Peptide spin systems

To avoid the interference from intramolecular REDOR dephasing, peptides with 15NH, labels could be mixed with peptides with 13COy labels in a 1 1 ratio before fibrillization, where i and j denote the residues with isotopic enhancement at the amide and carbonyl sites, respectively. If an in-register parallel (3-sheet is considered as the hypothetical structure, the most adequate model for the analysis of the 13C 15N REDOR data measured for the case of i = j should be a 15N-13C-15N spin system, where the NCN angle is ca. 127° and the two 15N-13C distances are 5.3 and 5.5 A (Fig. 4a). For the case of i = j + 1, the NCN angle should be 154° and the two 15N-13C distances 4.2 and 5.6 A. Other combinations of i and j for an... 

Frequency-selective REDOR (fsREDOR) is a very powerful technique developed for the study of 13C and 15N uniformly labeled peptides or proteins [92]. The basic idea of this technique is to combine REDOR and soft n pulses to recouple a selected 13C-15N dipole-dipole interaction in a multiple-spin system. Usually one could use Gaussian shaped pulses to achieve the required selective n inversions. Other band selective shaped pulses have been developed for a more uniform excitation profile [93]. In its original implementation, fsREDOR was used to extract the intemuclear distances of several model crystalline compounds [92], In the past few years, this technique has proven to be very useful for the study of amyloid fibrils as well. For the Ure2p10 39 fibril samples containing 13C and 15N uniformly... 

More recently, homonuclear correlation techniques relying on J-couplings have also been developed [86, 87, 89] and applied to the assignment of spin systems of amino-acid residues in uniformly labeled proteins and peptides [90]. These have, in some cases, a higher information content than the comparable dipolar-mediated experiments, as relayed correlations throughout the continuous 13C-13C network are more easily realized at high B0 fields [90]. 

TTie TOCSY 2D NMR experiment correlates all protons of a spin system, not just those directly connected via three chemical bonds. For the protein example, the alpha proton, Ft , and all the other protons are able to transfer magnetization to the beta, gamma, delta, and epsilon protons if they are connected by a continuous chain—that is, the continuous chain of protons in the side chains of the individual amino acids making up the protein. The COSY and TOCSY experiments are used to build so-called spin systems—that is, a list of resonances of the chemical shift of the peptide main chain proton, the alpha proton(s), and all other protons from each aa side chain. Which chemical shifts correspond to which nuclei in the spin system is determined by the conventional correlation spectroscopy connectivities and the fact that different types of protons have characteristic chemical shifts. To connect the different spin systems in a sequential order, the nuclear Overhauser effect spectroscopy... 

One problem with the REDOR method and other similar solid state NMR techniques is that unambiguous results only can be obtained when the spin system is based on isolated spin pairs, as shown in a recent publication . This means that the prevalent aggregates found in organolithium complexes cannot be studied by these methods. However, a recent 3D TEDOR study of uniformly labelled peptides gives some hope, provided that... 

Sequential resonance assignment involves the assignment of the spin systems to their position along the peptide chain. 

The common theme so far in our correlation experiments has been to allow spins to evolve during q under the influence of directly coupled nuclear spins. We have seen the power of COSY, HMQC, HMBC, and INADEQUATE to provide us with detailed structural information for ipsenol, caryophyllene oxide, and lactose. In this section, we will develop another method for showing correlations and apply it to molecules with distinct, isolated proton spin systems such as carbohydrates, peptides, and nucleic acids. 

There are various hybrid 2-D correlation experiments that combine features of two simpler 2-D experiments. A popular and useful example is the HMQC-TOCSY spectrum that correlates one-bond H—13C couplings (HMQC) but shows these correlations throughout an entire spin system (TOCSY). This experiment simplifies complex carbohydrate and peptide systems and allows ready assignments of systems of protons and carbons. 

What about the COSY-35 experiment (Fig. 9.34) We can now show with product operators why it simplifies the crosspeak fine structure. Consider again the AMX spin system of a peptide residue in D2O ND-CHa-CH H -Y. For the crosspeaks shown in Figure 9.33 (left) let s focus on the lower one F = Hp/F2 = Hq, (H -> Hq, coherence transfer). We start the t period with -ly and write down the terms that result from J coupling, keeping in mind that there are two / couplings affecting H Jap and Jppr. 

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... 

As is apparent from Figure 10.1, an a-helical structure imposes fairly rigid constraints on the relative positions of successive residues in a peptide chain. Thus there is a loss of entropy that must be overcome energetically in order for an a-helix to form. To explain the underlying biophysics of this system, John Schellman introduced a theory of helix-coil transitions that is motivated by the Ising model for one-dimensional spin system in physics [180, 170],... 

Fig. 3. (a) A typical spin system of three peptide planes with chemical shielding tensors and... 

Through SIMPSON numerical simulations. Fig. 4 compares the coherence transfer curves for a variety of the most popular and efficient heteronuclear dipolar recoupling schemes. Specifically, we evaluate the 07, POST-C7, off-resonance C7, R14, DCP, SPfCP, " SPECIFIC, and /DCP pulse sequences, realizing that many new powerful schemes enter the scene every year. For the present purpose, we address a three-spin system in a peptide backbone using parameters established for a... 

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