plasticity for their functionality. How to comprehensively capture dynamics correctly is of ubiquitous biological importance. Approaches commonly used to probe protein dynamics include model-free elucidation of site-specific motion by NMR relaxation, molecular dynamics (MD)-based approaches, and capturing the substates within a dynamic ensemble by recent eNOE-based multiple-structure approaches. Even though MD is sometimes combined with ensemble-averaged NMR restraints, these approaches have largely been developed and used individually. Owing to the different underlying concepts and practical requirements, it has remained unclear how they compare, and how they cross-validate and complement each other. Here, we extract and compare the differential information contents of MD simulations, NMR relaxation measurements, and eNOE-based multi-state structures for the SH3 domain of chicken α-spectrin. The data show that a validated, consistent, and detailed picture is feasible both for timescales and actual conformational states sampled in the dynamic ensemble. This includes the biologically important side-chain plasticity, for which experimentally cross-validated assessment is a significant challenge.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectrina/química , Domínios de Homologia de src , Animais , Galinhas , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas/metabolismo , Reprodutibilidade dos Testes , Espectrina/metabolismoRESUMO
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.