Proteins methyl-labelled samples

We have shown previously that the resolution of triple resonance experiments can be dramatically enhanced with random NUS in the indirect dimensions, and high-resolution 3D or 4D triple-resonance spectra of large proteins can be recorded within a few days, which would otherwise require months of instrument time with US [32, 35]. This works rather well with triple resonance experiments where peaks have similar intensities and there is not much of a sensitivity and dynamic range issue. In fact, if there are primarily strong signals, such as methyl peaks in ILV labeled samples, a straight Fourier transformation of the NUS data where the... 

IspG is a protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, Oldfield et al. have specifically assigned the hyperfine interactions in a reaction intermediate (144), which was created as a result of unusual 4Fe-4S cluster containing protein IspG catalysed reduction of 2-C-methyl-erythritol-cyc/o-2,4-diphosphate (143) to ( )-l-hydroxy-2-methyl-but-2-enyl-4-diphosphate (145) and then to dimethylallyl diphosphate (146) and isopentenyl diphosphate (147) (Scheme 39). " ... 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

Fig. 1. H- - C CT-HSQC spectrum of a sample of 1.5mM Val, Leu, lie (51) methyl-protonated maltose-binding protein (MBP), 2mM /3-cyclodextrin, 20 mM sodium phosphate (pH 7.2), 3mM NaN(, 200pM EDTA, 0. 1 mg/ml Pefabloc, 1 /ig//d pepstatin and 10% D20 recorded at 37°C, on a Varian Unity-1- 500-MHz spectrometer. Acquisition times of 28 and 64 ms were employed (/, t2) along with a relaxation delay of 1.5 s, fora total measuring time of 3 h. (a) Aliphatic region of the H- - C correlation map of MBP, illustrating the selectivity of labelling. Small amounts of residual protonation are observed at the Cy positions of a number of Pro/Arg residues, the Cp positions of Asp and Ser (aliased) residues, and the Cy2 methyl positions of lie. In all cases, intensities of these cross-peaks are less than 10% of the methyl peaks, (b) Methyl region of the H- - C HSQC. Reproduced with permission from Kluwer Academic Publishers Goto et al.H...

This approach, also known as ChIP-on-chip, has been the most used technique to establish histone-maps. Briefly, as we have been detailing, the chromatin fragments are incubated in the presence of an antibody that recognizes a specific histone modification (e.g., methylation on histone 3 or acetylation on histone 4, etc.) (Figure 4). Next, the protein-DNA complex is immunoprecipitated. After reversal of the cross-link, ChIP-enriched DNA and control DNA are amplified by PCR and labeled with fluorescent dyes (Cy3 and Cy5). Finally, the samples are hybridized onto a specific microarray. The ratio of the Cy5 to Cy3 intensities measured for each DNA sequence in the array is a measure of the amount of a specific histone bound to the DNA. 

---