13 research outputs found
Including Protons in Solid-State NMR Resonance Assignment and Secondary Structure Analysis: The Example of RNA Polymerase II Subunits Rpo4/7
International audience1 H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1 H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1 H and 13 C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13 C, 15 N, and 1 H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1 H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1 H secondary chemical shifts (SCS) alone by using the sum of 1 H α and 1 H N SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13 C chemical shifts, here either by using TALOS or in terms of 13 C SCS
Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation
International audienceModulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures
Solid-State NMR Studies of Large Protein Assemblies
Amongst other applications, solid-state nuclear magnetic resonance (NMR) spectroscopy can provide atomic-resolution data for the determination of protein structures and dynamics. This is true in particular for protein assemblies that cannot be crystallised or are too large for solution-state NMR. While understanding the dynamics of a protein is of great importance for a full appreciation of its molecular machinery, this thesis has a focus on the structural aspects of a number of large protein assemblies.
The first study, presented in Chapter 2, builds on previously determined chemical shifts of a mutant form of amyloid-β, which is compared to a brain-seeded form of the wild-type. This comparison suggests that the determined mutant fold can also be adopted by the wild-type, with small conformational adaptations which accommodate the E22 deletion in the Osaka mutant. In addition, this chapter illustrates how other mutants could conform to this model. The stabilisation of the N-terminal part of the protein via an intermolecular salt bridge to K28 may represent a common structural motif for the mutants that are related to early-onset Alzheimer’s disease. This feature may connect to the observed increased toxicity of the mutant forms compared to wild-type Amyloid-β1-40, where the salt bridge involving K28 is intramolecular instead of intermolecular.
A continuation of amyloid studies follows in Chapter 3, but for a different kind of amyloid: in recent years the idea that a number of peptide hormones and neuropeptides are transiently stored in aggregated form has accumulated support. These reversible, functional amyloids are believed to be packed into dense-core vesicles, which function as temporary depots of messenger peptides in secretory cells. Somatostatin (SST) is such a peptide hormone that occurs physiologically both aggregated and as a soluble monomer. The structure of human SST-14 in the context of a fibril was determined to atomic resolution using magic-angle spinning (MAS) solid-state NMR spectroscopy. In addition to scanning transmission electron microscopy data, the complete backbone resonance assignment is presented in this chapter. Subsequently, dipolar-based experiments that provide spectrally unambiguous long-range distance restraints are combined with a prediction of secondary-structure elements by secondary chemical-shift calculations and dihedral-angle restraints. The collective data culminate in the molecular structure presented in this chapter.
In Chapter 4, both 13C- and 1H-detected experiments are presented. Both approaches are compared in general, and more specifically in the context of several proteins related to the hepatitis B virus (HBV). HBV is a small enveloped DNA virus whose genomic information encodes only a few genes: the envelope proteins S, M and L (collectively known as hepatitis B surface antigen/HBsAg), the core protein (Cp), the polymerase (P), and the X protein (HBx). This chapter presents structural studies of the envelope protein S and the core protein Cp in its full-length (including C-terminal domain (CTD)) and reduced (without CTD) forms.
Proton detection is applied to probe interactions between protein and nucleic acids (ATP analogues and the deoxyribonucleotides of DNA) in combination with phosphorus-detected experiments in Chapter 5. Protein-nucleic acid interactions play important roles not only in energy-providing reactions such as ATP hydrolysis, but also in reading, extending, packaging or repairing genomes. While they can often be analysed in detail with X-ray crystallography, complementary methods are necessary to visualise these interactions in complexes which are not crystalline. This chapter describes how solid-state NMR can detect and classify protein-nucleic acid interactions via site-specific 1H- and 31P-detected spectroscopy. The sensitivity of 1H chemical-shift values for non-covalent interactions involved in these molecular recognition processes is exploited to directly probe the chemical bonding state, a characteristic that cannot be directly obtained from an X-ray structure. Despite its rather challenging size, the method is applied to study interactions in the 669 kDa dodecameric DnaB helicase in complex with ADP:AlF4-:DNA.
Finally, Chapter 6 investigates proton-detection in solid-state NMR rather from a more methodological point of view in the context of MAS and resolution. Spectral resolution is key to unleash the structural and dynamic information contained in NMR spectra. The advent of ever faster MAS, today exceeding 100 kHz, is what enabled proton detection in solid-state NMR. In this respect, it is valuable to evaluate the benefit of a continued investment in faster spinning. To address this question, MAS up to 150 kHz is used to investigate a protein complex of archaeal RNA polymerase subunits 4 and 7. Using a rotor with an outer diameter of 0.5 mm and a sample content of approximately 170 µg, the total linewidth of Rpo4/7 improves by a factor of 1.23 ± 0.05 by going from 100 to 150 kHz, and signal intensity increases by a factor 1.48 ± 0.13 in the same MAS range. With some further considerations demonstrated in this chapter, the conclusion is that continued investment in faster MAS is indeed meaningful
Alternative salt bridge formation in Aβ-a hallmark of early-onset Alzheimer's disease?
Affiliations ECOFECTInternational audienceRecently the 3D structure of the Osaka mutant form (E22Δ) of Amyloid-β1-40 has been determined. We here compare the NMR chemical-shift with the published shifts of a brain-seeded form of wild-type Aβ and suggest that the determined mutant fold is accessible to the wild-type protein as well, with small conformational adaptations which accommodate the E22 residue missing in the Osaka mutant. In addition, we illustrate how other mutants could also conform to this model. The stabilization of the N-terminal part of the protein via an intermolecular salt bridge to Lys28 may represent a common structural motif for the mutants which are related to early-onset Alzheimer disease. This feature might connect to the observed increased toxicity of the mutant forms compared to wild-type Aβ1-40, where the salt bridge involving Lys28 is intramolecular
Including protons in solid-state NMR resonance assignment and secondary structure analysis : the example of RNA polymerase II subunits Rpo4/7/7
1H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1H and 13C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13C, 15N, and 1H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1H secondary chemical shifts (SCS) alone by using the sum of 1Hα and 1HN SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13C chemical shifts, here either by using TALOS or in terms of 13C SCS
Nucleotide Binding Modes in a Motor Protein Revealed by 31 P‐ and 1 H‐Detected MAS Solid‐State NMR Spectroscopy
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100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids
International audienceWe sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ∼50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and H N back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170-110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several H N protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra
Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis B Virus Capsid C-Terminal Domain
Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083
Making the invisible visible: fast magic-angle-spinning NMR reveals the evasive hepatitis B virus capsid functional C-terminal domain
Experimentally determined protein structures often feature missing domains. One example is the C terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulated nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS is a game changer that allows to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain’s behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow to shed light on other flexible protein domains still lacking their structural and dynamic characterization
Structural Studies of Self-Assembled Subviral Particles: Combining Cell-Free Expression with 110 kHz MAS NMR Spectroscopy
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