Datasets depicting mobility retardation of NCS proteins observed upon incubation with calcium, but not with magnesium, barium or strontium

Abstract

In this data article we show the specificity of the Ca²⁺-induced mobility shift in three proteins that belong to the neuronal calcium sensor (NCS) protein family: Hippocalcin, GCAP1 and GCAP2. These proteins did not display a shift in mobility in native gels when incubated with divalent cations other than Ca²⁺ – such as Mg²⁺, Ba²⁺, and Sr²⁺, even at 10× concentrations. The data is similar to that obtained with another NCS protein, neurocalcin delta (Viviano et al., 2016, “Electrophoretic Mobility Shift in Native Gels Indicates Calcium-dependent Structural Changes of Neuronal Calcium Sensor Proteins”, [1]).

Specifications Table

Subject area	Biology
More specific subject area	Electrophoretic Techniques
Type of data	Figure
How data was acquired	Electrophoresis: Bio-Rad miniPROTEAN
Data format	Analyzed
Experimental factors	For electrophoresis, standard protocols were used.
Experimental features	Divalent cations were tested in their ability to shift NCS proteins on Native gel
Data source location	Stratford, New Jersey, USA
Data accessibility	Data is within this article

Value of the data

• 1.NCS proteins primarily bind and respond to calcium.
• 2.However, they have been shown to bind other metal ions, particularly Mg²⁺ [2].
• 3.Binding of Mg²⁺ was shown to affect the function of GCAP1 [3].
• 4.The data presented reveal that the mobility retardation on native gels is specifically induced only by calcium and not by other tested divalent cations – Mg²⁺, Ba²⁺, and Sr²⁺, even at 10× concentrations.
• 5.It may be possible to correlate binding of small ligands to structural changes detectable as electrophoretic mobility shift.

1. Data

Bacterially expressed proteins – Hippocalcin (HPCA), Guanylate Cyclase Activating Proteins 1 and 2 (GCAP1 and GCAP2) – were purified in their myristoylated forms. The proteins were incubated with the divalent cation (Ca²⁺, Mg²⁺, Ba²⁺, or Sr²⁺) and were subjected to electrophoresis in native gels. Representative images are provided in Fig. 1A. Data was compiled from at least two independent preparations of the proteins with three independent replicates from each preparation. The mobility values were determined. The data (mean+SEM) is presented as a bar graph (Fig. 1B). Results from Student t-tests are also presented for each group against the control group (**, P<0.05; **, P<0.01; ***, P<0.001; ns – not significant). Mobility retardation is observed with the addition of calcium but not with any of the other divalent cations, even at concentrations ten times that of calcium.

Fig. 1.

Effects of divalent cations on mobility of HPCA, GCAP1 and GCAP2 in native gels.

2. Experimental design, materials and methods

In order to determine if divalent cations such as magnesium, strontium or barium could induce a mobility shift in the same way that calcium can [1], analyses were carried out with the proteins HPCA, GCAP1 and GCAP2. Proteins were expressed in E. coli ER2566 as described for NCALD in [1]. Briefly, cells grown overnight were inoculated (1% inoculum) into fresh LB medium and grown to an optical density of 0.6 at 600 nm. IPTG (1 mM final concentration) was then added for induction. For myristoylation, cells with yeast N-Myristoyl Transferase were used and myristic acid was supplemented. Cells were collected 2.5 h after induction, sonicated and the protein was purified. The purified protein was then washed with calcium-depleted Tris–Cl (20 mM; pH 7.5) to remove any residual calcium. Calcium removal was through the use of Chelex-100 resin (BioRad Laboratories, CA, USA) using standard procedures [4]. Proteins were then individually incubated in the presence of divalent cations (at indicated concentration within parentheses): calcium (39 µM) or magnesium (~400 µM), strontium (~400 µM) or barium (~400 µM). Electrophoreses in native gels and analyses were carried out as described [1].

Appendix A. Supplementary material

Supplementary material