Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca²⁺-ATPase Probed by Fluorescence Quenching

Abstract

Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.

Dynamics of the ssDNA Recognition by the RepA Hexameric Helicase of Plasmid RSF1010: Analyses Using Fluorescence Stopped-Flow Intensity and Anisotropy Methods

“…The results indicate that there is no fluorescence change, as compared to the free ssDNA, in the first binding step, that is, in the formation of (H-ssDNA) 1 , indicating that in the first intermediate, the marker is in an environment similar to the bulk solution 45,46. This is very different from the large fluorescence changes observed for the first intermediate in the complex formation between the E. coli DnaB hexamer and the etheno derivative of the ssDNA 20-mer, d ε A(p ε A) 19 (see Discussion) 29.…”

“…Nevertheless, the subsequent conformational transition, (H-ssDNA) 1 ↔(H-ssDNA) 2 , induces significant quenching of the fluorescein emission. This is particularly pronounced for the two longer oligomers, 5′-Fl-dT(pT) 24 and 5′-Fl-dT(pT) 29 ,with F 2 ≈0.2, as compared to the free nucleic acid, indicating that the marker is placed in a very different environment in (H-ssDNA) 2 than in (H-ssDNA) 1 45,46. Emission intensity of the marker is also quenched in the intermediates (H-ssDNA) 3 and (H-ssDNA) 4 for all examined ssDNAs, although to a lesser extent than in (H-ssDNA) 2 .…”

RNAi– induced silencing of the plasma membrane Ca2+– ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

“…The enzymatic activity of the PMCA was measured in cortical neurons 48 h after exposure to the control RNA or PMCA2 siRNA oligonucleotides (Fonseca et al. 2002).…”

Fluorescence turn-on detection of iodide, iodate and total iodine using fluorescein-5-isothiocyanate-modified gold nanoparticles

“…The result provides clear evidence that the fluorescence of FITC was rarely quenched by low concentrations of I À . 34 Spectrum 'c' in Fig. 1A shows that the fluorescence intensity of 0.4 mM FITC at 517 nm was almost completely quenched upon the addition of 1.5 nM AuNPs ($9.0 Â 10 11 particles/mL).…”