Isotope-editing techniques

With a 13C label at the methide center, the presence of reactive methide intermediate can be verified and complex reaction products can be inventoried and eventually identified. The only limitations are the synthesis and cost involved in incorporation of the 13C label. As a rule we, only use 13C-labeled dimethylformamide and NaCN as starting materials because of their low cost and availability. Another limitation of enriched 13C-NMR monitoring is dilution of the enriched label to natural abundance levels. Currently, we are developing isotope-editing techniques that utilize unnatural 13C double labels to solve this problem. 

If a ligand is available in isotope-labeled form, then the use of standard isotope-edited techniques (preferentially with at least one heteronuclear dimension) will allow straightforward access to its structure in the bound state without having to solve the much more complex problem of the protein structure. 

Gemmecker, G. Isotope filter and editing techniques. In BioNMR in Drug Research (Methods and Principles in Medicinal Chemistry), Zerbe, O. (eds.), Wiley-VCH, Weinheim, 2003, Vol. 16, 373-390. 

In addition to traditional X-ray techniques to study silk (Bram etal., 1997 Lotz and Cesari, 1979 Riekel et al., 1999a Warwicker, 1960), other structural tools have helped unravel various aspects of silk protein conformation. These include solid-state NMR (Asakura et al., 1983, 1988, 1994 Beek et al., 2000, 2002) studies of native and regenerated silk together with and studies of isotopically edited silks, which have dramatically improved the model of structure distribution within silk fibers (Beek et al., 2000, 2002). 

The basic idea of isotope filtering/editing techniques is to selectively observe the subspectra of the labeled or unlabeled components only. A prerequisite for the application is of course the different isotopic composition of the different components. In an ideal case, we will have one compound which is completely labeled with the NMR active iso-tope(s) of one (or several) elements, and the other compound completely devoid of these isotopes (see Fig. 17.1). 

In the isotope edited/ filtered spectra of a protein-ligand complex, the species actually observed is generally the complex itself. This is an important difference from transferred NOE or saturation difference techniques, where the existence of an equilibrium between free and bound species - and a certain rate of exchange between them - is essential (Chapts. 13 and 16). The general conditions for isotope filtering/editing are therefore identical to those required for standard protein NMR sample concentrations are usually limited by availability and solubility of the components to the order of 1 mM. Considerably lower concentrations will reduce the sensitivity of the experiments to unacceptable levels,... 

Size restrictions are again similar to that encountered in protein NMR up to ca. 20-30 kDa complex size, line widths will usually be acceptable to allow detection of the XH resonances of the labeled or unlabeled component, including NOESY transfer steps. Beyond that, deuteration of the protein component becomes essential to reduce excessive line broadening. For the observation of the (15N-) labeled component (i.e., isotope editing), the use of TROSY techniques will further extend the size limit (Chapt. 10) however, this approach does not work for unlabeled components. 

In the case of a symmetric (protein-protein) homodimer, the preparation of molecules with differently labeled monomers is often far from trivial, and special approaches have been described in the literature [8, 9]. However, when successful, differential isotopic labeling of a symmetric homodimer, in combination with isotope editing/filtering techniques, offers a unique access to the NMR investigation of the monomer interfaces. 

The most straightforward isotope-editing method for selecting protons bound to a heteronucleus and suppressing all others is the simple acquisition of a spectrum with an indirect heteronuclear dimension (in the literature the term isotope editing is often used as a synonym for these techniques). This can be accomplished by a simple 2D HMQC or HSQC shift correlation, or a more elaborate 3D technique including an additional NOESY or TOCSY step (3D X-edited NOESY/TOCSY etc.), or even 4D experiments with a second heteronuclear shift dimension [13, 14]. 

Obviously, this approach cannot be used for selecting the nonisotope-labeled components. In the following we will consider isotope filtering/editing techniques that do not use heteronuclear chemical shift evolution. 

How gases are transferred, distilled, or otherwise processed in vacuum lines is briefly discussed under the different elements. A more detailed description can be found in the recently published Handbook of Stable Isotope Analytical Techniques, edited by de Groot (2004). 

Pearman, G. I., in World Meteorological Organization Global Atmosphere Watch, Report of the Seventh WMO Meeting of Experts on Carbon Dioxide Concentration and Isotopic Measurement Techniques, WMO TD 669, No. 88, Rome, Italy, 1993, edited by G. I. Pearman, PP 104-104. 

Grdning, M. (2004) International stable isotope reference materials. In Handbook of Stable Isotope Analytical Techniques, Vol. 1, edited by de Groot, PA. Amsterdam, The Netherlands Elsevier, pp. 874-906. 

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