NOE contacts

In principle, there are different ways to characterize the complexes On one hand, in favourable cases, it is possible to measure the NMR parameters of the receptor in the free and bound states, i.e., differences in chemical shift and NOE-contacts, before and after the formation of the complex. On the other hand, it is usually more feasible to observe and detect changes in the ligand NMR signals before and after the binding process. In this latter case, the use of STD (saturation transfer difference) and TR-NOE (transferred NOE) experiments is of paramount importance. 

In a multidomain protein whose domains have fixed orientations relative to each other, a unique alignment tensor will represent the preferred orientation of all the domains in the anisotropic environment. Therefore, structure refinement with dipolar couplings is performed as in one-domain proteins (Sect. 8.4). Several examples are reported in the literature of cases with conformational ambiguity due to the lack of NOE contacts between the domains. One example is the determination of subdomain orientation of the riboso-mal protein S4 z)41 [97]. In this work the lack of NOE contacts between the domains produces an ambiguity in interdomain orientation. The authors use two different anisotropic media to obtain dipolar couplings (DMPC/DHPC bicelles and Pfl filamentous bacteriophages). They conclude that subdomain orientation in solution is similar to the one present in the crystal structure. 

In each case, there was only one set of conformations in the final stage that correctly reproduced the observed NOESY contacts (see Table IV). These are the Aj conformations for X-S and the A2 conformations for G-X. Apparently the other conformations represent models that are trapped in local energy minima. Since they do not satisfy the observed NOE contacts, these other conformations (Bj, Cj and B2) are ignored in the remainder of the analysis. 

By reversing the order of polarization transfer steps, NOE contacts of signals hidden under the bulk of other resonances can be established in a ID TOCSY-NOESY [39] experiment (fig. 1(d)). The necessary requirement is that an efficient TOCSY transfer can be made to a particular proton from... 

The NOESY and TOCSY polarization transfers can also be arranged so that two NOESY steps are interrupted by one TOCSY transfer. This is useful for situations when a proton which is intended as a starting point for a ID TOCSY-NOESY experiment cannot be selectively excited, nevertheless it has a NOE contact to an isolated proton. The ID NOESY-TOCSY-NOESY sequence [72] (fig. 4(b)) is obtained by appending another NOESY step to the ID NOESY-TOCSY pulse sequence of fig. 1(c). The last NOESY step can be either selective or nonselective depending whether a selective 180° pulse is applied after the nonselective 90° pulse at the end of the TOCSY transfer. 

As illustrated on this example, interpretation of NOESY spectra of polysaccharides might be complicated by the presence of spin-diffusion and higher order elfects. ID NOESY-NOESY experiment can provide some assistance in identification and assignment of spin-diffusion signals and thus prolong networks of spins experiencing NOE contacts beyond immediate neighbours normally detected in NOESY experiments. 

Figure 9.3 (a) Duplex 3 4 consisting of two different strands with complementary H bonding sequences, (b) Duplex 5 5 consisting of two identical single strands with a self-complementary H bonding sequence. Interstrand NOE contacts revealed by NOESY are indicated by arrows. 

Lee, W., Revington, M. J., Arrowsmith, C. and Kay, L. E. (1994). A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts... 

Fig. 19. Filtered NOESY spectra with intermolecular NOE contacts between the protein CaM and the bound peptide C20W. The intermolecular NOEs can be detected exclusively due to the different labelling of the peptide and the protein.

Eig. 33. NOE-Contact plot ofCaM complexed to the peptide C20W. The thick diagonals indicate a-hehces, the antidiagonal /3-sheets. 

NOESY walks. For the assignment of non-labile H NMR resonances in B-DNA it is standard practice to employ two major NOE pathways, specifically the sugar HI <-> base H6/H8 and sugar H2 /H2" <-> base H6/H8 pathways (13). In all of the a-containing decamers we observe that the Hr network remains intact, as in the control. However, in each case the Hl /Hl" pathway is broken between the a-nucleotide and the subsequent residue. This effect is due to a flip in the orientation of deoxyribose ring of the a-nucleotide, which places the H2" proton too far away from the base moiety in the following residue to make an NOE contact (Figure 7 also see Models below). 

The most important technique for determining binding constants in modem supramolecular chemistry is undoubtedly NMR, particularly H NMR. Several factors contribute to the popularity of NMR the quality of information regarding the host-guest interactions (shifts in spectra, nOe-contacts, etc.) and availability being the most important ones (see also NMR Spectroscopy in Solution, Techniques). 

Figure 4 NMR spectrum of the calix[4]arene 5 capsule. Assignments are indicated in the inset. Arrows indicate NOE contacts. (Reproduced from Ref. 8. American Chemical Society, 1997.)...

Figure 6 NMR structure for the complex between 19c and M-/JGIcNAc." The biphenyl units are highlighted in cyan, and the substrate is shown in yellow. Intramolecular and intermolec-ular NOE contacts are shown as green and red broken lines, respectively. The water-solubilizing external substituent groups are omitted. (Reproduced from Ref. 49. Royal Society of Chemistry, 2009.)...

For mixing times larger than 200 ms contacts between protons which are more distant than 4.5 A are detectable. This is due to a process that is referred to as spin diffusion in which NOE contacts are mediated by an additional proton which acts as a kind of relay station. In this way, NOE contacts between the T-ribose protons and the aromatic protons (C6H/C8H) of both, the same and the 3 -neighboured nucleotide, can be observed. Thus, complete assignment paths between T and aromatic protons of aU the residues within one strand can be drawn as long as the helical geometry is not perturbed. There are also contacts between protons of nucleotides in the two different strands of the duplex, e.g. between C2H of an adenosine and the T-ribose proton of the 3 -neighboured nucleotide of the opposite strand [4]. 

In Fig. 19.5 all the NOE contacts between the protons of adjacent nucleotides detected for 300 ms mixing time NOESY spectra are compiled. 

The small number of the observed internucleotide NOEs between C72 and A73 suggests a weak stacking. However, there are cross peaks of the C2 proton of A73 with both, the aromatic C8H and the I -ribose proton of G1 of the opposite strand. The former cross peaks indicate an overlap of the purine base plane of A73 with that of Gl, which corroborates the before-mentioned interlocking effect of the purine base in position 73 and provides a structural explanation for the stabilizing effect of 3 -dangling purine nucleotides. At the same time, the C8 proton of A73 is moved away from the aromatic protons of C72 making the lack of the aromatic-aromatic cross peaks conceivable. Some NOE contacts have been... 

Basic data obtained from NMR studies consist of distance and torsion angle restraints. Once resonances have been assigned, nuclear Overhauser effect (NOE) contacts are selected and their intensities are used to calculate interproton distances. Information on torsion angles are based on the measurement of coupling constants and analysis of proton chemical shifts. Together, this information is used to formulate a nonlinear optimization problem, the solution of which should provide the correct protein structure. Typically, a hybrid energy function of the following form is employed ... 

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