| Popis: |
In NMR studies of polypeptides and proteins, there is a serious dynamic range problem associated with digitizing the signal from the solute (typically about 1 to 5 mM) in the presence of the HZ0 solvent (with an effective ‘H concentration of about 110 M). In many cases, this problem is overcome by selective presaturation of the HZ0 solvent resonance (I, 2). Although preirradiation remains the most practical and simple method for HZ0 solvent suppression, it has significant disadvantages. Saturated solvent protons can exchange into amide and other labile proton sites, resulting in significant reduction of resonance intensities. A second artifact arises from the unavoidable irradiation of C”H nuclei, which results in nuclear-Overhauser effects and spin diffusion during the preirradiation period. Although these spin-diffusion effects are minimal in small peptides, in proteins (and especially in large proteins) the associated negative NOES can greatly attenuate the intensities of proton resonances which are affected by spin diffusion from these irradiated C”H nuclei. D:ifferential attenuation of proton resonances due to saturation transfer and/or preirradiation-associated spin-diffusion effects prevents accurate determination of internuclear distances from intensities of NOESY cross peaks. Although saturationtransfer experiments can be useful for quantitative investigations of amide proton exchange rates (3-9)) pulse sequences used to obtain NOE data must provide minimal saturation transfer. Saturation-transfer effects also occur in pulse sequences that provide suppression by dephasing the HZ0 solvent coherence with homospoils or trim pulses ( 10, I1 ) . In order to minimize saturation-transfer effects, most protein NMR work is carried out in the pH range of about 2 to 5 and low temperatures. Under these conditions, the pH-dependent rate of amide proton exchange in peptides is slow and there is minimal saturation transfer ( 12-18). In the more physiological range of pH 6 to 8, where amide proton exchange rates become significantly faster, alternative methods for avoiding the dynamic range problem involve frequency-selective excitation of amide |
