In situ NMR measurement of macromolecule-bound metal ion concentrations
Many nucleic acids and proteins require divalent metal ions such as Mg2+ and Ca2+ for folding and function. The lipophilic alignment media frequently used as membrane mimetics also bind these divalent metals. Here we demonstrate the use of 31P NMR spectrum of a metal ion chelator (deoxycytidine diphosphate) to measure the bound [Mg2+] and [Ca2+] in situ for several biological model systems at relatively high divalent ion concentrations (1–10 mM). This method represents a general approach to measuring divalent metal ion binding in NMR samples where the amount and type of metal ion added to the system is known.
KeywordsDivalent ion concentration 31P NMR dCDP Bicelles Dickerson dodecamer βγ-Crystallin Ca2+ dependent protein
The authors acknowledge Dr. Philip Dennison for excellent management of the UCI NMR facility and Dr. Beniam Berhane for his help with biomolecular mass spectrometry. We thank Dr. Cassandra Burke and Jan Bierma for assistance with sample preparation. We acknowledge Prof. Juli Feigon for her helpful suggestions. This work was supported by NSF Grant CHE1308231 to RWM.
- Cohn M, Hughes TR Jr (1962) Nuclear magnetic resonance spectra of adenosine di- and triphosphate. II. Effect of complexing with divalent metal ions. J Biol Chem 237:176–181Google Scholar
- Gupta RK, Moore RD (1980) 31P NMR studies of intracellular free Mg2+ in intact frog skeletal muscle. J Biol Chem 255:3987–3993Google Scholar
- Gupta RK, Benovic JL, Rose ZB (1978) The determination of the free magnesium level in the human red blood cell by 31P NMR. J Biol Chem 253:6172–6176Google Scholar
- Hill AV (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J Physiol 40:iv–viiGoogle Scholar