Sequence-specific assignments

Direct and indirect methods are used to make sequence specific assignments. The direct method is to use a number of larger than two-dimensional hetero NMR spectra to generate the heteronuclei-based interresidue backbone atom correlations. One of the schemes of the direct method is shown in Fig. 12. These three-dimensional NMR experiments need not only C- and N-enriched samples, but also very expen- 

The indirect method is based upon two-dimensional nuclear Over-hauser effect spectroscopy (NOESY) experiments. For each residue, the (amide proton), H , and are called backbone protons. Based upon the statistics of short proton-proton distances in protein crystal structures, Wuthrich and co-workers summarized the relationships between the 

TABLE III Statistics of Short h- h Distances in Protein Crystal Structures (after Wuthrich ) 

Type Distance (A) NOESY peak intensity Probability (%) (7-i) = l 

In Table III, denotes the distance from one H (amide proton) to the other residue s H , with the distance from one H to the other residue s H°, and d f the distance from one H to the other residue s H . When two NOESY evidences of df, d /, and d f are found at the same time, the probability increases as shown in Table IV. 

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The sequence-specific assignment, however, can be made from NOE spectra (see Figures 18.17 and 18.18b) that record signals from H atoms that are close together in space. In addition to the interactions between H atoms that are far apart in the sequence, these spectra also record interactions between H atoms from sequentially adjacent residues, specifically, interactions from the H atom attached to the main chain N of residue number i -r 1 to H atoms bonded to N, Ca, and Cp of residue number i (Figure 18.19a). 

Despite the vast amount of data on the pharmacological properties, very little about the conformation of the proteins has b n known until recent NMR studies. 2D-NMR results have provided detailed information about the secondary structure of several related anemone toxins. ATX I from Anemonia sulcata (3,4) and AP-A from Anthopleura xanthogrammica (5,6) have been studied by Gooley and Norton, and more recently Widmer et al. have further purified the A. sulcata toxins and obtained complete sequence specific assignments for ATX la (7). Our laboratory, on the other hand, has studied the structures of RpII and RpIII from RadiarUhiis paumotensis (8). 

Sequence-specific assignments were obtained by analysis of the NOESY and COSY spectra obtained under the similar conditions in H2O. The three important classes of NOE cross peaks for this purpose are designated d j, d j, and In... 

In the last section, we will focus on the suite of novel triple-resonance TROSY experiments designed for accomplishing the sequence-specific assignment of backbone nuclei in high molecular weight proteins. 

Enrichment of the 15N content has become part of various powerful research techniques. For example, uniform labeling with 1SN was used for sequence-specific assignments and secondary structure determination of certain proteins by NMR60 and tracing of complicated processes including the increase of DON in soil61,62. 

KinaseX samples that are residue type-specifically labeled with II Thr, [ H]Lys and I111 Met have been also produced and NOEs between these residues and inhibitors have been observed (data not shown). Since there is one threonine, one lysine and one methionine in the ADP binding pocket of kinaseX, sequence-specific assignments for these residues can be obtained directly by the observation of protein-inhibitor NOEs. 

Using DQF-COSY and TOCSY we can link all of the protons within a single spin system, which corresponds to a single amino acid residue. We can classify each spin system as a pattern of chemical shifts unique to one amino acid or as a member of a class AMX or five spin. In order to get sequence-specific assignments, however, we have to have some way to correlate protons in one residue to protons in the next residue in the sequence. For unlabeled proteins this is done by NOE interactions certain protons in one residue are constrained by the peptide bond to be close in space to certain protons in the next residue. These NOE correlations are called sequential or z, i + 1 because they correlate a proton in residue z with a proton in the next residue in the sequence, residue z + 1. Specifically, we expect to see NOE correlations between Ha of residue z and Hn of residue z + 1 (Fig. 12.15) and sometimes between the protons of residue z and the Hn of the next residue. Because the DQF-COSY and TOCSY spectra correlate protons within a residue, we can move from... 

SEQUENCE-SPECIFIC ASSIGNMENT USING HOMONUCLEAR 2D SPECTRA... 

Strip plots can only be constructed when the crosspeaks have already been assigned in the 2D HSQC spectrum. In a 15N-labeled protein, sequence-specific assignments come from sequential NOE (a,N, /3,N and N,N) crosspeaks located in the 3D HSQC-NOESY spectrum. The walk through the protein backbone is done in the same way as with unlabeled proteins, except that overlap in NOESY spectra is greatly reduced by spreading the crosspeaks out in the 15N dimension of a 3D spectrum. 

Once we have sequence-specific assignments, the strips from the HNCACB matrix can be arranged in order of residue number. Figure 12.69 (right) shows the strip plot for A-Cro... 

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