In-Cell Protein Structures from 2D NMR Experiments

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 μM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures

Real-time NMR monitoring of biological activities in complex physiological environments

Biological reactions occur in a highly organized spatiotemporal context and with kinetics that are modulated by multiple environmental factors. To integrate these variables in our experimental investigations of 'native' biological activities, we require quantitative tools for time-resolved in situ analyses in physiologically relevant settings. Here, we outline the use of high-resolution NMR spectroscopy to directly observe biological reactions in complex environments and in real-time. Specifically, we discuss how real-time NMR (RT-NMR) methods have delineated insights into metabolic processes, post-translational protein modifications, activities of cellular GTPases and their regulators, as well as of protein folding events.Fil: Smith, Matthew J.. Ontario Cancer Institute; CanadáFil: Marshall, Christopher B.. Ontario Cancer Institute; CanadáFil: Theillet, Francois Xavier. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Binolfi, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Selenko, Philipp. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Ikura, Mitsuhiko. Ontario Cancer Institute; Canadá. University of Toronto; Canad

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson’s disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c’s activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits

In-cell structural biology by NMR: the benefits of the atomic-scale

International audienceIn-cell structural biology aims at extracting structural information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promises and is already providing new facts and outlooks of interest at both fundamental and applied levels. NMR spectroscopy has important contributions on this stage: it brings information on a broad variety of nuclei at the atomic scale, which ensures its great versatility and uniqueness. Here, we detail the methods, the fundamental knowledge and the applications in biomedical engineering related to in-cell NMR. We finally propose a brief overview of the main other techniques in the field (EPR, smFRET, cryo-ET…) to draw some advisable developments for in-cell NMR. In the era of large-scale screenings and deep learning, both accurate and qualitative experimental evidences are as essential as ever to understand the interior life of cells. In-cell structural biology by NMR spectroscopy can generate such a knowledge, and it does so at the atomic scale. This review is meant to deliver comprehensive but accessible information, with advanced technical details and reflections on the methods, the nature of the results and the future of the field. 5.1.1. In-cell EPR 5.1.2. In-cell FRET microscopy 5.1.3. Mass-spectrometry 5.1.4. Cryo-ET 5.2. The specific benefits of in-cell NMR 5.3. The future technical challenges of in-cell NMR 6. Conclusion Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations Reference

In-cell NMR: Why and how?

International audienceNMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies. NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR.. . All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success. We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the ''why", oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the ''how", where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology

Multidisciplinary Approaches to Study O-Antigen: Antibody Recognition in Support of the Development of Synthetic Carbohydrate-Based Enteric Vaccines

International audienceEnteric infections, including bacterium-induced diarrhoeal diseases, represent a major health burden worldwide. In developed countries, infectious diarrhoea contributes primarily to morbidity. It remains the second leading cause of death in children below 5 years of age living in the developing world (Cheng et al. 2005; You et al. 2010). It is anticipated that improved living conditions will contribute to diminish the transmission of enteric pathogens and lower the incidence of enteric diseases. In the meantime, the introduction of vaccines could play an active part in reducing the vulnerability of the target populations to the predominant enteric pathogens. Along this line, Shigella, ETEC, cholera, and typhoid fever were identified by WHO since the early 1990s as the highest bacterial disease priorities for the development of new or improved enteric vaccines. Substantial progress was made (Levine 2006). In this context, polysaccharide-based parenteral vaccines have been investigated with some success. The licensure of the purified capsular Vi polysaccharide against typhoid fever was an important achievement, especially since recent evidence of herd protection conferred by the vaccine has highlighted the benefit of large-scale use in endemic countries (Khan et al. 2010). Moreover, encouraging investigational studies on a Vi polysaccharide–protein conjugate vaccine, which could be introduced into the infant immunization schedule, were reported (Canh et al. 2004; Cui et al. 2010)

Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α

Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10

Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to identify their phospho-dependent binding partners

The combined expression of a handful of pluripotency transcription factors (PluriTFs) in somatic cells can generate induced pluripotent stem cells (iPSCs). Here, we report the structural characterization of disordered regions contained in four important PluriTFs, namely Oct4, Sox2, Nanog and Esrrb. Moreover, many post-translational modifications (PTMs) have been detected on PluriTFs, whose roles are not yet characterized. To help in their study, we also present a method i) to produce well-characterized phosphorylation states of PluriTFs, using NMR analysis, and ii) to use them for pull-downs in stem cell extracts analyzed by quantitative proteomics to identify of Sox2 binders

The power of pure shift HαCα correlations: a way to characterize biomolecules under physiological conditions

International audienceIntrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water-exchange, which disfavors classical amide 1H-detection, while 13C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of non-exchangeable 1Hα so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the SHACA-HSQC, using extensive hetero- and homo-nuclear decoupling applicable to aqueous samples (≥ 90% H2O) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to 5-fold increased S/N compared to the standard 1H,13C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumorsuppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any 1Hα-detected triple resonance experiment, but may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries