Use of113Cd NMR to probe the native metal binding sites in metalloproteins: An overview

Ian M. Armitage, Torbjörn Drakenberg, Brian Reilly

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

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 for113Cd chemical shifts from metalloproteins. The updated113Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the113Cd 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 metal-loenzyme 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, Cd NMR, in conjunction with C and P 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 coopera-tivity 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 D 9k 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 Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The Cd 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 heteronuclear1H- 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 a-helix and b-sheet and the first native protein identified with bound Cd. The113Cd NMR studies also enabled the characterization of the affinity of the individual sites for113Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

Original languageEnglish (US)
Pages (from-to)117-144
Number of pages28
JournalMetal Ions in Life Sciences
Volume11
DOIs
StatePublished - 2013

Keywords

  • Alkaline phosphatase
  • Calbindin
  • Calcium-binding proteins
  • Calmodulin
  • Metallothionein

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