A constant-time 2D overbodenhausen experiment for inverse correlation of isotopically enriched species

Santoro, Jorge; King, Garry C.

doi:10.1016/0022-2364(92)90250-b

Cited by 110 publications

(128 citation statements)

References 10 publications

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“…Temperature and NaCl concentration were varied as specified in figure legends. Complete 1 H, 15 N, and 13 C resonance assignments for Ltn40 (20 mM sodium phosphate, pH 6.0) were obtained at 40°C by using the following experiments: 15 N-1 H HSQC (26), 3D HNCA (27,28), 3D HNCO (27,29), 3D HN(CO)CA (27), 3D HNCACB, 3D HN(CA)CO, 3D HCCONH, 3D SE C(CO)NH (30), 3D HCCH TOCSY (31), and 2D 13 C constant time HSQC (32). NMR data were processed with NMRPipe (33), and XEASY (34) was used for resonance assignments and analysis of NOE spectra.…”

Section: Methodsmentioning

confidence: 99%

Interconversion between two unrelated protein folds in the lymphotactin native state

Tuinstra¹,

Peterson²,

Kutlesa

et al. 2008

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

261

428

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Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric threestranded ␤-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-␤-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures.chemokine ͉ conformational change ͉ NMR spectroscopy

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Section: Methodsmentioning

confidence: 99%

Interconversion between two unrelated protein folds in the lymphotactin native state

Tuinstra¹,

Peterson²,

Kutlesa

et al. 2008

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

261

428

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“…All NMR experiments were acquired at 600 MHz on a Varian Unityplus spectrometer+ Homonuclear 2D NOE spectra (mixing time 50, 150, and 400 ms) in D 2 O were recorded at 20 8C, 30 8C, and 35 8C (400 t 1 values with 32 scans each, recycling delay 2+0 s)+ DQF-COSY spectra were acquired similarly+ TOCSY experiments were run with MLEV-17 mixing and cycling (Bax & Davies, 1985) (mixing time 30 and 75 ms)+ Twodimensional NOE experiments in H 2 O were collected at 10 8C using the SSnoesy pulse sequence (Smallcombe, 1993)+ Translational diffusion constants, D t , were determined with the water-sLED sequence (Altieri et al+, 1995) and subsequent fitting of the intensities of the aromatic signals to (Haner & Schleich, 1989;Lapham et al+, 1997)+ Here, A and A 0 are peak intensities at a given gradient strength G z and without a gradient, respectively; g is the gyromagnetic ratio of 1 H; d is the duration of the gradient and ⌬ is the time between gradients+ One-bond heteronuclear correlations were obtained with 1 H 15 N-HMQC or CT-1 H 13 C-HSQC experiments (Santoro & King, 1992)+ Ribose spin systems were elucidated with 3D-HCCH-COSY and -TOCSY experiments (Nikonowicz & Pardi, 1993)+ The latter experiment also detected long range H2-H8 correlations Marino et al+, 1994)+ To complete assignments and extract structural information, 3D-1 H 13 C 1 H-NOESY-HMQC experiments (Marion et al+, 1989;Nikonowicz & Pardi, 1993) were collected for all labeled 43mer samples (mixing time 150 ms) and optimized for either observation of aromatic or ribose 13 C 1 H moieties+ For A43mer and G43mer samples, 2D…”

Section: Nmr Experimentsmentioning

confidence: 99%

Structure of the phylogenetically most conserved domain of SRP RNA

Schmitz

Behrens

Freymann

et al. 1999

RNA

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“…The 'H-I'N HMBC spectrum was recorded on a 13C-and "N-enriched sample to allow a precise alignment with the 'H-"C HSQC spectrum of BCX. The constant-time 'H-"C HSQC spectrum of the aromatic region of uniformly '3C/"N-enriched BCX was recorded using spectral widths of 6,250 Hz (1 2.5 ppm) and 6,200 Hz (49 pprn), and acquisition times of 164 ms and 16.3 ms in 'H and 13C, respectively (Santoro & King, 1992;Vuister & Bax, 1992). The I3C transmitter was set at 124.5 ppm.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange

et al. 1996

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Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 'H, I3C, and I5N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 'HE' of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 X low5 s-' at pH* 7.04 and 30 "C) is retarded by > 106-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at -6.5, as measured from the pH dependence of the "N-and 'H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N'*H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 "C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.

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A constant-time 2D overbodenhausen experiment for inverse correlation of isotopically enriched species

Cited by 110 publications

References 10 publications

Interconversion between two unrelated protein folds in the lymphotactin native state

Interconversion between two unrelated protein folds in the lymphotactin native state

Structure of the phylogenetically most conserved domain of SRP RNA

Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange

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