Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

“…All NMR spectra were acquired on a 720-MHz Varian Unity Plus spectrometer (National High Magnetic Field Laboratory, Tallahassee, Florida)+ Quadrature detection was achieved by implementation of the States method (States et al+, 1982) in all experiments, with the exception of the excitation sculpting NOESY experiment (Callihan et al+, 1996), for which the States-TPPI method (Marion et al+, 1989) was utilized+ All NMR data were acquired in the phase-sensitive mode+ Two-dimensional experiments were processed using Varian VNMR software, and assigned using SPARKY visualization software+ Twodimensional spectra were apodized with a Gaussian function, and zero-filling was used in both the directly detected and indirectly detected dimensions+ Exchangeable protons were observed at 4 8C with samples in aqueous buffer including 10% 2 H 2 O to obtain lock+ NOESY spectra with 150-ms mixing times were collected using excitation sculpting for solvent signal suppression (Callihan et al+, 1996)+ Spectra were referenced to the residual solvent peak at 5+01 ppm+ NOESY spectra acquired for nonexchangeable protons were collected at 20 8C using a lowpower irradiation presaturation pulse during the relaxation delay for residual solvent suppression, and were referenced at that temperature to the H 2 HO resonance at 4+80 ppm+ NOESY spectra were collected at several mixing times to observe buildup and decay of particular NOEs+ Sequential assignment from aromatic-anomeric NOEs were made from NOESY spectra collected with 350-ms mixing times+ TOCSY spectra utilizing a clean MLEV composite spin lock pulse scheme were collected at 20 8C to identify pyrimidine H5-H6 correlations and strong H19-H29 couplings+ DQF-COSY spectra, acquired at 20 8C, were collected to identify riboses with C29-endo character based on observable J H19-H29 couplings+ Approximate values for observable H19-H29 couplings were measured from one-dimensional traces of DQF-COSY spectra+ The accepted values of 1-2 Hz for C39-endo ribose H19-H29 couplings and 7-8 Hz for C29-endo were used to assess ribose conformation+ One-dimensional proton-decoupled phosphorous NMR spectra were conducted on a Varian Inova 500 spectrometer (phosphorous frequency 202+3 MHz) at 20 8C and referenced to an external standard of 85% phosphoric acid, H 3 PO 4 at 0 ppm (3+46 ppm upfield of trimethyl phosphate)+ All spectra were collected with 2,000 transients+ The identical uBP and cBP NMR samples were used for phosphorous detection as were used for detection of nonexchangeable protons+ The NMR sample for the unbulged duplex (ubBP) was prepared identically to the samples for uBP and cBP+…”

Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer

“…The RNA samples for the NMR studies were generated by in vitro transcription using T7 RNA polymerase and synthetic DNA templates (Milligan et al+, 1987;Milligan & Uhlenbeck, 1989)+ The uniformly 13 C/ 15 N-labeled sample of the ⌬-33 RNA was prepared with 13 C/ 15 N-labeled NTPs as described (Batey et al+, 1992;Nikonowicz et al+, 1992)+ Pre-RNAs containing a 7-nt 39-tail sequence (59-GCAGGUC-39) relative to the RNA shown in Figure 1B were transcribed and cleaved with a hammerhead ribozyme added in trans to produce a homogeneous-length RNA as described (Shields et al+, 1999)+ After the cleavage reaction, the product RNA was separated from the pre-RNA by denaturing polyacrylamide gel electrophoresis purification, followed by a DEAE-Sephacel (Phar- macia) anion-exchange chromatography+ The final step in the production of the NMR sample was to dialyze the RNA extensively in the NMR buffer: 20 mM NaH 2 PO 4 , pH ϭ 6+8, 30 mM NaCl, 2 mM MgCl 2 using a Centricon-3 concentrator (Amicon)+ Generally, 10-12 mL of buffer were passed over the RNA to insure complete equilibration of pH and Mg 2ϩ ions+ For the 1:1 RNA-theophylline complex, one equivalent of theophylline (Sigma-reference grade) was added to the sample+ The sample was then lyophilized to dryness and finally resuspended in 350 mL of 90% H 2 O/10% D 2 O or 99+99% D 2 O+ For the NMR studies of the caffeine:RNA complex, 10 equivalents of caffeine were added to the purified RNA sample+ After prolonged storage at 4 8C, samples were heated to 35 8C for 5 min in the NMR tube and then allowed to cool slowly to the experimental temperature+ NMR spectra of the manganese ion titration of RNA-theophylline complex A two-dimensional ( 1 H, 13 C) HSQC spectrum was collected on the uniformly 13 C/ 15 N-labeled RNA/theophylline complex in 20 mM NaH 2 PO 4 , pH ϭ 6+8, 30 mM NaCl, 2 mM MgCl 2 + A Mn 2ϩ titration was performed by adding 5 mL aliquots of a concentrated MnCl 2 solution (in D 2 O), and identical HSQC spectra were collected at total Mn 2ϩ concentrations of 5,10,20,25,30,35, and 40 mM+ The concentration of the RNAtheophylline complex was therefore reduced by ;10% in the 40 mM added Mn 2ϩ spectrum relative to the initial spectrum+ Each spectrum was acquired in ;2 h with 512 complex points for 6,000 Hz in the 1 H dimension (t 2 ), and 190 complex points for 7,500 Hz in the indirect-13 C dimension (t 1 ) and used the States-TPPI method for quadrature detection in t 1 (Marion et al+, 1989)+…”

Structure in solution of a four‐helix lipid binding protein

“…Two-dimensional DQF-COSY spectra (Piantini et al, 1982), TOCSY spectra with the DIPSI-2 mixing sequence Bax & Davis, 1985), and NOESY spectra (Jeener, 1971;Anil-Kumar et al, 1981) in 90/10 H20/'H20 at pH 4.0, of bLTP at 298 K and 310 K, respectively, were recorded and processed according to the States-TPPI scheme (Marion et al, 1989). Mixing times in the NOESY spectra were 50, 100, or 200 ms; for the 50-ms mixing time, NOE magnetization transfer via zero quantum coherence was minimized by the method of Otting (1990).…”