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The X half-filter element ( 88JACS2388 and 96ENC2239 ) plays an important role in multidimensional NMR experiments specifically designed to study protein-ligand complexes, where the protein is usually 13C and/or 15N-labeled and the ligand is at natural abundance. Two data sets are recorded which differ only by the phase of a editing 90º X pulse. For instance, sumation of these data selects 1H-12C magnetization and rejects 1H-13C magnetization, whereas subtraction selects 1H-13C magnetization and rejects 1H-12C magnetization in 13C-labeled proteins.REQUIREMENTS
Several options to implement such filter are possibles:
- Non-refocused X half-filter in which antiphase 1H-X magnetization (IxSz) is selected.
- Refocused X half-filter in which in-phase 1H-X magnetization (Ix) is selected.
- Double X half-filter in which independent X half-filters are incorporated into the same experiment.
- Time-shared X and S half-filter in which two half-filter applied on different nuclei (13C and 15N) are combined in a single element.
This block can be easily inserted in conventional 2D 1H experiments (NOESY, TOCSY ...) replacing the original preparation (w1-filtering/editing) or detection (w2-filtering/editing) schemes (see, for instance: 2D X-filtered NOESY experiment and 2D X-filtered TOCSY experiment). For instance, replacing the initial 90º 1H pulse by a X-half filter in a NOESY experiment results in a 2D w1-X-filtered/edited NOESY experiment. The basic sequence is repeated twice, once with the phase of the last 90º X pulse set to x and once with the phase to -x. Data addition or data subtraction allows to select for selection or filtering purposes. On the other hand, if the filter is placed instead of the last 90º 1H pulse, a 2D w2-X-filtered/edited NOESY is obtained. Alternatively, the above X half-filter can be incorporated into the same experiment (the so-called double half-filter) giving a 2D w1,w2-X-filtered/edited NOESY experiment.
In such experiment, two editing pulses are present and, therefore, four different data acquisitions are performed that can be combinated in four different ways:
In addition of the above possibilities, the X-half filter is equivalent to the 1D HMQC experiment:
- Selection of 1H-13C magnetization in both F1 and F2 dimensions that allows to study intramolecular NOEs of the 13C-labeled protein.
- Selection of 1H-12C magnetization in both F1 and F2 dimensions that allows to study intramolecular NOEs of the unlabeled ligand.
- Selection of 1H-12C magnetization in the F1 dimension and 1H-13C magnetization in the F2 dimension that allows to study intermolecular NOEs between the protein and the ligand.
- Selection of 1H-13C magnetization in the F1 dimension and 1H-12C magnetization in the F2 dimension that also allows to study intermolecular NOEs between the protein and the ligand.
Thus, it is very easy to incorporate a variable evolution period between the two 90º X pulses. A series of widely used 3D and 4D experiments result of this approach:
- 3D HMQC-NOESY experiment.
- 3D NOESY-HMQC experiment.
- 3D HMQC-NOESY-HMQC experiment.
- 4D HMQC-NOESY-HMQC experiment.
Easy implementation on AVANCE spectrometers.EXPERIMENTAL DETAILS
The interpulse delay is optimized to 1/(2*1JCH) and the last 90º X pulse must be cycled with the receiver.SYNTAX
The most standard way to implement a refocused X half-filter in a pulse program is:RELATED TOPICSin which:
...
d2
(p3 ph1):f2
p2 ph2
(p3 ph3):f2
d2
...
- d2 is the evolution period optimized to 1/(2*1JXH)
- p2 is the 180º 1H pulse (in microseconds) applied at a power level pl1 from the f1 channel.
- p3 is the 90º 13C pulse (in microseconds) applied at a power level pl2 from the f2 channel.
- All phase may be specified at the end of pulse program. In particular, ph3 must be cycled according to the receiver phase (p31=0) (ph3=0 0 or 0 2).
However, during the relaxation delay a series of perturbations can be applied in order to achieve an specific goal.
Several related isotope filters have been described ( 00PROG323 ):
