1,721,010 research outputs found
A Unique Tool for Cellular Structural Biology: In-cell NMR
Full text linkConventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When this is done, important structural and functional features of macromolecules, which depend on their native network of interactions within the cell, may be lost. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches and focus on the applications to protein folding, interactions, and post-translational modifications
Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor
Full text linkIn-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells
Backbone resonance assignment of human DJ-1 in the reduced state and in the cysteine sulfinic acid state
Full text linkDJ-1 is a highly conserved soluble protein that is associated to several cellular pathways. In humans, DJ-1 has been implicated in several pathologies such as cancer, Parkinson’s disease and amyotrophic lateral sclerosis. Several roles have been attributed to DJ-1, including defense against oxidative stress, chaperone activity and proteasome regulation. The recent finding that DJ-1 acts as a protein and DNA deglycase further confirms the protective function of DJ-1 and suggests a common mechanism of action in the various pathways in which DJ-1 is involved. Cysteine 106, located in the putative active site of DJ-1, is critical for the biological activity of DJ-1 and is easily oxidized to cysteine-sulfinate. While such oxidation modulates DJ-1 activity, the underlying molecular mechanism has not yet been elucidated. Cysteine oxidation does not perturb the protein structure, therefore changes in protein dynamics in solution could modulate its function. Here, we report a revised and completed (98%) backbone assignment of reduced DJ-1, together with the backbone assignment of oxidized DJ-1. Chemical shift perturbation is observed in several regions across the sequence, while no changes in secondary structure are observed. These data will provide the starting point for further characterization of the changes in the backbone dynamics of DJ-1 upon oxidation in solution at physiological temperature
Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells
Full text linkIn-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection
Structural insights of proteins in sub-cellular compartments: In-mitochondria NMR
No full textAbstractMany eukaryotic proteins exert their physiological function in specific cellular compartments. Proteins of the inter-membrane space (IMS) of mitochondria, for example, are synthesized in the cytoplasm and translocate to the IMS, where they are further processed to their mature form. In-cell Nuclear Magnetic Resonance (NMR) has proven to be an ideal approach to investigate eukaryotic proteins at the atomic level, inside the cytoplasm. Here we show that proteins inside intact mitochondria isolated from human cells can be structurally characterized by NMR (in-mitochondria NMR). By this approach, we characterized the folding and maturation state of two human proteins in the IMS, SOD1 and Mia40. Both observed proteins were in the folded state. Mia40 was in the oxidized, functional state, while SOD1 disulfide bond formation was promoted by increasing the level of the SOD1 chaperone, CCS, in the IMS
Protein interaction patterns in different cellular environments are revealed by in-cell NMR
Full text linkIn-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular
environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present,
while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins
In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation
Full text linkCellular structural biology methods are needed to characterize biological processes at atomic resolution in the physiological environment of the cell. Toward this goal, solution in-cell NMR is a powerful approach because it provides structural and dynamic data on macromolecules inside living cells. Several approaches have been developed for in-cell NMR in cultured human cells, which are needed to study processes related to human diseases that rely on the delivery of exogenous macromolecules to the cells. Such strategies, however, may not be applicable to proteins that are sensitive to the external environment or prone to aggregate and can introduce artifacts during protein purification or delivery.
As a complementary approach, direct protein expression for in-cell NMR in human cells was developed. This strategy is especially useful when studying processes like protein folding, maturation, and post-translational modification, starting right after protein synthesis. Compared with the protein expression techniques in mammalian cells commonly used in cellular biology, the low sensitivity of NMR requires higher protein levels. Among the cell lines used for high-yield protein expression, the HEK293T cell line was chosen, as it can be efficiently transfected with a cost-effective reagent. A vector originally designed for secreted proteins allows high-level cytosolic protein expression. For isotopic labeling, commercially available or homemade labeled media are employed. Uniform or amino acid type-selective labeling strategies are possible. Protein expression can be targeted to specific organelles (e.g., mitochondria), allowing for in organello NMR applications. A variant of the approach was developed that allows the sequential expression of two or more proteins, with only one selectively labeled.
Protein expression in HEK293T cells was applied to recapitulate the maturation steps of intracellular superoxide dismutase 1 (SOD1) and to study the effect of mutations linked to familial amyotrophic lateral sclerosis (fALS) by in-cell NMR. Intracellular wild-type SOD1 spontaneously binds zinc, while it needs the copper chaperone for superoxide dismutase (CCS) for copper delivery and disulfide bond formation. Some fALS-linked mutations impair zinc binding and cause SOD1 to irreversibly unfold, likely forming the precursor of cytotoxic aggregates. The SOD-like domain of CCS acts as a molecular chaperone toward mutant SOD1, stabilizing its folding and allowing zinc binding and correct maturation. Changes in protein redox state distributions can also be investigated by in-cell NMR. Mitochondrial proteins require the redox-regulating partners glutaredoxin 1 (Grx1) and thioredoxin (Trx) to remain in the reduced, import-competent state in the cytosol, whereas SOD1 requires CCS for disulfide bond formation. In both cases, the proteins do not equilibrate with the cytosolic redox pool. Cysteine oxidation in response to oxidative stress can also be monitored.
In the near future, in-cell NMR in human cells will likely benefit from technological advancements in NMR hardware, the development of bioreactor systems for increased sample lifetime, the application of paramagnetic NMR to obtain structural restraints, and advanced tools for genome engineering and should be increasingly integrated with advanced cellular imaging techniques
New structural and functional insights from in-cell NMR
No full textIn recent years, it has become evident that structural characterization would gain significantly in terms of biological relevance if framed within a cellular context, while still maintaining the atomic resolution. Therefore, major efforts have been devoted to developing Cellular Structural Biology approaches. In this respect, in-cell NMR can provide and has provided relevant contributions to the field, not only to investigate the structural and dynamical properties of macromolecules in solution but, even more relevant, to understand functional processes directly in living cells and the factors that modulate them, such as exogenous molecules, partner proteins, and oxidative stress. In this commentary, we review and discuss some of the main contributions to the understanding of protein structural and functional properties achieved by in-cell NMR.</jats:p
In-cell NMR: a topical review
Full text linkClassical structural biology approaches allow structural characterization of biological macromolecules in vitro, far from their physiological context. Nowadays, thanks to the wealth of structural data available and to technological and methodological advances, the interest of the research community is gradually shifting from pure structural determination towards the study of functional aspects of biomolecules. Therefore, a cellular structural approach is ideally needed to characterize biological molecules, such as proteins, in their native cellular environment and the functional processes that they are involved in. In-cell NMR is a new application of high-resolution nuclear magnetic resonance spectroscopy that allows structural and dynamical features of proteins and other macromolecules to be analyzed directly in living cells. Owing to its challenging nature, this methodology has shown slow, but steady, development over the past 15 years. To date, several in-cell NMR approaches have been successfully applied to both bacterial and eukaryotic cells, including several human cell lines, and important structural and functional aspects have been elucidated. In this topical review, the major advances of in-cell NMR are summarized, with a special focus on recent developments in eukaryotic and mammalian cells
X-ray fluorescence microscopy artefacts in elemental maps of topologically complex samples: Analytical observations, simulation and a map correction method
No full textXRF spectroscopy is among the most widely used non-destructive techniques for elemental analysis. Despite the known angular dependence of X-ray fluorescence (XRF), topological artefacts remain an unresolved issue when using X-ray micro- or nano-probes. In this work we investigate the origin of the artefacts in XRF imaging of topologically complex samples, which are unresolved problems in studies of organic matter due to the limited travel distances of low energy XRF emission from the light elements. In particular we mapped Human Embryonic Kidney (HEK293T) cells. The exemplary results with biological samples, obtained with a soft X-ray scanning microscope installed at a synchrotron facility were used for testing a mathematical model based on detector response simulations, and for proposing an artefact correction method based on directional derivatives. Despite the peculiar and specific application, the methodology can be easily extended to hard X-rays and to set-ups with multi array detector systems when the dimensions of surface reliefs are in the order of the probing beam size
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