Selective isotopic labelling

Abstract To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues. 

Staunton D, Schlinkert R, Zanetti G, Colebrook SA, Campbell LD (2006) Cell-free expression and selective isotope labelling in protein NMR. Magn Reson Chem 44 S2-9... 

Tab. 1.1 Chemical structures of metabolites involved in selective isotope labeling strategies (from Ref. [14] with kind permission)...

These monomers produce a single disyndiotactic polymer characterized by alternating erythro and threo relationships between adjacent substituents, 18 or 19. In such a polymer it is not possible—unless one turns to selective isotopic labeling—to determine whether the erythro and threo relationship refers to the substituents A and B of the same monomer unit or to those of two successive monomeric units. 

By definition, it is necessary to recognize geometric isomers, isotopomers and stereoisomers as distinct species. Moreover, there is the pragmatic issue that regio-selectivity, isotopic labeling and stereo-chemical investigations are three very important avenues of mechanistic enquiry. 

R. Bernstein, S.M. Thornberg, R.A. Assink, A.N. Irwin, J.M. Hochrein, J.R. Brown, D.K. Derzon, S.B. Klamo, and R.L. Clough, The origins of volatile oxidation products in the thermal degradation of polypropylene, identified by selective isotopic labeling. Polym. Degrad. Stab., 92, 2076-2094 (2007). 

Due to the wave-particle duality of neutrons, they can be reflected and refracted in a manner similar to light. Reflected neutron beams can interfere with each other to produce a reflected beam intensity that is characteristic of the reflecting material (Lekner, 1987). Detailed analysis of the reflectivity is able to able to provide information on the structural organization normal to the surface on which the beam is incident. Neutron reflectometry is particularly useful (vis a vis x-ray reflectometry), since selective isotopic labeling can be used to highlight particular regions of interest in a surface structure. This is especially valuable for monolayers on surfaces. 

Methylases specific for each of the four bases of sRNA (97) and for adenine and cytosine of DNA (98) are available. Selective modification of a nucleic acid or its oligonucleotide fragments before digestion with a specific nuclease gives additional dimensions for selective degradation. Use of C-methyl labelled S-adenosylmethionine permits selective isotopic labelling by these enzymes. 

Another way of overcoming spectral overlap for proteins is selective isotope labeling of one or several amino acid types, which results in less-crowded H- N spectra with cross-peaks from the labeled residues only. The same is possible for RNA where nucleotides can be selectively isotope enriched. In addition, methods for segmental labeling of parts of the biomolecules have been developed for proteins (48, 49) and for RNA (50). 

Dynamic line shape combined with selective isotopic labeling has found numerous applications, in particular, in polymer science (mainly for biopolymers (both... 

Beimani, Y.L., Marron, K.S., Mais, D.E., Flatten, K., Nadzan, A.M., and Boehm, M.F., Synthesis and characterization of a highly potent and selective isotopically labeled retinoic acid receptor ligand, ALRT1550, J. Org. Chem., 63, 543, 1998. 

As mentioned above, the determination of atomic level structure, i.e., the backbone torsion angles for an oriented protein fiber, is possible by using both solid-state NMR method described here and specifically isotope labeling. This is basically to obtain the angle information. Another structural parameter is distance between the nuclei for atomic coordinate determination. The observation of Nuclear Overhauser Enhancements (NOEs) between hydrogen atoms is a well known technique to determine the atomic coordinates of proteins in solution [14]. In the field of solid-state NMR, REDOR (rotational echo double resonance) for detection of weak heteronuclear dipole interactions, such as those due to C and N nuclei [15, 16] or R (rotational resonance) for detection of the distance between homonuclei, are typical methods for internuclear distance determination [17,18]. The REDOR technique has been applied to structure determination of a silk fibroin model compound [19]. In general, this does not require orientation of the samples in the analysis, but selective isotope labeling between specified nuclear pairs in the samples is required which frequently becomes a problem. A review of these approaches has appeared elsewhere [16]. 

Muir et al. [217,218] also reported a convenient in vitro chemical ligation strategy that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product [219]. 

In the rotational-echo double-resonance (REDOR) [60—62] technique, the distance between two heteronuclei is determined by comparing the signal intensity in two closely related experiments. The interpretation of the experimental results assumes the existence of isolated spin pairs, and there is thus usually a requirement for selective isotopic labelling at the two sites, the distance between which is of interest. 

In an extension to experiments which measure internuclear distances, Levitt and co-workers and Griffin and co-workers have presented ingenious methods which allow the measurement of torsional angles [148, 149]. The methods involve the creation of MQC between a pair of nuclei (selective isotopic labelling is required), which may be homonuclear, e. g. or heteronudear, e. g. A spin-... 

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