Doubly Sensitivity-Enhanced 3D HCCH-TOCSY of13C-Labeled Proteins in H2O Using Heteronuclear Cross Polarization and Pulsed Field Gradients
✍ Scribed by Sybren S. Wijmenga; Elles Steensma; Carlo P.M. van Mierlo
- Publisher
- Elsevier Science
- Year
- 1997
- Tongue
- English
- Weight
- 231 KB
- Volume
- 124
- Category
- Article
- ISSN
- 1090-7807
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✦ Synopsis
Singly and doubly sensitivity-enhanced versions are de-( 7) which employs B 0 field gradients as spoiling pulses to scribed of the three-dimensional cross-polarization driven suppress the water signal shows good H 2 O suppression, and HCCH-TOCSY experiment (3D CP HCCH-TOCSY), therefore allows CP HCCH-TOCSY experiments to be rewhich employ enhanced cross-polarization sequences with corded in H 2 O. gradient coherence selection for both the 1 H r 13 C transfer
The CP HCCH-TOCSY sequence introduced by Wang step and the 13 C r 1 H transfer step. The sequences are demand Zuiderweg (7) does not select coherence pathways via onstrated on a 13 C/ 15 N doubly labeled sample of flavodoxin the use of gradients; instead, the artifacts are suppressed II of Azotobacter vinelandii (179 amino acid residues; 20 via conventional phase cycling and the use of trim pulses. kDa) dissolved in 90% H 2 O/10% D 2 O. The presented imple-However, it has now been recognized that coherence-pathmentations of 3D CP HCCH-TOCSY experiments are simway selection via gradients is the foremost method to supple and robust with regard to missettings and therefore easy press artifacts and to obtain excellent water suppression (8). to use and set up.
In addition, when coherence-pathway selection via gradients The assignment of resonances of larger biomolecules reis used in combination with an enhanced INEPT sequence, quires a combination of several multidimensional hetero-eINEPT, an increase in sensitivity of a factor of 2 1/2 per nuclear experiments [see, for example, (1, 2)]. The generindividual transfer step can be achieved (9). Single enhanceally used protocol is to obtain main-chain resonance assignment of the final transfer step by an eINEPT sequence is ments from three-dimensional triple-resonance experiments now routinely applied in 2D and 3D NMR experiments (8). and to subsequently extract the side-chain resonances from However, the sensitivity improvement in nD NMR experi-3D HCCH-COSY or 3D HCCH-TOCSY experiments [see, ments can be as high as 2 ( n 01)1/2 , when each transfer step for example, (1)]. Originally, the 3D HCCH-TOCSY exin the nD experiment is modified into an enhanced sequence periment was based on INEPT (RINEPT) sequences (3,4). (10, 11). Double enhancement using eINEPT sequences has Since the 3D HCCH-TOCSY experiments are essential for been demonstrated in a number of 3D NMR experiments the assignment procedure, improved implementations are of (10-13). major importance. The cross-polarization driven HCCH-Since the cross-polarization sequence can be modified into TOCSY experiment, in which the 1 H r 13 C and 13 C r 1 H an enhanced sequence ( 14), sensitivity improvements of the magnetization transfers are accomplished via heteronuclear 3D CP HCCH-TOCSY experiment are also possible. The cross polarization (5-7), is such an improvement. First, double-enhancement approach has been demonstrated in a heteronuclear cross polarization, CP, is more effective with 3D HCCH-TOCSY experiment by Sattler et al. (10, 11), regard to the 1 H r 13 C and 13 C r 1 H magnetization transfers in which an enhanced CP sequence transfers magnetization then INEPT(RINEPT) (6). Second, to avoid chemical-shift from 1 H r 13 C and an e(R)INEPT sequence transfers magchanges associated with isotope effects and with sample hannetization from 13 C r 1 H. Considerable sensitivity improvedling, it is desirable to use one 13 C/ 15 N-labeled sample disment as compared to the singly enhanced and unenhanced solved in H 2 O for both the triple-resonance and the HCCHversions of the experiments were obtained. Sattler et al. (11) TOCSY experiments. The CP HCCH-TOCSY sequence also noted good water suppression, given their gradient and pulse settings, but did not discuss the water suppression method in detail.