FIELD-DEPENDENT AL-27 NMR-STUDIES OF THE TRANSFERRINS - AN APPROACH FOR THE STUDY OF METAL-ION BINDING-SITES IN LARGER PROTEINS

The aluminum/carbonate derivatives of three transferrins-ovotransferri n and its half-molecules, serotransferrin and lactoferrin-have been st udied by Al-27 NMR spectroscopy at four magnetic fields. In the case o f ovotransferrin, one observes two Al-27 signals, corresponding to Al3 + bound to both metal ion-binding sites in the protein. However, for b oth serotransferrin and lactoferrin, these signals are degenerate at e very field used in this study. In each case, only the central (m = 1/2 --> -1/2) quadrupolar transition is observed; its detection is facili tated by the high molecular weights of these proteins. Moreover, for e ach transferrin-bound Al-27 signal, increasing the magnetic field lead s to a downfield shift in peak position (termed the second-order dynam ic frequency shift) and a decrease in line width. From the field depen dence of the chemical shift, we have obtained values for the quadrupol e coupling constant (chi) of Al3+ bound to the metal ion-binding sites of each protein ranging from chi= 3.3 to 4.1 MHz. The values of chi f or the half-molecules of ovotransferrin and the corresponding sites in the intact protein are virtually identical. Using these chi values an d the field dependence of the line width, we calculated the rotational correlation time (tau(c)) of bound Al3+ in each protein. For the inta ct transferrins, tau(c), ranged from almost-equal-to 40 to 60 ns, whil e significantly shorter values of tau(c) were found for the half-molec ules of ovotransferrin (tau(c) almost-equal-to 14-17 ns). This report constitutes the first complete field-dependent investigation of a quad rupolar nucleus in isotropic motion under far from extreme narrowing c onditions and demonstrates how quadrupolar metal ion NMR may be used t o obtain physical information about the nature of the metal-binding si tes of larger proteins.