It is of interest to note that the Dd/DT values fo r the chemical shifts of the Cys6 amide proton are significantly less for the cis than the trans isomer; for cis-LVP, in particular, they are in the range observed for amide protons which are either in an intramolecular hydrogen bond or shielded from solvent. These results were interpreted by assuming that the conformational properties of the cis and trans isomers are different with the macrocyclic hexapeptide ring of the cis isomer existing in a higher population of structured species. Characterization of the hydration accessibility of the cis isomer by 2D WATERGATE-NOESY/ROESY, however, will be extremely difficult because of the very low S/N of the cis isomer in the cross sections through the 2D NOESY/ROESY (Fig. 1B,C) despite a total accumulation time of 90 hours.

From the above it is evident that, although, 2D-WATERGATE NOESY and ROESY experiments result in an excellent alleviation of the water resonance and definition of the baseline in the NH region, the method suffers form very low sensitivity, especially in the case of the minor cis-isomer, and complications due to proton exchange even at low temperatures (273K). Furthermore, integrating cross peaks in spectra with different mixing times and determining the build-up rates of ROE/NOE effects is a formidable task.

The sensitivity problem can be alleviated by the use of selective excitation techniques such as the off-resonance 1D-ROESY experiment (7,8) which reduces the dimensionality of the spectra and, thus, the experimental time. In this case an off-resonance spin-lock field is applied where the transmitter frequency is shifted far to low (or high) frequency from the spectral region. The angle q formed by the effective spin-lock axis and the Z axis in the rotating fra me, represents an adjustable parameter with which one can vary the contribution of the longitudinal and transverse relaxation rates during the spin lock period. It is obvious that for q equal to O^o or 90^o the standard NOESY and ROESY are obtained, respectively. The off-resonance 1D ROE experiment, however, is not complicated by Hartmann-Hahn transfers (7). Interestingly, for a rigid isotropic rotational motion with wt_c > 5, which is typical for large biomolecules, a particu lar angle q = 35.5^o can be used so that dipolar cross relaxation between protons of the macromolecule vanishes and thus exchange phenomena can be selectively observed.

To evaluate the potentiality of this approach a series of 1D selective off-resonance ROESY experiments, at different q angles and varying selectivity of the 180 RE-BURP shaped DANTE sandwich, were recorded. The spectrum of Fig. 2b (q=O^o) is not identical t o the row at the water frequency (parallel to the F2 axis) of the 2D NOESY spectrum of Fig. 1B. This effect can be attributed to reduced selectivity of the 180^o RE-BURP shaped pulse, of total width of 13 ms, which results in sufficient excitation of the CHa protons. This is clearly illustrat ed in the spectrum of Fig.2 (b) which is due to a superposition of NOEs with hydration water (negative peaks) and NOEs with CaH protons (positive peaks). This is particularly acute for Cys6 which sho ws the largest positive peak since the a-proton resonance is very close to the water signal.