Amide dipolar coupling

However, if side-chain carbon assignments are wanted, C(CC)(CO)NH experiments [33] that start directly with carbon magnetization and transfer it further to the amide proton for detection are available. If protonated substituents, for example methyl groups, have been introduced into the otherwise perdeuterated protein, the usual HC(C)(CO)NH-TOCSY pulse sequence can be used to obtain the proton chemical shifts. These protons can provide a small number of NOEs that, together with residual dipolar couplings and the secondary structure identification from chemical shifts, make the determination of the global fold of large proteins possible. 

Fig. 8.1 Orientation of two dipolar coupling vectors in a protein segment. The vectors connect the amide Hn and 15N atoms. In this case the interaction vector coincides with the chemical bond. The axis system of the alignment tensor is designated as A, Aw Aa. The angles ( n, y>n, and 02, define the orientation of the two dipolar vectors with respect to the alignment tensor. (Reproduced with permission from N. Tjandra, Structure 1999, 7, R205-R211.)...

Fig. 8.2 Ori entations of an amide NH dipolar coupling bond-vector of the protein ubiquitin. Each cone of orientations is compatible with two different alignment directions adopted by the protein in two different alignment media. The central lines defining each cone correspond to the orientations obtained from the measured dipolar couplings. The outer lines include orientations that are possible if the dipolar coupling values are either increased or decreased by 1 Hz. The angle at which the two cones intersect is defined by ft. The solid dot at the cone intersection determines the orientation of the dipolar coupling vector. (Reproduced with permission from B. E. Ramirez and A. Bax, J. Am. Chem. Soc. 1998, 720, 9106-9107.)...

The two-bond HNC dipolar coupling is observable in a 15N-HSQC experiment in which the J coupling between the carbonyl atom C and the 15N amide is active. The doublet components in the 15 N dimension that represent the C N coupling are displaced with respect to one other in the H dimension as in an E.COSY [39] because of this two-bond coupling. 

NMR data [95]. This new method requires two sets of dipolar couplings from two different protein orientations. Together with the backbone dipolar couplings that are typically used (i.e., amide NH, C N, CaC, CaHa and the two-bond HNC ), CaCp dipolar couplings are also needed. Provided that the orientation of one peptide plane is known independently, the dipolar coupling data give rise to two possible orientations for the subsequent peptide plane, where the conformations about the alpha carbon in these two orientations are mirror images. One of the conformations can be ruled out because of chirality. 

Conformationally heterogeneous states of proteins can be determined using NMR-derived structural data and suitable molecular dynamics techniques.100 Residual dipolar couplings have been shown to represent motions in ubiquitin slower than its correlation time.101 Using an extensive set of residual dipolar couplings, namely, 36 sets of amide NH, 6 sets of HNC and NC as well as 11... 

Concatenation of the 3H—15N HSQC (or HMQC) sequence with a JH—JH NOESY gives rise to the 3D 15N-edited NOESY-HSQC (or 3D NOESY-HMQC) experiment.66-68 Here, two of the frequency dimensions represent the amide JH and 15N chemical shifts, while the third dimension provides information about the chemical shift of protons with which each amide proton is dipolar coupled (i.e., separated by <5.5 A). The spectrum is routinely viewed as narrow 2D (JH—JH) strips taken at the 15N chemical shift of each crosspeak in the JH—15N HSQC spectrum (see Figure 14). 

Two-dimensional PISEMA spectra experimentally obtained from powder samples are analyzed by comparison with simulations to determine Euler angles that define the orientation of the CSA tensor in the molecular frame, while the magnitudes of the CSA tensor is measured from a ID chemical shift powder pattern. Some examples of PISEMA spectra of powder samples simulated using SIMPSON program 5 are shown in Fig. 6. PISEMA spectra correlating the N chemical shift and the ll 5r dipolar coupling interactions associated with an amide site of a peptide (Fig. 6(A)) and with the N -H bond of the imidazole ring in histidine (Fig. 6(B)) were simulated. 

Fig. 6. Simulated 2D PISEMA spectra of powder samples. Correlation of dipolar coupling and chemical shift interactions associated with an amide N-H bond (A) and the histidine side chain Njt-H bond (B). For the amide N-H bond, the simulations were performed using CSA principal values of 33 = 64, 22 = 77, and Sii = 217 ppm, an N-H bond length of 1.07 A, and the Euler angles ( = () and yg=17°) to define the relative orientations of the chemical shift and dipolar coupling tensors. For the histidine side chain Nrt-H bond, the following parameters were used in the simulations CSA principal values of 533 = 77, 522 = 203, and 5 =260 ppm,...

Fig. 16. Simulated amide H- N dipolar couplings as a function of the residue...

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