Use of 113Cd NMR to Probe the Native Metal Binding Sites in Metalloproteins: An Overview
- Ian M. ArmitageAffiliated withDepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Email author
- , Torbjörn DrakenbergAffiliated withDepartment of Biophysical Chemistry, Lund University
- , Brian ReillyAffiliated withDepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota
Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with 113Cd NMR methods, we will present the results from a thorough literature search for 113Cd chemical shifts from metalloproteins. The updated 113Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the 113Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca2+ binding proteins, and metallothioneins.
In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, 113Cd NMR, in conjunction with 13C and 31P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceeded the availability of the X-ray crystal structure.
In the case of the calcium binding proteins, we will focus on two proteins: calbindin D9k and calmodulin. For calbindin D9k and its mutants, 113Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with 113Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The 113Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment.
In the case of metallothionein, we will reflect upon how 113Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear 1H-113Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The 113Cd NMR studies also enabled the characterization of the affinity of the individual sites for 113Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.
Keywordsalkaline phosphatase calbindin calcium-binding proteins calmodulin metallothionein 113Cd NMR methods 113Cd NMR chemical shifts from metalloproteins
- Use of 113Cd NMR to Probe the Native Metal Binding Sites in Metalloproteins: An Overview
- Book Title
- Cadmium: From Toxicity to Essentiality
- pp 117-144
- Print ISBN
- Online ISBN
- Series Title
- Metal Ions in Life Sciences
- Series Volume
- Series ISSN
- Springer Netherlands
- Copyright Holder
- Springer Science+Business Media Dordrecht
- Additional Links
- alkaline phosphatase
- calcium-binding proteins
- 113Cd NMR methods
- 113Cd NMR chemical shifts from metalloproteins
- Industry Sectors
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- Editor Affiliations
- 3. Inorganic Chemistry, Chemistry, Universität Basel
- 4. Inst. Anorganische Chemie, Universität Basel
- 5. Institute of Inorganic Chemistry, University of Zürich
- Author Affiliations
- 1. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street S.E., Minneapolis, MN, 55455, USA
- 2. Department of Biophysical Chemistry, Lund University, 124, SE-22100, Lund, Sweden
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