Peptide-Based Drug Design

NMR of Peptides 95
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.
It is worth noting here that despite an extensive database of chemical
shifts being available for peptides and proteins (BioMagResBank
(http://www.bmrb.wisc.edu/) (54)), chemical shifts by themselves cannot be
used to make spectral assignments. This is illustrated in Fig. 3, whichshows
that the overlap in the range of chemical shifts seen for the �H protons in the
20 natural amino acids among known proteins is too large to be of diagnostic
value.
Once the NMR spectra of a peptide are assigned the range of techniques
mentioned earlier can be used to retrieve information that is valuable in the drugdesign
process. Clearly the 3D structure of the peptide is of vital importance,
and we examine it first. Indeed, as is illustrated in Fig. 4, the assignment and
structure determination processes are often completed together.
4.2.2. Peptide Structure Determination
Figure 4 shows the general workflow required for determining the structure
of a small peptide by NMR, including the assignment step. Typically, structure
determination requires the recording of 6–12 2D spectra under various conditions
of temperature, pH, or spectral parameters such as mixing times. As is
implied by the figure, temperature variation is often useful to remove ambiguities
due to overlap, as the amide chemical shifts are differentially sensitive
to temperature. Exchange of the sample into D2O provides information about
hydrogen-bonding networks.
It is convenient to illustrate the principles involved in the assignment and
structure determination process by reference to a structure of a small bioactive
peptide solved recently in our laboratory. Figure 5 shows the sequential connectivity
diagram (an overlay of 2D NOESY and TOCSY spectra) for Vc1.1, a 16residue
conotoxin-based peptide that is currently in clinical trials as a treatment
for neuropathic pain (55–57). In general, completion of this sequential walk,
together with assignment of the side-chain protons, provides a basis for assessing
whether a full 3D structure determination is warranted. At this stage it is also
important to check the spectra for evidence of multiple conformations, which
can occur due to the presence of cis-trans isomerization near proline residues.
In the case of Vc1.1 the data are of good quality, and a full 3D structure was
determined (58).