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involved, varies considerably from protein to protein. Therefore, identification of
metal coordination parameters including numbers and types of ligands and metal-
ligand geometry and mapping the structural and dynamic changes upon metal
binding are of significance towards understanding biological functions of
metalloproteins. Today, NMR spectroscopy is one of the leading techniques for
this purpose. Applicability of homonuclear metal NMR and heteronuclear 1 H-metal
HMQC to monitor directly protein-metal interactions rely greatly on the properties
of the nuclei. Some of the metal (e.g., 113 Cd) NMR has been used extensively to
identify coordination spheres of the metal ions. Moreover, the coupling constants
between the NMR active metals and nuclei of the protein provide insight into the
identity and geometry of the metal ligands [ 71 ]. Many metal (e.g., 43 Ca and 67 Zn)
NMRs are less powerful and hardly used owing to the fact that these nuclei have the
spin quantum number I greater than 1/2 which leads to lower sensitivity, poor
resolution, and broadening due to large quadrupolar moments although ultrahigh
fields improve it. Several reviews have systematically summarized the application
of heteronuclear NMR spectroscopy in biological and medicinal chemistry as well
as in the study of metalloproteins [ 23 , 71 - 73 ]. Here, we will highlight some of the
recent progresses as a snapshot of using metal NMR to identify metal coordination.
Cadmium is one of the most widely used metal nuclei for probing metal-protein
interactions, despite its toxic properties. It has two NMR active nuclei, 113 Cd and
111 Cd (spins of 1/2), with the former being slightly more sensitive and therefore
usually used as a preferred nucleus. At natural abundance, the sensitivity of 113 Cd
is very low (ca. 7.6-fold of 13 C), and therefore isotopic enrichment (ca. 96%) of
113 Cd is usually needed to ensure reasonable quality of spectra to be acquired in
a relatively short period of time (a few hours for ca. 0.5 mM samples). 113 Cd or
1 H- 113 Cd NMR spectroscopy has been utilized in the study of a variety of
metalloproteins where the native Zn 2+ ,Ca 2+ ,Mg 2+ ,Mn 2+ ,Fe 2+ , and Cu 2+ can be
substituted by 113 Cd, given that the adaptable ligand coordination number and
geometry of Cd 2+ is similar to Zn 2+ and the ionic radius of Cd 2+ (0.97 ˚ ) is similar
to that of Ca 2+ (0.9 ˚ )[ 27 , 74 - 77 ]. Moreover, the substitution of the native zinc
from metalloenzymes and DNA-binding proteins by cadmium caused almost no
changes in their structures and functions [ 78 , 79 ].
113 Cd chemical shifts are very sensitive to the nature, number, and geometric
arrangement of the coordinated ligands [ 71 ], as shown in Fig. 3 . Such wide chemical
shift dispersion not only provides information about the types and numbers of ligand
at a particular metal site, but also discriminates multiple metal sites with identical
ligand coordination environments. 113 Cd NMR and 1 H- 113 Cd HMQC have been
employed exclusively in identification of metal-thiolate clusters in a family of
small proteins, e.g., metallothionines [ 27 , 48 , 71 , 80 ]. Both homonuclear 1D 113 Cd
decoupling studies (Fig. 4a )and2D 113 Cd- 113 Cd COSY (Fig. 4b )of 113 Cd 7 -MTs
established the existence of two metal-thiolate clusters in this protein, while 1 H- 113 Cd
HMQC (Fig. 4c ) was used to identify sequence-specific cysteine-cadmium coordi-
nation bonds. The chemical shift patterns for the two clusters Cd 3 Cys 9 and Cd 4 Cys 12
of human MT3 as shown in Fig. 4 a showed seven resonances at analogous positions
compared with MT1/2 with chemical shift ranging from 600 to 690 ppm [ 27 ].
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