Abstract
Many chemical and biological processes rely on the movement of monovalent cations and an understanding of such processes can therefore only be achieved by characterising the dynamics of the involved ions. It has recently been shown that 15N-ammonium can be used as a proxy for potassium to probe potassium binding in bio-molecules such as DNA quadruplexes and enzymes. Moreover, equations have been derived to describe the time-evolution of 15N-based spin density operator elements of 15NH4+ spin systems. Herein NMR pulse sequences are derived to select specific spin density matrix elements of the 15NH4+ spin system and to measure their longitudinal relaxation in order to characterise the rotational correlation time of the 15NH4+ ion as well as report on chemical exchange events of the 15NH4+ ion. Applications to 15NH4+ in acidic aqueous solutions are used to cross-validate the developed pulse sequence while measurements of spin-relaxation rates of 15NH4+ bound to a 41kDa domain of the bacterial Hsp70 homologue DnaK are presented to show the general applicability of the derived pulse sequence. The rotational correlation time obtained for 15N-ammonium bound to DnaK is similar to the correlation time that describes the rotation about the threefold axis of a methyl group. The methodology presented here provides, together with the previous theoretical framework, an important step towards characterising the motional properties of cations in macromolecular systems.
Highlights
Monovalent cations such as potassium and sodium regulate many enzymes via binding to either active sites or to allosteric sites [1,2,3]
The dynamics and movements of these ions are crucial factors to understand the regulation of enzymes by monovalent cations and experimental insight into the dynamics of cations becomes important in order to characterise many biological processes
Solution NMR spectroscopy is a powerful technique to probe the dynamics of nuclear spin, where in particular nuclear spin-relaxation rates have been used to report on the dynamics of small ions [4,5,6,7] to large macromolecular complexes [8,9,10,11]
Summary
Monovalent cations such as potassium and sodium regulate many enzymes via binding to either active sites or to allosteric sites [1,2,3]. The obtained nuclear spin-relaxation rates often report on both the rotational correlation time of the nuclear spin as well as on chemical exchange between different magnetic environments and a separation of the different contributions to the observed nuclear spin-relaxation rate is important. It was shown that 15NH4+ can be used as a proxy for potassium to probe potassium-binding sites in nucleic acids and enzymes [17,18,19,20] This method relies on several characteristics of the ammonium ion: (i) The ionic radius of the ammonium ion is similar to the ionic radius of potassium, 1.44 Å versus 1.33 Å [21,22], such that ammonium generally binds to potassium binding-sites in macromolecules. This method relies on several characteristics of the ammonium ion: (i) The ionic radius of the ammonium ion is similar to the ionic radius of potassium, 1.44 Å versus 1.33 Å [21,22], such that ammonium generally binds to potassium binding-sites in macromolecules. (ii) Under physiological conditions the chemical exchange of the ammonium protons with the bulk solvent is so fast that free ammonium is not observed in NMR correlation spectra, the protection of the ammonium ion, for example by a protein environment, slows the exchange of the protons with the bulk solvent to such an extend that these are observed in two-dimensional 15N-1H correlation spectra [19]. (iii) protein-bound ammonium ions appear to have a fast internal correlation time such that the line broadening due to the 15N-1H dipolar-dipole interactions is limited
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