Assignment of Large Proteins 15 to 25 kDa

In this case, it is absolutely necessary to label the protein with both C and N. The larger number of residues increase the complexity of N separated three-dimensional spectra such that it is difficult to obtain the assignments by use of dipolar coupling between protons on adjacent residues because of the potentially misleading dipolar coupling to nonadjacent residues. Consequently, it is necessary to employ scalar coupling across the peptide bond to obtain the assigmnents. 

This greatly simplifies the assignment task because scalar coupling only occurs through chemical bonds. 

Doubly labeled protein is obtained in a similar manner to N-labeled protein. The bacterial cell line expressing the protein are simply grown in minimal media containing N ammonia and glucose. The cost of this media is considerably more than the N-labeled media, approximately 1000 per liter. It is often advantageous to have a separate sample that is labeled with alone because the coupling increases the line width of the resonances, decreasing the resolution of the separated NOESY and TOCSY spectra. Thus both a N-labeled and a C- N-labeled sample are desirable. 

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. 

There are several problems with this approach. First, because prolines lack an amide proton, they do not appear in the spectrum. Consequently, they break the sequential linking of the a-carbon chemical shifts. Second, if two or more residues have the same a-carbon shift (i.e., they are degenerate), it is not possible to unambiguously determine which residue should be the preceding one. Consequently, it is necessary to perform additional experiments that associate other inter- and intra- 

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