132,755 research outputs found
Solid-state NMR spectroscopy on complex biomolecules
No full textBiomolecular applications of NMR spectroscopy are often merely associated with soluble molecules or magnetic resonance imaging. However, since the late 1970s, solid-state NMR (ssNMR) spectroscopy has demonstrated its ability to provide atomic-level insight into complex biomolecular systems ranging from lipid bilayers to complex biomaterials. In the last decade, progress in the areas of NMR spectroscopy, biophysics, and molecular biology have significantly expanded the repertoire of ssNMR spectroscopy for biomolecular studies. This Review discusses current approaches and methodological challenges, and highlights recent progress in using ssNMR spectroscopy at the interface of structural and cellular biology
Binding hotspots of BAZ2B bromodomain:histone interaction revealed by solution NMR driven docking
No full textBromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.</p
Selective Membrane Transport Systems studied by Solid-State NMR Spectroscopy
No full textMembranes border cells from their environment. Thus, they belong to the basic prerequisites for life as we know it. Within cells, membranes are used to establish compartments dedicated to specific tasks such as providing local proximity of components, assembling specialized molecular machinery and confining their numerous functions to salutary consistency. Biological membranes are functionalized by associated proteins which account for a large fraction of all proteins encoded by the genome. Not surprisingly, many of the available drugs act via their interaction with these membrane proteins. Despite their large biological and pharmaceutical importance only very few membrane proteins are understood on the molecular level. Often structure and function of these proteins depend on their lipidic environment. Then, traditional techniques like x-ray diffraction or solution-state NMR are not well suited due to the large molecular size and the inhomogeneous character of membrane-protein systems. Solid-state NMR (ssNMR) spectroscopy on the other hand is a prime technique to investigate structure and dynamics of membrane proteins at atomic resolution even in the presence of a lipid bilayer. This thesis reports on progress made in ssNMR methodology, sample preparation, and data analysis enabling the study of two distinct proteins involved in selective membrane transport. First, ssNMR spectroscopy was used to establish structure-function relations of the chimeric potassium (K+) channel KcsA-Kv1.3 which serves as model system for regulated ion transport across membranes essential for cellular excitability. The results provide detailed insight into structure and dynamics of K+ channel activation and inactivation gating and reveal modulating effects of ligands, ions, and the lipidic environment on the functionally relevant conformations of the K+ channel. Second, functional hydrogels formed by FG-repeat domains of the yeast nucleoporin Nsp1p were characterized by ssNMR. These polypeptides constitute an essential part of the nuclear pore complex controlling all molecular trafficking between nucleus and cytoplasm in eukaryotic cells. The acquired data provide structural details of FG-hydrogels as well as their gelation kinetics and link amyloid-like protein-protein interactions to the selectivity barrier of the nuclear pore complex
CASD-NMR: critical assessment of automated structure determination by NMR
No full textNMR spectroscopy is currently the only technique for determining the solution structure of biological macromolecules. This typically requires both the assignment of resonances and a labor-intensive analysis of multidimensional nuclear Overhauser effect spectroscopy (NOESY) spectra, in which peaks are matched to assigned resonances. Software tools that fully automate the NOESY assignment and the structure calculation steps have the potential to boost the efficiency, reproducibility and reliability of NMR structures
Solid-state NMR on larger biomolecules
No full textIn the last years, remarkable progress has been made to probe molecular structure of biological systems using Magic Angle Spinning solid-state NMR (ssNMR). Prominent examples relate to research areas that have remained challenging to classical structural biology methods such as membrane proteins1,2 and protein fibrils (see, e.g., Ref. 3,4,5). In addition, ssNMR continues to contribute to a structural understanding of basic biological processes including enzyme catalysis or photosynthesis and is capable of studying far more complicated heterogeneous biomolecular systems such as bacterial cell walls6 or inclusion bodies7,8. Clearly, these advancements would have been impossible without methodological and instrumental progress in the field of ssNMR and the pioneering work of Griffin, Opella, Cross, Torchia and others in the field of biomolecular ssNMR. Yet, a decade ago, it was still unclear whether one would be able to obtain sequential assignments of larger proteins, not to mention the determination of their 3D structures from ssNMR data. Since then, ssNMR progress has been substantial and improvements in the field of solutionstate NMR continue to cross fertilize and speed up developments in solid-state NMR. Finally, the revolutionary developments in biochemistry and molecular biology in combination with isotope-labelling, and in more general sense, the ability to design biomolecular sample preparations for ssNMR studies has played a critical role. With further increasing molecular size, for example relating to proteins comprising several hundred amino acids, new challenges and opportunities lay ahead of us
Characterization of membrane protein function by solid-state NMR spectroscopy
No full textMembrane proteins are an important class of biological molecules whose association with lipid bilayers and intrinsic molecular mobility can complicate their structural study by high-resolution methods. As different experimental techniques require different membrane mimetics, it can be challenging to relate membrane protein structure to function. This review presents examples of the use of solid-state nuclear magnetic resonance spectroscopy (ssNMR) to correlate structure and function in membrane proteins with diverse biological roles, including signaling, transport, and enzymatic reactions. The types of ssNMR experiments, as well as sources of complementary information and implications for biology, will be discussed. An outlook towards extending ssNMR studies to cellular preparations will be given
Solid-state NMR on complex biomolecules: novel methods and applications
No full textSolid-state NMR (ssNMR) represents a versatile technique in providing atomic-resolution information without the need for crystals or fast molecular motion required for X-ray crystallography and solution-state NMR, respectively. Recent past has witnessed the ability of this technique in providing detailed structural information for many challenging biomolecules such as membrane proteins, amyloid fibrils,nucleoprotein complexes, and protein gels. However, other challenges still persist resulting in poor spectral resolution and sensitivity due to the presence of strong anisotropic interactions which are not averaged out by fast molecular motions as in the case of solution-state NMR. Hence further methodological advances are required to extend the use of ssNMR to study structure–function relationships in larger biomolecular systems. This thesis focuses on methodological aspectsand the application of solid-state NMR for the structural characterization of membrane proteins and amyloid fibrils. This thesis in the start discusses the application of fractional deuteration as a novel isotope-labeling scheme in ssNMR on a chimeric potassium channel KcsA-Kv1.3 embedded in lipid bilayers and its potential advantages in reducing spectral crowding, resolution enhancement in 1H MAS based ssNMR experiments and the establishment of structural constraints detecting long-range intra as well as intermolecular correlations in standard ssNMR correlation experiments. Secondly, a ssNMR-based hybrid strategy which employs structural constraints obtained from uniformly labeled [13C,15N] as well as fractionally deuterated [2H,13C,15N] versions of the chimeric potassium channel KcsA-Kv1.3 are used to construct 3D molecular structures. The approach is then used to characterize the channel before and after inactivation embedded in lipid bilayers. Furthermore, the structural stability as well as the influence of different lipid bilayer environment on the conformation and function of the potassium channel KcsA-Kv1.3 is probed using ssNMR spectroscopy. Later part of the thesis focuses on the structural characterization of amyloid forming proteins by ssNMR. Dedicated ssNMR methods are employed to investigate the overall fibril arrangements and to generate structural models of polyglutamine peptides that contain polyglutamine expansions of varying lengths for amyloid forming proteins, such as in the Huntingtin protein involved in Huntington’s disease. The last section of the thesis deals with the secondary structure characterization of a liposomal vaccine against Alzheimer’s disease using high resolution ssNMR spectroscopy in combination with other biophysical techniques. In particular, the secondary structure of a series of peptides used as antigen and their vaccine variants upon incorporation into liposomes has been investigated. Furthermore, the key parameter that control peptide conformation by modulation of the peptide lipidation pattern, spacer and liposome composition are studied
A NMR guided approach for CsrA–RNA crystallization
No full textStructure determination of protein–nucleic acid complexes remains a challenging task. Here we present a simple method for generating crystals of a CsrA–nucleic acid complex, guided entirely by results from nuclear magnetic resonances spectroscopy (NMR) spectroscopy. Using a construct that lacks thirteen non-essential C-terminal residues, efficient binding to DNA could be demonstrated. One CsrA dimer interacts with two DNA oligonucleotides, similar to previous findings with RNA. Furthermore, the NMR study of the CsrA–DNA complex was the basis for successfully homing in on conditions that were suitable for obtaining crystals of theCsrA–DNAcomplex.Our resultsmay be useful for those caseswhereRNAin protein–nucleic acid complexes may be replaced by DNA
Probing molecular motion by double-quantum (13C,13C) solid-state NMR spectroscopy: Application to ubiquitin
No full textWe demonstrate the use of two-dimensional (13C,13C) double-quantum spectroscopy to detect molecular dynamics by solid-state NMR. Data collected on tyrosine-ethylester (TEE) are in line with previously determined (1H,13C) order parameters. Application of these experiments to microcrystalline ubiquitin reveals the presence of dynamics on millisecond or faster time scales and differences in local mobility depending on microcrystal preparation. In addition, solid-state NMR-based structure calculation indicates conformational variability of loop regions between different solid-phase ubiquitin preparations. Our data relate preparation-dependent changes observed in NMR spectral parameters such as chemical shifts and through-space correlations to differences in ubiquitin dynamics and conformation and suggest a prominent role of molecular mobility in microcrystalline ubiquitin
Characterization of a Cyclic Nucleotide-Activated K+ Channel and its Lipid Environment by Using Solid-State NMR Spectroscopy
No full textVoltage-gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain-related mobility, structural characterization has proved challenging. We analyzed high-resolution solid-state NMR data on different isotope-labeled protein constructs of a bacterial cyclic nucleotide-activated K+ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage-sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope-labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane-embedded proteins, even in the presence of local disorder
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