Isotopic labeling double labeled proteins

A second approach that can be adopted to overcome the intrinsic requirement for cysteine at the N-terminus of C-terminal fragment utilizes the enzyme subtiligase, a double mutant of subtilisin, which is able to join two unprotected peptides. Thioester-modified proteins were shown to present good substrates of subtiligase [65]. However, although this approach could be potentially useful for general isotope labeling, the efficiency of this process remains to be proven. 

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]. 

The most common technique used for heteronuclear distance measurement is rotational-echo double resonance - REDOR [239,240,241]. It is now relatively commonplace to label proteins with the spin-1/2 isotopes and N, and C-N distance constraints can be derived using REDOR [242, 243] and used to determine elements of secondary structure. More importantly for molecules of interest to the pharmaceutical industry is the measurement of distances between and the quadmpolar deuterium ( H) nucleus [244]. Introduction of the H isotope is relatively straightforward and the measurement of distances at natural abundance over several bonds can be used to provide torsional constraints [245]. The measurement of distances (up to 6.8 A)... 

Later, Ong et al. introduced a method termed SILAC (stable isotope labeling by amino acids in cell). In SILAC, two cell-culture populations are grown under identical conditions, except that one is supplied with the labeled amino acids (e.g. arginine with six atoms) and the other is with the non-labeled. After five or six doublings, two kinds of amino acids have fully incorporated into proteins. Every peptide pair is separated by the mass shift by the labeled amino acid. This approach cannot be applied to tissues or body fluids, and is... 

DNP enhancement was also used to investigate 40 nmol of a 25-residue signal peptide bound to the lipid-reconstituted 600-residue protein translocation complex SecY translocon [229]. Although double quantum filtering was mandatory to suppress the large natural abundance SecY background, a decent 2D-spectrum of the peptide could be obtained within 20 h of measurement time. For three of four isotope labeled amino acids the spin system could be identified by a sequential walk. The corresponding secondary chemical shifts were indicative of an a-helical secondary structure of the peptide in its bound form. 

The development of NMR techniques for double-labeled material began in the late 1980s, and there are dozens of different types of experiments that exploit scalar couplings for assignment purposes. These experiments are called triple resonance experiments because they involve three different nuclear spins, H, C, and N (Ikura et al., 1990 Bax and Grzesiek, 1993). To perform experiments of this type, it is usually necessary to isotopically enrich the protein to 99% for the and N atoms. The goal of these experiments is to correlate intra- and inter-residue chemical shifts with the amide proton and nitrogen chemical shifts. 

Fig. 4. Double-label gel profiles of isolated mitochondria obtained during petite induction. Log-phase cultures of 55-R5-3C and 1121 were grown on 2% galactose for 5-6 doublings at 18°C (A), or at 28°C in the presence of 3 mg per ml of chloramphenicol (B), and were labeled with [ H]leucine or [ C]leucine in the presence of 200 Mg per ml cycloheximide. After labeling, mitochondria were isolated and the protein was separated on 10% SDS polyacrylamide gels. After electrophoresis, the gels were sliced and counted. The data are normalized to the total radioactivity recovered from the gels after correction for spillover of the two isotopes. (From Weislogel and Butow. )...

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