Yuki Toyama, Ichio Shimada
Solution NMR spectroscopy is a particularly powerful technique for characterizing the functional dynamics of biomolecules, which is typically achieved through the quantitative characterization of chemical exchange processes via the measurement of spin relaxation rates. In addition to the conventional nuclei such as 15 N and 13 C, which are abundant in biomolecules, fluorine-19 (19 F) has recently garnered attention and is being widely used as a site-specific spin probe. While 19 F offers the advantages of high sensitivity and low background, it can be susceptible to artifacts in quantitative relaxation analyses due to a multitude of dipolar and scalar coupling interactions with nearby 1 H spins...
June 25, 2024: Journal of Biomolecular NMR
Dallas Bell, Florian Lindemann, Lisa Gerland, Hanna Aucharova, Alexander Klein, Daniel Friedrich, Matthias Hiller, Kristof Grohe, Tobias Meier, Barth van Rossum, Anne Diehl, Jon Hughes, Leonard J Mueller, Rasmus Linser, Anne-Frances Miller, Hartmut Oschkinat
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...
June 21, 2024: Journal of Biomolecular NMR
Shibani Bhattacharya, Kristen M Varney, Tassadite Dahmane, Bruce A Johnson, David J Weber, Arthur G Palmer
Deuterium (2 H) spin relaxation of 13 CH2 D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B0 dependence of the 2 H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ωD ) and J(2ωD ). In this study, the linear relation between 2 H relaxation rates at B0 fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping...
June 10, 2024: Journal of Biomolecular NMR
Alexandra Locke, Kylee Guarino, Gordon S Rule
A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2 O and 2 H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes...
May 24, 2024: Journal of Biomolecular NMR
Benxun Pan, Canyong Guo, Dongsheng Liu, Kurt Wüthrich
In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19 F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19 F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan"...
March 30, 2024: Journal of Biomolecular NMR
Arthur Giraud, Lionel Imbert, Adrien Favier, Faustine Henot, Francis Duffieux, Camille Samson, Oriane Frances, Elodie Crublet, Jérôme Boisbouvier
Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples...
March 28, 2024: Journal of Biomolecular NMR
Qianqian Wang, Zhiwei Miao, Xiongjie Xiao, Xu Zhang, Daiwen Yang, Bin Jiang, Maili Liu
The fast motions of proteins at the picosecond to nanosecond timescale, known as fast dynamics, are closely related to protein conformational entropy and rearrangement, which in turn affect catalysis, ligand binding and protein allosteric effects. The most used NMR approach to study fast protein dynamics is the model free method, which uses order parameter S2 to describe the amplitude of the internal motion of local group. However, to obtain order parameter through NMR experiments is quite complex and lengthy...
March 26, 2024: Journal of Biomolecular NMR
Whitney N Costello, Yiling Xiao, Frederic Mentink-Vigier, Jaka Kragelj, Kendra K Frederick
With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein...
March 23, 2024: Journal of Biomolecular NMR
Giorgia Toscano, Julian Holzinger, Benjamin Nagl, Georg Kontaxis, Hanspeter Kählig, Robert Konrat, Roman J Lichtenecker
We present an economic and straightforward method to introduce 13 C-19 F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13 C, 2,3,5,6-2 H4 ] 4-fluorophenylalanine from the commercially available isotope sources [2-13 C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins...
March 21, 2024: Journal of Biomolecular NMR
Bharat P Chaudhary, Jochem Struppe, Hem Moktan, David Zoetewey, Donghua H Zhou, Smita Mohanty
N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein...
February 29, 2024: Journal of Biomolecular NMR
Kieran T Cockburn, Brian D Sykes
The focus of this project is to take advantage of the large NMR chemical shift anisotropy of 19 F to determine the orientation of fluorine labeled biomolecules in situ in oriented biological systems such as muscle. The difficulty with a single fluorine atom is that the orientation determined from a chemical shift is not singlevalued in the case of a fully anisotropic chemical shift tensor. The utility of a labeling approach with two fluorine labels in a fixed molecular framework where one of the labels has an axially symmetric chemical shift anisotropy such as a CF3 group and the other has a fully asymmetric chemical shift anisotropy such as 5-fluorotryptophan is evaluated...
February 26, 2024: Journal of Biomolecular NMR
Ganesh P Subedi, Elijah T Roberts, Alexander R Davis, Paul G Kremer, I Jonathan Amster, Adam W Barb
A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[15 N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y)...
February 26, 2024: Journal of Biomolecular NMR
Ved Prakash Tiwari, Debajyoti De, Nemika Thapliyal, Lewis E Kay, Pramodh Vallurupalli
Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange...
January 3, 2024: Journal of Biomolecular NMR
Pauline Defant, Christof Regl, Christian G Huber, Mario Schubert
Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose...
December 20, 2023: Journal of Biomolecular NMR
Sungsool Wi, Conggang Li, Karen Pham, Woonghee Lee, Lucio Frydman
A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) 15 N-15 N correlations on uniformly 15 N-labeled GB1 powders. A major feature exploited in these 15 N-15 N correlations was AL FRESCO's remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These 15 N-15 N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings...
December 16, 2023: Journal of Biomolecular NMR
Francisco Mendoza-Hoffmann, Canyong Guo, Yanzhuo Song, Dandan Feng, Lingyun Yang, Kurt Wüthrich
For the A2A adenosine receptor (A2A AR), a class A G-protein-coupled receptor (GPCR), reconstituted in n-dodecyl-β-D-maltoside (DDM)/‌‌‌‌‌cholesteryl hemisuccinate (CHS) mixed micelles, previous 19 F-NMR studies revealed the presence of multiple simultaneously populated conformational states. Here, we study the influence of a different detergent, lauryl maltose neopentyl glycol (LMNG) in mixed micelles with CHS, and of lipid bilayer nanodiscs on these conformational equilibria...
December 10, 2023: Journal of Biomolecular NMR
Alexander R Davis, Elijah T Roberts, I Jonathan Amster, Adam W Barb
Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E...
November 21, 2023: Journal of Biomolecular NMR
Sina Kazemi, Anna Lopata, Andreas Kniss, Lukas Pluska, Peter Güntert, Thomas Sommer, Thomas F Prisner, Alberto Collauto, Volker Dötsch
Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations...
November 15, 2023: Journal of Biomolecular NMR
Alexander Klein, Suresh K Vasa, Rasmus Linser
1 H-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...
November 9, 2023: Journal of Biomolecular NMR
Leonardo Querci, Deborah Grifagni, Inês B Trindade, José Malanho Silva, Ricardo O Louro, Francesca Cantini, Mario Piccioli
The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed Cα /C' connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized 13 C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems...
October 18, 2023: Journal of Biomolecular NMR
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