Electron paramagnetic resonance spectroscopy of nitroxide-labeled calmodulin

Abstract

Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca(2+))(4-)form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2-3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca(2+) to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca(2+) binding. The midpoint of the Ca(2+)-mediated transition determined by EPR occurred at a higher Ca(2+) concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca(2+))(4-)CaM], contains a compact intermediate corresponding to [(Ca(2+))(2-)CaM], and the present results suggest that the spin probes are reporting on Ca(2+) binding to the last two sites in the N-terminal domain, i.e. for the [(Ca(2+))(2)-CaM] → [(Ca(2+))(4-)CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca(2+)-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.

Fig. 1

Ca²⁺-mediated activation of phosphodiesterase by CaM and SL-CaM. CaM (A) and SL-CaM (B) were each present at a saturating concentration (3.0 μM), and the data are given in a normalized form as (V – V_o) / (V_max – V_o), where V is the measured velocity, V_o the basal activity in the presence of 2 mM EGTA, and V_max the maximal activity achieved at 1 μM Ca²⁺. The results of two independent experiments are shown, and each point denotes the mean of duplicate assays. Results in the presence of 2 mM EGTA with no added Ca²⁺ are shown as closed symbols, and the enzymic activity in the presence of 2 mM EGTA with no added Ca²⁺, but with CaM or SL-CaM, was invariably higher than the basal activity in the absence of CaM or SL-CaM. Midpoints (pCa²⁺ for CaM and SL-CAM are 6.84 and 6.73, respectively. The abscissa is – log₁₀ (Ca²⁺⁾_f with Ca²⁺ in units of M.

Fig. 2

Ca ²⁺-induced changes in the mean residue ellipticity at 222 nm for CaM and SL-CaM. The CD changes were determined in both the forward (A, C), i.e. from 0 -1.2 mM Ca²⁺, and reverse (B, D), i.e. from 1.2 mM Ca²⁺, directions for CaM (A, B) and SL-CaM (C, D) in 50 mM Tris-HCl, pH 7.5 (0.26 mg/mL). The closed symbols in panels A and C denote the presence of 2 mM EGTA, and forward titrations were done with small aliquots of 0.01 M CaCl₂ in the same Tris-HCl buffer. The conditions were such that in going from the starting solution (0 mM Ca²⁺) to the final solution (1.2 mM Ca²⁺), the volume change was 4 %; appropriate corrections were made to [Θ]_{222 nm}. The closed symbols in panels B and D refer to solutions in the Tris-HCl buffer that also contained 1.93 mM EGTA and 3.55 mM CaCl₂; these were titrated with 0.5 mM EGTA in the Tris-HCl buffer. As above, corrections were made to the small dilution of protein. p(Ca²⁺)_f midpoints of the Ca²⁺-mediated transition are 6.68 and 6.76, respectively. The Ca²⁺ concentration is given as – log₁₀ (Ca²⁺)_f with Ca²⁺ in units of M.

Fig. 3

EPR spectra of SL-CaM ± Ca²⁺. SL-CaM (29 μM) in 50 mM Tris-HCl, pH 7.5 containing 1.0 mM Ca²⁺ (heavy line and marked as +Ca²⁺) or 10 mM EGTA (lighter line and highlighted by –Ca²⁺). A typical 100 Gauss scan is shown.

Fig. 4

Spectral intensity of SL-CaM EPR spectra as a function of free Ca²⁺ concentration. The sum of the area of the three Lorentzian bands, obtained as described in Section 2.4, is in arbitrary units and was thus normalized to 1.0 in 2 mM EGTA (0 mM Ca²⁺); it is designated by a square. The arrow indicates the transition midpoint observed using CD (222 nm); that for the data shown is pCa²⁺ = 6.40. Ca²⁺ had no effect on the intensity of the free spin label (data not shown). The abscissa is – log₁₀ (Ca²⁺⁾_f with Ca²⁺ in units of M.

Fig. 5

EPR resonance peak heights at various free Ca²⁺ concentrations. The peak-to peak heights in arbitrary units are shown for the center field peak (A) and the low and high field peaks (B). The transition midpoint determined at 222 nm by CD is indicated by the arrows, and the pCa²⁺ values for the peak height results are 6.36 (h₋₁), 6.39 (h₀), and 6.36 (h₊₁). The peak heights of the free spin label were invariant to Ca²⁺ concentration (data not shown). The Ca²⁺ concentration is shown as – log₁₀ (Ca²⁺⁾_f with Ca²⁺ in units of M.

Fig. 6

Structures of apo-CaM (left) and (Ca²⁺)₄-CaM (right) with the nine methionine side chains highlighted (yellow) and identified. The structures were drawn with Chimera [70], and coordinates are from the Protein Data Bank [12], referring to reports based on NMR [6] and protein crystallography [5], respectively. The two views were chosen to optimize visualization of the methionines, and the amino (N) and carboxy (C) termini are noted. The pair-wise distances between the methionines are given in Supplement Table 2 for each of the structures.