Sequential assignment, peptides

Scheme 1 Peptide Sequence Sequential NOEs and Observable Through-Bond Couplings Useful for Sequential Assignment of the Peptide...

The basis of the sequential assignment approach is to use a combination of TOCSY/COSY spectra to assign peaks to residue type and then link those residue types sequentially together using NOESY spectra (53). A schematic representation of some of the key NOESY connectivities used in assigning the spectra of peptides is given in Fig. 2. 

For example, ambiguities in the sequential assignment of peptide or protein spectra can often be eliminated through use of relayed NOESY-type experiments (Wagner, 1984), where the mixing process consists of... 

Fig. 9 Strips from CBCANH and the CB(CACO)NH of calmodulin in the complex with the peptide C20W. The two strips are taken at the same and H chemical shifts, thus connecting two sequential NH resonances. By detecting identical Cp and C chemical shifts, the sequential assignment can be completed.

To determine the conformation of our complex CaM/C20W, the C20W peptide needs to be assigned as well. The techniques that were used for the protein caimot be applied, since the peptide is not labelled with and C. Thus, the traditional assignment techniques based on COSY, NOESY, and TOCSY are applied for the assignment [71]. The COSY and TOCSY spectra reveal the peak patterns of the amino acids, whereas the NOESY provides the sequential assignment of the peptide. The pulse sequences need to filter all... 

A common area in which 2D methods have been employed to help provide resonance assignments is in the study of peptides or small proteins, specifically when the same amino acids occur more than once in the peptide sequence. The so-called sequential assignment process is used to define the position of a specific amino acid residue within a peptide and relies upon the observation of NOEs between protons in adjacent residues (Fig. 8.47). Typically, these will be between an alpha proton and the amide NH of the following residue. The identification of neighbouring amino acids in this way can be used to string these units together, which may then be mapped onto the usually known peptide sequence. Once all residues have been sequentially identified, this can provide the basis for... 

Sequential assignment of residues in a peptide chain requires correlation across the peptide bond that separates the proton spin systems of adjacent residues. This sequential information can be provided by dipolar couplings using NOESY or ROESY experiments, or by heteronuclear scalar couplings using HMBC experiments. 

ROESY spectrum, as in the case of folded peptides and small proteins. Sequential assignment of these molecules therefore can be ambiguous and requires a... 

It is also possible to determine the sequence of a peptide using scalar coupling to the carbonyl carbon. This is best done with standard or selective HMBC experiments, where only carbonyl carbons are excited and indirectly detected during The sequential assignment is then unambiguous and does not require any knowledge about the conformation of the peptide (Figure 6). A clear distinction between the... 

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

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... 

Solid-state NMR also can be applied to membrane proteins in lipid bilayers, and recent advancements in magic angle spinning solid-state NMR show promise for stmcture determination. Although the stmctures of small crystalline proteins (89) and membrane bound peptides (90) have been determined, the stmcture of a polytopic membrane protein has yet to be reported. The major necessity that is required to push the technique forward is the de novo sequential chemical shift assignment of the amino acid residues, and in the last few years, several groups have reported successful strategies (91, 92). 

Figure 4 Homonuclear 2D spectra of the 8mer peptide EWTLYWR in 90 % H2O, 10 % D2O. (a) Representation of coherence transfer pathways for COSY (soiid arrows), TOCSY (dotted arrows) and NOESY (dashed arrows) experiments, (b) Section of COSY spectrum displaying the backbone H -H -correlations, additionaiiy the side-chain H -H -correlations of R8 is visible in the upper right-hand corner. The H -H -correlation of El cannot be detected because of soivent-exchange broadening of the N-terminal amino group, (c) Section of TOCSY spectrum that displays the correlations of the backbone with all protons within the amino acid side chain, (d) Section of ROESY spectrum that displays correlations between backbone H -H intraresidual as well as to the neighboring (i-1) amino acid. The cross peaks to the (i-1) amino acid have higher intensities. Thus, a "sequential walk" is possible (arrows) that allows identification of the position of amino acids within the peptide chain. Additionally the H of El can be assigned (first arrow on the left).

The methodology discussed above has been used in the resonance assignment and to determine the backbone conformation of a uniformly N-labeled M2 transmembrane peptide of nicotinic acetylcholine receptor (AChR), one of the major excitatory neurotransmitters in the brain. By comparing with spectra from selectively and specifically N-labeled M2 peptides and using the sequential (z to z-Fl) cross peaks in the spin exchange spectra, all the resonances from the M2 peptide were assigned. 

With the dissemination of multinuclear multidimensional NMR experiments, the automated NMR resonance assignment became feasible and indispensable, simultaneously.23,35,41,99 Typically, 10-20 individual multidimensional NMR spectra are measured for a large protein. The NMR-derived information quality definitely relies on the resonance assignment. The precious assignment has been based on and assessed by the sequential NOE continuity on the peptide sequence and consistency with the amino acid spin... 

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