Abstract：
:NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.
journal_name
J Biomol NMRjournal_title
Journal of biomolecular NMRauthors
Aranibar N,Borys M,Mackin NA,Ly V,Abu-Absi N,Abu-Absi S,Niemitz M,Schilling B,Li ZJ,Brock B,Russell RJ 2nd,Tymiak A,Reily MDdoi
10.1007/s10858-011-9490-8subject
Has Abstractpub_date
2011-04-01 00:00:00pages
195-206issue
3-4eissn
0925-2738issn
1573-5001journal_volume
49pub_type
杂志文章abstract：:A substantial time savings in the collection of multidimensional NMR data can be achieved by coupling the evolution of nuclei in the indirect dimensions. In order to save time, the sampling of the indirect dimensions is inherently incomplete. Therefore, many algorithms and samplings schemes have been developed aimed a...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-009-9309-z
更新日期：2009-05-01 00:00:00
abstract：:Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1008340210079
更新日期：2001-02-01 00:00:00
abstract：:Signal overlapping is a major bottleneck for protein NMR analysis. We propose a new method, stable-isotope-assisted parameter extraction (SiPex), to resolve overlapping signals by a combination of amino-acid selective isotope labeling (AASIL) and tensor decomposition. The basic idea of Sipex is that overlapping signal...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00295-9
更新日期：2020-03-01 00:00:00
abstract：:Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a singl...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-9936-5
更新日期：2015-06-01 00:00:00
abstract：:19F solid-state NMR is an excellent approach for measuring long-range distances for structure determination and for studying molecular motion. For multi-fluorinated proteins, assignment of 19F chemical shifts has been traditionally carried out using mutagenesis. Here we show 2D 19F-13C correlation experiments that all...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-020-00306-0
更新日期：2020-03-01 00:00:00
abstract：:NMR spectroscopy provides a powerful approach for the characterisation of chemical exchange and molecular interactions by analysis of series of experiments acquired over the course of a titration measurement. The appearance of NMR resonances undergoing chemical exchange depends on the frequency difference relative to ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00297-7
更新日期：2020-01-01 00:00:00
abstract：:Due to practical limitations in available 15N rf field strength, imperfections in 15N 180 degrees pulses arising from off-resonance effects can result in significant sensitivity loss, even if the chemical shift offset is relatively small. Indeed, in multi-dimensional NMR experiments optimized for protein backbone amid...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-006-9089-7
更新日期：2006-12-01 00:00:00
abstract：:Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00285-x
更新日期：2019-12-01 00:00:00
abstract：:In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignmen...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1026776721348
更新日期：2000-12-01 00:00:00
abstract：:We present heteronuclear three-dimensional gradient-NMR techniques for the resonanceassignment of exchangeable (-OH and -NH) protons in uniformly 13C isotopically enrichedoligosaccharides and for the measurement of 1H-1H nuclear Overhauser enhancementsinvolving these protons. These techniques are derived from conventi...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/A:1018671517876
更新日期：1997-01-01 00:00:00
abstract：:Prerequisite for chemical shift (CS) and CS tensor calculations are highly refined structures defining the molecular surroundings of the nuclei under study. Here, we present geometry optimizations with 13C and 15N CS constraints for large bio-molecules like peptides and proteins. The method discussed here provides bot...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00284-y
更新日期：2019-12-01 00:00:00
abstract：:Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic re...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-9930-y
更新日期：2015-06-01 00:00:00
abstract：:An 'isotopomer-selected NOE' (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the 'partner' hydroxyl, whereas a NOE for...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/A:1008237308320
更新日期：1998-07-01 00:00:00
abstract：:The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-020-00310-4
更新日期：2020-05-01 00:00:00
abstract：:The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13C-15N distances, which are usually measured using the rotational-echo double-resonance (R...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-018-0187-0
更新日期：2018-05-01 00:00:00
abstract：:Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] chemical shifts very often observed within intr...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-017-0138-1
更新日期：2017-11-01 00:00:00
abstract：:The kinetics of lanthanide (III) exchange for calcium(II) in the C-terminal EF-hand of the protein calbindin D9k have been studied by one-dimensional (1D) stopped-flow NMR. By choosing a paramagnetic lanthanide (Ce3+), kinetics in the sub-second range can be easily measured. This is made possible by the fact that (i) ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1020245031235
更新日期：2002-08-01 00:00:00
abstract：:Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequenc...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00274-0
更新日期：2019-11-01 00:00:00
abstract：:Relaxation measurements of side-chain 13CH2-groups of uniformly 13C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30 degrees C. Dipole-dipole cross-correlated relaxation effects in T1 experiments were suppressed by the combination of radio-frequency pulses and pulsed field gr...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1023833407515
更新日期：2003-07-01 00:00:00
abstract：:A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C') and 3J(H beta,C') in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schm...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF00203824
更新日期：1996-03-01 00:00:00
abstract：:HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14-17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution dem...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-010-9404-1
更新日期：2010-05-01 00:00:00
abstract：:The human alpha 3-chain type VI collagen C-terminal Kunitz domain fragment (alpha 3(VI)) has been studied by two dimensional 1H-1H and 1H-13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and a number of unusual H alpha chemical shifts, which ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF00228142
更新日期：1996-12-01 00:00:00
abstract：:Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00235-7
更新日期：2019-09-01 00:00:00
abstract：:The inclusion of explicit solvent water in molecular dynamics refinement of NMR structures ought to provide the most physically meaningful accounting for the effects of solvent on structure, but is computationally expensive. In order to evaluate the validity of commonly used vacuum refinements and of recently develope...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1014929925008
更新日期：2002-04-01 00:00:00
abstract：:A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3 mM sampl...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-014-9841-3
更新日期：2014-08-01 00:00:00
abstract：:One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assig...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/A:1018329420659
更新日期：1997-10-01 00:00:00
abstract：:Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-0003-z
更新日期：2016-01-01 00:00:00
abstract：:For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform (13)C, (15)N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected (15)N enriched amino acids and...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-007-9159-5
更新日期：2007-07-01 00:00:00
abstract：:The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-014-9840-4
更新日期：2014-08-01 00:00:00
abstract：:A large portion of the 13C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28 degrees C has been determined at natural isotope abundance. Sequence-specific 13C assignments are reported for 100% of the assignable C alpha, 96% of the C beta, 86% of the aromatic and 70% of the remaining peri...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF00208807
更新日期：1995-02-01 00:00:00
