Isotope editing

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. 

These low-temperature amide V IR and VCD isotope-edited results could be modeled with near-quantitative accuracy with DFT parameters by transferring ab initio FF, APT, and AAT parameters from computations for an a -helical heptapeptide model compound onto an o -helical 20-mer oligopeptide (Fig. 8 left). This simulation does not agree with data for the C-terminally labeled oligomer, because experimentally that end of... 

Stoner-Ma D, Melief EH, Nappa J et al (2006) Proton relay reaction in green fluorescent protein (GFP) polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP. J Phys Chem B 110 22009-22018... 

DPFGSE sequence with double tuned filters has been first proposed by Ogura et al. These filters demonstrate very high filtering efficiency for isotope-filtered, isotope-edited NOESY spectra.40... 

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). 

A. Starzyk, W. Barber Armstrong, M. Sridharan, and S. M. Decatur, Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2 trifluoroethanol An isotope edited FTIR study. Biochemistry 44, 369 376 (2005). 

In general, the concentrations of biomolecular NMR samples are in the 0.1-1 mM range, so that the direct detection of isotope-edited spectra of low y nuclei (13C, 15N) would suffer from very low sensitivity. In addition, the observation of the unlabeled moi-... 

Fig. 17.1 The concept of isotope filtering and isotope editing, in an idealized way (for a more realistic picture, see Fig. 17.21). In a complex consisting of two (or more) differentially labeled compounds (left), the NMR spectra could be consider-...

Isotope editing means selecting protons which are directly bound to an NMR-active heteronucleus (xH-13C, H-15N). 

Fig. 17.2 A realistic picture of the possibilities and limitations of isotope filtering and isotope editing, shown using the example of 15N. The selection of the isotope-labeled moiety is not significantly perturbed by the low level of 15N natural abundance (center). However, in the 15N-filtered case (right), only the 15N-bound protons of the...

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. 

When combining isotope filtering/editing with coherence transfer steps to multidimensional experiments, then further size restrictions apply. For example, isotope edited / filtered H TOCSY or COSY experiments are generally limited to systems of <10 kDa, because of their sensitivity to T2 relaxation. In larger systems, heteronuclear correlation spectroscopy can be used for the correspondingly labeled component, while structural information about both the labeled and unlabeled moiety can be extracted from isotope edi-ted/filtered NOESY spectra, respectively. 

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. 

In case (1), isotope editing will select all protons bound to the chosen NMR-active hetero-nuclear isotope, 15N or 13C. In first approximation (neglecting different line widths and relaxation rates for the two components), the intensity ratio between the desired signals of the labeled moiety and the unwanted signals of the other component can be described by... 

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]. 

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. 

With another immunophilin, FK binding protein (FKBP), experiments were performed using isotope editing of the [U-13C]-labeled inhibitor ascomycin (bound to unlabeled FKBP) [34], as well as by isotope filtering with unlabeled ascomycin derivatives (bound to labeled FKBP) [35],... 

For example, cells of E. coli can be grown on a minimal medium containing [15N] NH4C1. Since 13C can also be introduced in a similar way it is possible to incorporate both isomers simultaneously. Production of uniformly labeled protein containing 15N and / or 13C provides the basis for multidimensional isotope-edited spectra necessary for protein structure determination (next section) and for study of tautomerization of histidine rings (Eq. 2-6) 460/462-464 15N chemical shifts of groups in proteins are spread over a broad range (Table 3-3).465... 

Of great importance in the determination of protein structures is the use of 15N- or 13C-enriched samples to obtain isotope-edited spectra. For example in HSQC or in 15N-multiple quantum cohenence (HMQC) spectra we see only NH protons in a plot of H chemical shift in one dimension versus the 15N chemical shift of the attached... 

Sarkar et al. [32] report the use of MAS for single-bead NMR . A simple molecule on Wang resin 1 (Fig. 3) was fully 13C-labeled. A 13C-filtered H NMR spectrum was then recorded for a single bead. The isotope editing eliminates the interferences from solvents and contaminants, allowing the signal to be observed. The only information obtained is the resonance for the methyl protons directly attached to the labeled carbon, making this experiment mainly a novelty. 

Ligand- macromolecule complex Stoichiometry of complex Kinetics of binding Location of interacting sites Orientation of bound ligand Structure of complex Dynamics of complex Chemical shift titration Line width, titration analysis HSQC, isotope editing NOE docking 3D/4D NMR Relaxation time measurements... 

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