Charlotta Johansson

Analysis of myo-inositol phosphates by 2D 1H-n.m.r. spectroscopy

Abstract 2D 1 H-N.m.r. spectroscopy applied to phosphorylated myo -inositols can indicate the number and the sites of phosphorylation, and up to three isomers can be completely analyzed simultaneously. The resonance of the sole equatorial proton (H-2) is shifted furthest downfield (to ∼4.7 p.p.m.) when C-2 is phosphorylated; when C-5 is unphosphorylated, the H-5 resonance has the lowest chemical shift (∼ 3.3 p.p.m.). The number of sites of phosphorylation can be determined from the sum of the chemical shifts for the resonances in a given myo -inositol phosphate and, based on the data presented, an algorithm can be constructed that will identify any myo -inositol phosphate on the basis of the chemical shifts.

Metal-Ion NMR studies of Ion Binding

Publisher Summary This chapter covers the studies, where nuclear magnetic resonance (NMR) is used as a tool to investigate the interaction between an ion and another ion or a molecule or an aggregate. The work dealing with ion binding is discussed in the chapter. NMR studies of ion binding correspond to the alkali and alkaline-earth ions. Thus, the chapter describes the small ligands, such as alkali metals, alkaline-earth metals, transition metals, and group IIA and IIIA metals. A number of macromolecules have been studied, using a limited number of NMR sensitive nuclei, owing to the size and complexity of the systems. However, of the available nuclei, both the alkali and the alkaline-earth metals have been used extensively. The metal ions that bind to the macromolecules are alkali metals ( 23 Na and 7 Li), alkaline-earth metals ( 25 Mg and 43 Ca), transition metals ( 51 V), and thallium ( 205 Tl). The ion binding to aggregates and cells occurs through Gramicidin channels. Finally, the chapter gives an account of the whole-cell studies.

Calcium nuclear magnetic resonance

Publisher Summary A survey of the nuclear properties of naturally occurring isotopes of all elements in the periodic table reveals that nearly all of the 30 or so essential elements in biological systems have at least one potentially valuable isotope amenable for nuclear magnetic resonance (NMR) studies. Calcium has one isotope, 43 Ca, with a nuclear spin I of 7 / 2 , meaning that the nucleus also has an electric quadrupole moment. The nuclear parameter that determines the frequency at which NMR signals may be observed at a given magnetic field—namely, the magnetogyric ratio, is such that at a field of 11.7 tesla (T) the 43 Ca NMR signals will be observable at 33.5 MHz. At this field, H NMR signals would occur at 500 MHz. The low NMR frequency for 43 Ca also implies low relative spectroscopic sensitivity; if the sensitivity of H nuclei is put as 1.00, the sensitivity for an equal amount of 43 Ca nuclei would only be 0.0064. This chapter discusses the way 43 Ca NMR can be used to obtain unique information about biological systems: to identify and characterize Ca 2+ binding sites, to study compartmentalization and transport processes, to determine Ca 2+ binding constants, and to obtain rates of chemical exchange of Ca 2+ ions from a macromolecule under equilibrium conditions. It also describes the experimental requirements for observation of 43 Ca signals and the way relevant information can be extracted biochemically from the observed NMR spectra.

Mechanism of hydroxylamine mutagenesis : role of tautomerism, conformation and proton exchange on base pairing between the promutagen N6-methoxyadenosine and uridine

Abstract A study of the interaction of N 6 -methoxyadenosine (OMe 6 A, which exists as an equilibrium mixture of amino and imino tautomers) with the potentially complementary uridine in non-aqueous medium demonstrated that only the tautomer amino -OMe 6 A base pairs with uridine. The association constant, determined by 1 H NMR spectroscopy, was 101 M −1 at + 30°C, an order of magnitude greater than those for autoassociation of amino -OMe 6 A or uridine. Base pair formation between amino -OMe 6 A and uridine led to a shift of the amino-imino equilibrium of 10% in favour of the amino species which associates with uridine, with the concomitant decrease in population of the imino form. Base pairing was accompanied by intermolecular proton exchange between the N 6 -H of amino -OMe 6 A and the N(3)-H of uridine. The rate constants for these exchanges, as well as for tautomeric exchange of OMe 6 A in the presence of uridine, were measured by means of the saturation transfer technique. The mechanism of proton exchange is compared with, and shown to be different from, that previously observed for base pair formation between the imino tautomer of OMe 6 A and cytidine (Stolarski et al., Biochemistry 26 (1987), 4332 also linked with tautomeric exchange. The resulting proposed schemes of base pairing are dependent on the conformation of the exocyclic N 6 -methoxy group, and are compared and discussed with reference to published data on base pairing at the oligonucleotide duplex level, as well as known data on base pairing of the analogous promutagen N 4 -methoxycytidine. The overall findings further contribute to our understanding of the dual functionality of the promutagen OMe 6 A at the molecular level, and furthermore, provide an excellent model system for studying the role of tautomerism on proton exchange in base pairs.

PROTEIN ENGINEERING AND STRUCTURE/FUNCTION RELATIONS IN BOVINE CALBINDIN D9k

In an attempt to elucidate the relationships between structure, dynamics and function in the calmodulin superfamily of Ca2+-binding intracellular proteins we have undertaken a detailed study of bovine calbindin D9k. This protein has a size (Mr≃8,500) and tertiary structure similar to that of the globular domains of calmodulin and troponin C and binds two Ca2+ -ions strongly (K ≃107 - 108 M-1, depending on the ionic strength). The schematic structure of the molecule is shown in figure 1.

Neutralization of Surface Charges Markedly Affects the Properties of Bovine Calbindin D9k

Calbindin D9k constitutes an attractive model system for exploring the relationships between structure, dynamics and function in the EF-hand family of proteins. It is the smallest protein known (Mr ≈ 8,500; 75 a.a:s) with a pair of EF-hand Ca2+-binding sites and its structure resembles closely the globular domains of the homologous proteins calmodulin, parvalbumin and skeletal muscle troponin C. [1,7]. The schematic structure of calbindin is shown in Fig. 1.