Field-dependent aluminum-27 NMR studies of the transferrins: an approach for the study of metal ion binding sites in larger proteins

Abstract

The aluminum/carbonate derivatives of three transferrins-ovotransferrin and its half-molecules, serotransferrin and lactoferrin-have been studied by 27Al NMR spectroscopy at four magnetic fields. In the case of ovotransferrin, one observes two 27Al signals, corresponding to Al3+ bound to both metal ion-binding sites in the protein. However, for both serotransferrin and lactoferrin, these signals are degenerate at every field used in this study. In each case, only the central (m = */2 - J/2) quadrupolar transition is observed; its detection is facilitated by the high molecular weights of these proteins. Moreover, for each transferrin-bound 27Al signal, increasing the magnetic field leads to a downfield shift in peak position (termed the second-order dynamic frequency shift) and a decrease in line width. From the field dependence of the chemical shift, we have obtained values for the quadrupole coupling constant (x) of Al3+ bound to the metal ion-binding sites of each protein ranging from x= 3.3 to 4.1 MHz. The values of x for the half-molecules of ovotransferrin and the corresponding sites in the intact protein are virtually identical. Using these x values and the field dependence of the line width, we calculated the rotational correlation time (T~) of bound Al3+ in each protein. For the intact transferrins, rC ranged from =40 to 60 ns, while significantly shorter values of I, were found for the half-molecules of ovotransferrin (7, = 1&17 ns). This report constitutes the first complete field- dependent investigation of a quadrupolar nucleus in isotropic motion under far from extreme narrowing conditions and demonstrates how quadrupolar metal ion NMR may be used to obtain physical information about the nature of the metal-binding sites of larger proteins.