Improved WATERGATE Pulse Sequences for Solvent Suppression in NMR Spectroscopy

Liu, Maili; Mao, Xi‐an; Ye, Chaohui; Huang, He; Nicholson, Jeremy K.; Lindon, John C.

doi:10.1006/jmre.1998.1405

Cited by 512 publications

(399 citation statements)

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“…The NMR experiments were performed on an AV800MHz spectrometer (Bruker, Rheinstetten, Germany) with a 5-mm z-axis gradient TXI-HCN cryogenic probe at 283 K. The NMR data were analyzed by using the software TOPSPIN 1.3, felix2000 (Accelrys, San Diego, CA) and SigmaPlot 9.0. All NMR spectra were recorded in H 2 O/D 2 O (9:1) by using standard pulse sequences with WATERGATE water suppression (40) or jump-return-echo pulse sequences (41). Light-induced reaction initiation was achieved by using a laser installation (28,30) with direct coupling of a quartz fiber from a CW argon ion laser (Spectra-Physics, Darmstadt, Germany) into the NMR tube equipped with a quartz tip insert.…”

Section: Methodsmentioning

confidence: 99%

Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution

Buck

Fürtig²,

Noeske³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

130

140

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spectroscopy ͉ riboswitches ͉ dynamics ͉ purine ͉ caged compound S tructural transitions of proteins and RNA constitute an important aspect of cellular function and information transfer. In proteins, the kinetics of these structural transitions, mainly from an unfolded ensemble to a single unique folded state, can be investigated by hydrogen exchange experiments monitored by NMR spectroscopy (1). Such studies have profoundly influenced our understanding of protein-folding pathways. In hydrogen exchange experiments, labile hydrogen atoms become protected against exchange with the bulk solvent during folding. This acquired exchange protection indicates formation of a persistent structure at atomic resolution. Incorporation of structural information derived from exchange experiments into molecular dynamics (MD) simulations (2), including data from methods exhibiting time and atomic resolution together with -value analysis (3) derived from mutational work, has provided detailed structural information of intermediates populated during folding.RNA molecules can adopt a variety of secondary and tertiary conformations (4, 5). In general, the energetic difference between alternate RNA conformations is very small, and the equilibrium distribution is strongly affected through the binding of proteins (6), ions (7), or small metabolites (8-10), or by structural modifications (11). Alternate RNA structures are stabilized by different Watson-Crick base-pairing interactions (12). To date, RNA folding has been investigated by using x-ray footprinting (13), oligonucleotide hybridization, and classical biochemical methods in conjunction with mutational data (14). Although hydrogen exchange rates can be used as reporters of RNA ground-state dynamics and stability (15-17), RNA hydrogen exchange experiments conducted in a pulse-chase manner fail in most cases because of the high intrinsic exchange rates observed even in folded RNA structures.Here, we report on ligand-induced conformational changes of an RNA at atomic resolution by using real-time NMR methods. We investigated the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain (GSR apt ) of the Bacillus subtilis xpt-pbuX operon (18). Riboswitch RNAs have emerged as an important example of macromolecular structural transitions that lead to transcriptional or translational regulation of protein expression induced through the specific binding of a metabolite (reviewed in refs. 19 and 20). Riboswitches are located in the 5Ј-untranslated region (5Ј-UTR) of bacterial mRNA and consist of a highly specific metabolite receptor region (aptamer domain) coupled to a 3Ј-downstream sequence (expression platform). Gene expression is thought to be modulated in response to conformational differences between the ligand-bound and ligand-free conformations of the aptamer domain (21). Crystal structures of the ligand-bound aptamer domains belonging to the class of purine riboswitches (guanineand adenine-sensing riboswitches) have revealed the presence of complex tertiary stru...

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Section: Methodsmentioning

confidence: 99%

Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution

Buck

Fürtig²,

Noeske³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

130

140

View full text Add to dashboard Cite

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“…Quadrature detection in f1 was achieved using States-time-proportional phase incrementation or the echo-antiecho method. The solvent signal was suppressed by the WATERGATE W5 method (19,20) or by excitation sculpting (21). Two-dimensional 1 H-1 H TOCSY (22) spectra with mixing times of 40 and 80 ms, respectively, and two-dimensional 1 H-1 H NOESY (23) spectra with mixing times of 150 and 300 ms, respectively, were recorded at 283 K. The temperature was calibrated with methanol (24).…”

Section: Methodsmentioning

confidence: 99%

CD and NMR Studies of Prion Protein (PrP) Helix 1

Ziegler

Sticht

Marx

et al. 2003

Journal of Biological Chemistry

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The conversion of prion helix 1 from an ␣-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrP C of the prion protein to the pathogenic isoform PrP Sc . Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrP C 3 PrP Sc conversion.Prion protein, PrP, 1 is probably the disease-causing agent of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy in cattle or Creutzfeldt-Jakob disease in man (1). Its cellular form, PrP C , is a highly conserved cell surface glycoprotein of 230 amino acids expressed in all of the mammals studied so far as well as in several species of fish and birds (2, 3). The physiological function of PrP C is not yet fully understood. PrP C seems to be involved in the maintenance of proper presynaptic copper levels as well as in protecting neurons from oxidative stress (4, 5). In addition, the physiological function of PrP C could be associated with higher neurological functions such as learning and memory (5). According to the protein-only hypothesis, disease is caused by accumulation of a misfolded pathogenic isoform, PrP Sc , which is the result of an irreversible large scale conformational change of PrP C . Although PrP C is largely ␣-helical and soluble in polar solvents and sensitive to protease K digestion, PrP Sc consists mostly of ␤-sheets, is soluble only in nonpolar, denaturing solvents, and is resistant to digestion with protease K (6). PrP Sc forms fibrillar aggregates similar to other amyloid fibrils (7). Accumulation of PrP Sc aggregates is accompanied by astrocytosis and gliosis in central nervous tissue, which in turn result in vacuoles in the brains of patients.The solution structures of human PrP-(23-230), huPrP (8), mouse PrP-(121-231) (9), bovine PrP-(23-230) (10), and Syrian hamster PrP-(29 -231) (11) have been determined by NMR spectroscopy. They possess a high degree of structural conservation consistent with the high sequence identity of these proteins. Prion proteins consist of a flexible NH 2 -terminal domain spanning residues 23-124 (huPrP C -numbering scheme), which is largely disordered. This region includes an octapeptide sequence that is repeated four times from residues 60 to 92 and that is likely to bind copper (4). This part al...

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“…NMR spectra were recorded on a Bruker DRX 600 MHz instrument. A combination of through-space nuclear Overhauser effect (NOESY) and through-bond correlated (TOCSY) two-dimensional spectra were recorded at 17 °C using Double Pulsed Field Gradients Spin Echo water suppression (dpfgsew5) [14,15]. The data were processed using XWIN-NMR (Bruker) and analyzed using Sparky [16].…”

Section: Methodsmentioning

confidence: 99%