Medium And Longrange Noe Correlations

So far we have not learned anything from the NOE data except to assign all of the protons. This is an essential first step, but the real goal is to extract distances between specific pairs of protons from the NOE data. Sequential NOEs are not very useful because we already know that an amino acid residue is close in space to its nearest neighbor in the sequence. The real "mother lode" of the NOESY data consists of the medium-range (i ^ i + 2, i ^ i + 3 and i ^ i + 4) and long-range connectivities. These define the secondary structure

Figure 12.28

Figure 12.28

(a-helix and ^-sheet) elements and the precise spatial relationships between them, which constitute the tertiary structure. Figure 12.28 shows the NOESY spectrum of Heregulin-a EGF domain with the assignments indicated for the three fragments assigned earlier. The CSI data (Fig. 12.14) predicts these three sections (3-5, 19-23, and 33-37) to be in an extended or ^-strand conformation. Weak long-range crosspeaks are also indicated, connecting one strand with another (34-21,20-35,3-24,21-4, and 23-4). These long-range correlations indicate that the strands are antiparallel because increasing the residue number by one (34 to 35) decreases the residue number on the other strand by one (21 to 20). The precise alignment of the ^-strands can be determined from the Ha to Ha long-range NOE correlations. Figure 12.29 shows the F2 = Ha, F1 = H a region of the D2O NOESY spectrum. To see the Ha protons in the F2 dimension the experiment is performed in D2O because the Ha chemical shifts are close to the water resonance. The Ha to Ha NOE interactions are directly across the interface between strands in the ^-sheet, so the alignment of strands in unambiguous: Leu-3 aligns with Val-23 and Cys-20 aligns with Cys-34 (Fig. 12.30). Now we have a complete 2D picture of the major ^-sheet part of the protein. Figure 12.30 summarizes a large body of information that supports the ^-sheet structure. A large number of cross-strand Ha-Ha and Ha-HN NOEs are observed, and hydrogen bonds are implied by a slow exchange of Hn with Dn when the protein is dissolved in D2O. Finally, the temperature coefficient (change in HN chemical shift with a temperature change) measures the degree of exposure of the HN to solvent. Amide protons with a small coefficient are probably buried in the protein interior with little access to solvent. In proteins with a well-defined single conformation this can be used as a criterion for hydrogen bonding. In peptides, it is not as useful because the equilibrium between a "folded" peptide and a random coil form is shifted as the temperature is raised, and this also contributes to the temperature coefficient.

Deuterium exchange is measured by recording a series of fast TOCSY experiments (30 min each) immediately after dissolving the protein in D2O. As the amide protons

Figure 12.29

exchange with solvent they are replaced with 2H and disappear from the spectrum. Figure 12.31 shows the TOCSY spectrum in 90% H2O (left) and the first TOCSY spectrum acquired in D2O (right). Already the majority of HN-H crosspeaks has disappeared and those that remain are the "buried" HN protons. The rate of loss of the HN-Ha crosspeaks can be quantified by measuring the crosspeak volume at each time point and plotting against time (Fig. 12.32). The loss of signal is exponential, and curve-fitting yields the rate constant (or half-life) for the exchange. In the plots shown, the half-lives range from just slow enough to observe (Val-4, 19 min) to very long-lived (F21, 7.4 h). These can be compared to the inherent (random coil) exchange rates for each of the 20 amino acids to obtain a rate ratio or "protection factor" that indicates the degree of "burial" of the HN proton in the protein core.

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