Half-filter

Figure 6 X-half filters used for filtering or selecting 13C and 15N-attached protons. Thick and thin closed rectangles are 180° and 90° pulses, respectively, open rectangles are spin lock pulses. (A) A simple X-half filter (2). The delay t is equal to 0/(2[1JXH]) where 1JXH is the one-bond coupling between proton and either 13C (120 to 140 Hz) or 15N (95 Hz). The second 90° pulse is the editing...

This scheme is applied in the so-called X-half-filter technique (Fig. 17.4a, c), with the only difference of an additional 90 (X) pulse with constant phase [16, 17]. Instead of generating heteronuclear multiple quantum coherence 2 Iy Sy, which cannot readily be detected (in case of selecting the 1H-X pairs), one now always ends up with proton antiphase coherence, but with the same phase alternation ... 

The single filter elements (as shown in Figs. 17.4a-c) are often called X-halffilters since each of them acts only in one dimension of a 2D experiment, to be distinguished from Xfilters that select (or suppress) 1H-X pairs in both dimensions of a 2D experiment [17, 20, 21]. Of course, X-half filters can be employed twice in a 2D experiment, to yield isotope selection in both dimensions (see Sect. 17.3.3). 

Fig. 17.4 Common filter elements a X-half filter based on X pulse phase cycling [16, 17], b X-half filter with purge gradient [18], c X-half filter as in a, but with refocusing period for the hetero-nuclear antiphase magnetization [16, 17]. Sequences d [22], e [23] and f [18] show double filters based on single filter elements the delays r and r can be set to slightly different values to cover a broader range of ]J coupling constants (see text for a more detailed description).

The simplest double tuned filter can be constructed by a concatenation of two X-half filters and removal of redundant 180° pulse pairs (Fig. 17.4d) [22]. Alternatively, it can also be realized by keeping the 180° pulse pairs and adding short spin-lock periods (to dephase the 1H-13C magnetization which is orthogonal to the spin-lock axis, Fig. 17.4e) [23], or it is based on the gradient-purging scheme of Fig. 17.4b, resulting in the double filter shown in Fig. 17.4f [18]. 

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

Selecting the C-bound protons before performing a homonuclear two-dimensional experiment enables to measure small heteronuclear coupling constants [16]. Such an experiment with a sample of natural isotopic abundance was first published by Otting and Wuthrich in 1990, where the half-filter element with spin-lock purge pulse was used to select the C-bound protons in a small protein in aqueous solution [6]. Later applications illustrated the usefulness of the same half-filter element with smaller molecules [17, 18]. 

As an illustration of the use of the half-filter element for the measurement of small coupling constants, consider the TOCSY experiment... 

The purpose of the C(o i)-half-filter is to start the TOCSY experiment only with the magnetization of protons bound to C. No further C pulses are applied after the start of the evolution time t. For the description of the multiplet fine-structure of the resulting cross-peaks, it is instructive to consider a 3-spin system with the operators H, and C denoting the spins of two protons and one carbon. Starting from antiphase magnetiza-... 

Figure 4 shows a TOCSY spectrum with C(wi)-half-filter recorded with the small globular protein bovine pancreatic trypsin inhibitor (BPTI) using the pulse sequence of fig. 3. Although proton multiplets are usually difficult... 

In order to determine couplings to nuclei in natural abundance, it is necessary to suppress the signals of protons that are not bonded to a magnetically active heteronucleus. An (iix hetero half-filter that selects for such nuclei in F, via the phase cycle was used for this purpose. Presaturation of the protons bonded to 12C by the BIRD pulse allows a rapid pulse-sequence (2 scans per second). The resulting 2D spectra are TOCSY spectra in which the cross peaks show the desired E. COSY pattern. From the results shown, the only limitation seems to be the resolution obtained, although the authors do not hesitate to use a third heteronuclear frequency domain for improvement. 

Dalvit C, Cottens S, Ramage P, Hommel U, Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins, J. Biomol. NMR, 13 43-50, 1999. 

Otting G, et al. Editing of 2D IH NMR spectra using X half-filters, combined use with residue-selective 15N labeling of proteins. J. Magnet. Reson. (1969) 1986 70 500-505. 

Another possibility is to use half-edited/ half-filtered 2D experiments to detect NOEs that specifically involve interactions between protons attached to or and those that are not. This approach is used, for example, to detect intermolecular NOEs between a labeled protein and an unlabeled ligand. Examples of isotope editing/filtering are given in section 3.2.4. 

---