A number of reports in recent years have demonstrated the feasibility as heme proteins, where the native metal ion is diamagnetic of detecting quadrupolar metal ions bound tightly to rather large pro-(low-spin Fe 2/ ) (8). Among the aforementioned reports, the teins via the quadrupolar central transition (QCT) NMR approach. use of heteronuclear inverse (i.e., 1 H, X) techniques, in In this article, an in-depth investigation of several interesting properties which the observation of the protein-bound metal nucleus is of transferrin-bound 27 Al NMR signals, namely, their dependence on facilitated by detection via spin-spin coupled protons on the temperature, viscosity, and molecular size is presented. It is shown residues involved in coordinating the metal ion, has attracted that (1) decreasing temperature and (2) increasing viscosity by adding considerable attention. However, relaxation mechanisms reagents such as glycerol and ethylene glycol perturb only the linewcommon to I Γ
1 2 nuclei, such as chemical-shift anisotropy idths of transferrin-bound 27 Al signals, and, in fact, produce a decrease (CSA) (9), limit the practical application of this approach in signal linewidth. These effects are in accord with quadrupolar relaxto relatively small metalloproteins (i.e., MW Β£ 50 kD). ation theory, which predicts that the linewidth of the central transition of a half-integer quadrupolar nucleus should decrease with increasing
While 43 Ca (I Γ
7 2 ) NMR has been employed to probe the correlation time of the protein under nonextreme narrowing conditions. high-affinity Ca 2/ -binding sites in several calcium-binding Furthermore, it is demonstrated that these trends, which are comproteins (10), until recently, it was generally accepted that pletely opposite to those generally observed in NMR spectroscopy, the unfavorable relaxation properties of quadrupolar (I ΓΊ can be exploited to monitor ovotransferrin half-molecule reassociation 1 2 ) nuclei should render quadrupolar NMR useless in terms reactions. In combination with the peculiar properties of transferrinof its suitablity for investigating large macromolecules.
bound quadrupolar nuclei reported in the literature to date, the phe-However, recent 51 V (I Γ
7
2 ) (11-13), 27 Al (I Γ
5 2 ) (14nomena described here provide the basis for understanding the condi-17), 45 Sc (I Γ
7 2 ) (17, 18), and 69,71 Ga (I Γ
3 2 ) (17, 19) tions and experimental parameters which may facilitate the application of the QCT NMR technique to the study of other quadrupolar nuclei NMR studies of the transferrins, a class of large (MW Γ and proteins. α§ 1996 Academic Press, Inc.
80 kD) Fe 3/ -binding proteins (20), using the quadrupolar central transition (QCT) NMR technique largely dispel this widely held myth. The approach hinges on the detection of the central (m I Γ
1 2 r 0 1 2 ) transition of a half-integer quadru-