The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

Abstract

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca²⁺-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca²⁺-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pK_a of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca²⁺-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg²⁺-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca²⁺/Mg²⁺ affinity of the EF site, and accelerates Ca²⁺ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca²⁺ affinity.

1. Introduction

Oncomodulin (Ocm) or parvalbumin β (β-PA) is a small (11–12 kDa), acidic (pI < 5), globular vertebrate-specific Ca²⁺-binding protein of the EF-hand superfamily [1,2,3,4]. The EF-hand domain consists of a 12-residue Ca²⁺-coordinating loop flanked by two amphiphilic α-helices (“helix-loop-helix” motif) [5]. Ocm contains three EF-hand domains, named according to the flanking helices: AB, CD, and EF (see Figure S1). In contrast to the high-affinity CD and EF domains (their equilibrium Ca²⁺ dissociation constants reach 1 nM [6]), the truncated 10-residue loop of the AB domain (AB-loop) is inactive due to the lack of some critical residues (Figure S2). Meanwhile, the AB domain is essential for maintenance of structural stability of Ocm and its high affinity to Ca²⁺ [7,8].

The closest relative of Ocm, parvalbumin α (α-PA) [9], has similar structural properties [2,3]. While the CD and EF domain loops are the most conservative regions of both Ocm and α-PA, their AB domains exhibit relatively high variability, especially in the loop region (see Figures S2 and S3). The consensus sequences of AB-loops of Ocm and α-PA differ in 4 positions versus 2 and 1 positions for the CD and EF loops, respectively, indicating evolutionary specialization of Ocm/α-PA via modification of the AB-loop.

Despite structural similarities, Ocm and α-PA exhibit marked differences in their expression and functional profiles. Both parvalbumin (PA) isoforms were found in muscles in a species-specific manner, favoring muscle relaxation process (shown for α-PA [10,11]) and serving as major fish allergens (mostly Ocm; reviewed in [12]). While α-PA was found in both the outer and inner hair cells of cochlea, Ocm expression is restricted to the outer hair cells (2–3 mM) [13,14]. Targeted deletion of Ocm causes progressive cochlear dysfunction [15]. Ocm was found in placenta and in numerous cancers (reviewed in [16]), while α-PA is expressed in distal convoluted tubule [17]. Neuronal expression of Ocm is highly limited, and becomes noticeable in α-PA knockout mice [18]. Ocm exhibits hormone-like and growth factor activities (reviewed in [19]). It is expressed and secreted by macrophages and neutrophils and serves as a growth factor for neurons [20,21]. Chicken β-PA is expressed by epithelial cells in thymus and circulates in the blood, stimulating development of cell-mediated immunity (see in [19]). Ocm and α-PA are present in cutaneous mucus of some amphibians and fish genera, participating in bacterial defense via Ca²⁺ chelation and serving as chemoattractants for thamnophiine snakes [22]. In spite of the wide spectrum of biological activities of Ocm and α-PA, their targets and molecular mechanisms of action remain obscure. As Ca²⁺ binding induces only minor changes in tertiary structure of α-PA [23], it is considered as a Ca²⁺ buffer (as observed in muscles [10,11]). Meanwhile, the marked calcium sensitivity of tertiary structure of β-PA [24,25,26] argues that Ocm could serve as a Ca²⁺ sensor. Such activity was reported for rat Ocm, which recognizes and inhibits glutathione reductase under Ca²⁺ excess [27]. Importantly, Ca²⁺ binding induces in β-PA the structural rearrangements, which consistently involve the AB domain [24,25,26]. Therefore, the AB domain of Ocm might be crucial for Ca²⁺-dependent target recognition.

Positions 3, 5, 8, and 9 of the AB-loop are highly conservative in both Ocm and α-PA (see Figure S4). Positions 5, 8, and 9 of both PA lineages mainly contain Ala, Ser, and Phe residues, respectively. The only difference between the highly conservative residues of Ocm and α-PA is position 3, which mostly contains Cys in Ocm and Phe in α-PA. Therefore, the highly conservative Cys residue of Ocm could be critical for functional specialization of the protein.

Studies of reactivity of the conservative Cys of rat Ocm and functional consequences of its disulfide dimerization gave surprisingly conflicting results [28,29]. Meanwhile, direct structural data evidence exposure of the Cys18 to solvent upon Ca²⁺ removal [24] (Figure S1), in line with elevated reactivity of the apoprotein with Ellman’s reagent [28]. The analogous effect was reported for carp [30], perch [31], and silver hake [32] β-PAs. The exposure of Cys18 of rat Ocm is accompanied by disruption of the hydrogen bonds between carbonyl oxygen of Cys18 and side chain nitrogen atoms of Arg75, disappearance of the salt bridge between side chains of Arg75 and Glu81, along with formation of the hydrogen bond between side chains of Arg75 and Glu25 [24] (Figure S1). The evident Ca²⁺-induced changes in both solvent accessibility of Cys18 of rat Ocm and its interactions with neighboring groups point out that minor structural modifications of this residue could affect the Ca²⁺ binding process and/or structural properties of the Ca²⁺-bound/free protein states. To explore this possibility, we have studied influence of the homologous C18S substitution, serving as a model of the thiol oxidation, on conformational stability and metal-binding properties of recombinant rat Ocm. Our analysis of thiol–disulfide equilibrium of the Cys18 showed that its redox potential is close to the physiological level, which implies accumulation of disulfide dimer of Ocm under oxidizing conditions, as reported for some other EF-hand proteins. Overall, the data presented favor the view that the highly conservative Cys18 of rat Ocm likely serves as a redox sensor.

2. Materials and Methods

2.1. Materials

Molecular biology grade HEPES (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), ultra-grade H₃BO₃/glycine/Tris (tris(hydroxymethyl)aminomethane), and MES (2-(N-morpholino)ethanesulfonic acid) were from Calbiochem (San Diego, CA, USA), Fluka (Munich, Germany), Sigma-Aldrich Co. (St. Louis, CA, USA), and Amresco (Solon, OH, USA), respectively. Pharma grade KCl, ultra-grade TCA (trichloroacetic acid), LC/MS grade acetonitrile, reagent-grade formic acid, and GSH (reduced L-glutathione)/GSSG (oxidized L-glutathione) were purchased from PanReac AppliChem. (Darmstadt, Germany). Biotechnology grade DTT (DL-dithiothreitol) was purchased from DiaM (Moscow, Russia). Biotechnology grade 2-ME (2-mercaptoethanol) and molecular mass markers for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were from Helicon (Moscow, Russia). Analytical grade bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid)/DTNB (5,5′-dithiobis(2-nitrobenzoic acid)), ultra-grade KOH/EDTA (ethylenediaminetetraacetic acid)/EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), standard solutions of CaCl₂ and MgCl₂ (Ca²⁺ content of 0.0005%) were purchased from Sigma-Aldrich Co. (St. Louis, CA, USA). Standard solutions of EDTA/EGTA potassium salt were prepared as described in [6]. Standard solution of Mg(NO₃)₂ was purchased from Fluka (Munich, Germany). Molecular biology grade glutaric aldehyde and ultra-grade CuCl₂ were from Amersham Biosciences (Uppsala, Sweden) and Riedel-de Haën (Buchs, Switzerland), respectively. Biochemistry grade guanidinium chloride and Coomassie Brilliant Blue R-250 were products of Merck (Darmstadt. Germany). Toyopearl SuperQ-650M was purchased from Tosoh Bioscience (Griesheim, Germany). Sephadex G-25 and PD MidiTrap^TM G-25 were products of Pharmacia LKB (Uppsala, Sweden) and GE Healthcare (Chicago, IL, USA), respectively. d-10-camphorsulfonic acid was acquired from JASCO, Inc. (Tokyo, Japan). Other chemicals were reagent grade or higher.

All buffers and other solutions were prepared using ultrapure water (Millipore Simplicity 185 system). To avoid contamination of protein samples with Ca²⁺, only plastics or quartz ware were used instead of glassware. Degassed buffers were used for preparation of DTT solutions just before their usage to avoid DTT oxidation. Thermo SnakeSkin dialysis tubing (3.5 kDa MWCO) and Millipore Amicon Ultra centrifugal filters (3.0 kDa MWCO) were used for dialysis and concentration of protein solutions, respectively.

2.2. Preparation of Recombinant Rat Ocm

Recombinant wild type rat (Rattus norvegicus) oncomodulin (“rWT Ocm”) was isolated and purified as described earlier [33]. C18S substitution was introduced into rat Ocm by site-directed mutagenesis [34] of the plasmid encoding rWT Ocm via use of the oligonucleotide with mismatch in codon 18 (“TGC” triplet is replaced by “AGC”). C18S Ocm was expressed and purified similarly to rWT Ocm, but with the following modifications. The reducing agents were not used. The Toyopearl SuperQ-650M column (0.9 cm × 7.5 cm) was equilibrated with 20 mM Tris-HCl, 75 mM KCl, pH 8.5 (buffer B). The protein sample was dialyzed against buffer B and loaded onto the column. The latter was washed by buffer B and then by 20 mM Tris-HCl, 90 mM KCl, pH 8.5 buffer. The yield was 40–60 mg of protein per liter of cell culture for both Ocm samples. Purity of the protein samples was confirmed by native and SDS-PAGE, and characteristic UV absorption and fluorescence spectra (rat Ocm contains Tyr and Phe residues, but lacks Trp). The purified rWT Ocm samples were exhaustively dialyzed at 4 °C against distilled water with 1 mM 2-ME (avoided in the case of C18S Ocm), freeze dried, and stored at −20 °C. Before experiments, the dry rWT Ocm samples were dissolved in distilled water, reduced by addition of freshly prepared 5 mM DTT and purified from 2-ME and DTT by passage through PD MidiTrap G-25 column equilibrated with a buffer of choice. The reduced protein was used for experiments as soon as possible. Molecular masses of the Ocm samples were determined by liquid chromatography–mass spectrometry. The sample of 0.5 μM Ocm in 15:75 (v/v) mixture of deionized water and acetonitrile (10 mM formic acid) was passed through C18 sorbent (granule diameter of 2.7 µm and pore size of 300 Å), 15–100% acetonitrile gradient for 220 min, using EASY-nLC 1000 UHPLC chromatograph (Thermo Scientific, Waltham, MA, USA). The mass spectra were collected by Orbitrap Elite mass spectrometer (Thermo Scientific) in a positive ion mode (m/z from 600 to 2000) at detector voltage 1.8 kV, capillary temperature of 240 °C, and nebulizing nitrogen flow of 1 L/min. Ocm concentrations were calculated spectrophotometrically using the molar extinction coefficient at 280 nm, ε_{280 nm}, derived from the molar extinction coefficients at 205 nm estimated according to the work in [35]: 2409 M⁻¹cm⁻¹ and 2721 M⁻¹cm⁻¹ for apo- and metal-loaded rWT Ocm, respectively; 2202 M⁻¹cm⁻¹ and 2575 M⁻¹cm⁻¹ for apo- and metal-loaded C18S Ocm, respectively.

2.3. Ellman’s Assay

Quantitation of thiol groups of rWT Ocm (1 Cys residue) was performed according to the modified Ellman’s test [36]. The dry rWT Ocm sample was dissolved in distilled water (100 μL), reduced by addition of freshly prepared 5 mM DTT and purified from 2-ME and DTT by passage through PD MidiTrap G-25 column equilibrated with 50 mM Tris-HCl, 1 mM EDTA, pH 8.0 buffer. rWT Ocm (20 µM) was unfolded by 6 M guanidinium chloride and treated by 1 mM DTNB for 10 min at 20 °C. The kinetics of the reaction was monitored by absorbance at 412 nm with a Cary 100 spectrophotometer (Varian Inc., Palo Alto, CA, USA) in a double-beam mode versus the reference solution without the protein. The molar extinction coefficient of 5-thio-2-nitrobenzoic acid at 412 nm of 13,700 M⁻¹cm⁻¹ (manual to Thermo Scientific cat. #22582) was used. Efficacy of the procedure used was confirmed by the analogous examination of reduced L-glutathione solution.

2.4. Removal of Metal Ions from Ocm

The contaminating Ca²⁺/Mg²⁺ ions were removed from rWT Ocm using precipitation by TCA [37] of the preliminary reduced protein (see above) (10–50 mM HEPES-KOH with/without 100 mM KCl, pH 7.0–8.2 or 10-20 mM H₃BO₃-KOH, pH 8.4–8.8 or 20 mM glycine-KOH, pH 9.2 or 10 mM Tris-HCl, pH 7.3–7.4 buffer), followed by purification from Ca²⁺/Mg²⁺ and TCA by gel filtration method [38] using Sephadex G-25 column equilibrated with a respective buffer. To protect the reduced rWT Ocm from oxidation, 5 mM of freshly prepared DTT solution was added prior to the precipitation by TCA and before the gel filtration. In case of C18S Ocm, the dry protein sample was dissolved in the same buffer and purified from metal ions in the identical manner, but without addition of DTT. This procedure was used for metal removal from rWT/C18S Ocm for preparation of disulfide dimer of rWT Ocm and its redox studies, CD measurements of apo- and Mg²⁺-loaded Ocm, DSC measurements of Mg²⁺-loaded Ocm, chemical cross-linking and DLS measurements, bis-ANS fluorescence, and metal affinity studies. Only the gel filtration method [38] was used for spectrophotometric pH titrations of Ocm, CD measurements of Ca²⁺-loaded Ocm, DSC measurements of apo- and Ca²⁺-loaded Ocm, and kinetic studies of metal association/dissociation.

2.5. Spectrophotometric pH Titration of Ocm

Estimation of pK_a value of Cys18 of Ocm at 20 °C was carried out spectrophotometrically mainly as described in ref. [39]. UV absorption spectra of rWT/C18S Ocm (23 μM) were measured with a Cary 100 spectrophotometer (Varian Inc., Palo Alto, CA, USA), equipped with NESLAB RTE-7 circulator (Thermo Scientific, Waltham, MA, USA). Buffer conditions: 30 mM MES, 30 mM HEPES, 30 mM H₃BO₃, 30 mM glycine, 100 mM KCl, 1.5 mM EDTA (apo-Ocm), or 1 mM CaCl₂ (Ca²⁺-bound Ocm). The protein solution was titrated by small aliquots of KOH stock solution. To avoid solution contamination with Ca²⁺ during the titrations, pH values were independently measured under identical conditions. The protein absorbance at 240 nm was corrected for a minor light scattering contribution via extrapolation with power function of the long wavelength region of Ocm absorption spectrum. Contribution of deprotonated Tyr residues to the absorbance at alkaline pH values [40] was subtracted from the experimental data.

2.6. Preparation of Disulfide Dimer of rWT Ocm

Disulfide dimer of rWT Ocm (“dOcm”) was prepared by Cu²⁺-induced oxidation of the protein reported earlier [28]. Two-hundred micromolar CuCl₂ was added to the metal-depleted rWT Ocm (50 μM) in 50 mM HEPES-KOH, 100 mM KCl, pH 7.4 buffer (refer to Removal of metal ions from Ocm section), followed by incubation at 25 °C for 24 h. Metal ions were removed from the protein sample using the same metal removal procedure (TCA treatment, followed by gel filtration), but without DTT usage. dOcm solution was frozen and stored at −20 °C. The SDS-PAGE analysis of the sample under non-reducing/reducing conditions confirmed total conversion of monomeric rWT Ocm into the disulfide dimer (data not shown).

2.7. Redox Dependence of Disulfide Dimerization of rWT Ocm

The redox potential of Cys18 of rWT Ocm was measured mainly as described in [41]. Metal-free/Ca²⁺-loaded dOcm solution (1.5 μM) in 50 mM HEPES-KOH, 100 mM KCl, 1 mM EDTA/CaCl₂, pH 7.4 buffer was incubated with 0.2 mM GSSG and various concentrations of GSH (25 μM to 1.6 mM). The dissolved oxygen was removed from the solutions using a degassing station from TA Instruments (pressure of 25 mm Hg for 20 min, stirring at 700 rpm), followed by saturation of the solutions with argon by bubbling 99.993% argon for 7–10 min. The reaction solutions were sealed under argon, incubated at 35 °C for 62 h, and analyzed by non-reducing SDS-PAGE (15% resolving gel). The gels were stained with Coomassie Brilliant Blue R-250 and scanned using Molecular Imager PharosFX Plus System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The weight fractions of Ocm forms were calculated by Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The redox equilibrium in the system of a protein with one thiol group and GSH/GSSG couple can be described by the scheme [I]:

K
M–M + 2 GSH ↔ 2 M + GSSG,

[I]

where M–M and M denote disulfide dimer and monomer of the protein, respectively, and K is an apparent dimer dissociation constant (K = [M]²[GSSG]/[M-M][GSH]²). Combining the equation for K with the material balance equation 2[M–M] + [M] = M₀ (M_o is a total concentration of the protein monomer) it is easy to obtain weight fraction of the dimeric protein, f_D, which is expressed as follows,

$$f_D=\frac{2\left[M-M\right]}{M_0}=\frac{K•{10}^x+4M_0-\sqrt{{(K•{10}^x)}^2+8M_{0}K•{10}^x}}{4M_0}$$

(1)

where x equals to log([GSH]²/[GSSG]).

The experimental dependence of f_D on x for dOcm sample was fitted by Equation (1) using OriginPro v.9.0 (OriginLab Corp., Northampton, MA, USA) software and K as a fitting parameter (the changes in [GSH] and [GSSG] due to reaction [I] were neglected).

Considering that at the middle of the transition between M–M and M (scheme [I]) their weight fractions are equal, the x value at this point can be expressed as follows.

$$x_{1/2}=\log \frac{M_0}{K}$$

(2)

At pH below pK_a value of GSH (8.65 at 25 °C and ionic strength, I, of 0.1 M [42]), the equilibrium between GSSG and GSH is described by the following scheme [43].

GSSG + 2 H⁺ + 2 e⁻ ↔ 2 GSH

[II]

The respective redox potential of the GSSG/2GSH redox pair, E_h, is defined by Nernst equation (activity coefficients of GSSG and GSH are commonly assumed to be close to 1) [42]:

$$E_h=E_0-\frac{RT}{2F}\ln \frac{{[GSH]}^2}{[GSSG]}=E_0-\frac{RT}{2F}\ln 10•x$$

(3)

where E_o is a standard reduction potential for the GSSG/2GSH couple and R, T, and F are gas constant, absolute temperature, and Faraday constant, respectively. The E_o value at the experimental conditions used here (pH 7.4 and I of 0.12 M) was estimated by linear interpolation of the E_o values at pH 7 and 8 and I of 0 and 0.25 M (25 °C [44]), which gives −297 mV. Thus, at 35 °C Equation (3) can be rewritten as

$$E_h=-297mV-30.6mV•x$$

(4)

The experimental x values were converted into the scale of redox potential of the GSSG/2GSH redox pair using Equation (4).

2.8. Circular Dichroism (CD) Measurements

Far-UV CD studies were carried out at 20 °C using J-810 spectropolarimeter (JASCO, Inc.), equipped with a Peltier-controlled cell holder. The instrument was calibrated with an aqueous solution of d-10-camphorsulfonic acid and purged with nitrogen. The quartz cell with pathlength of 1 mm was used. Ocm concentration was 7–13 µM. Buffer conditions: 10 mM H₃BO₃-KOH, pH 8.8, 1.5 mM EDTA or 1 mM CaCl₂ for apo- and Ca²⁺-loaded Ocm, respectively, and 10 mM Tris-HCl, pH 7.3 with 1 mM EGTA, 1 mM MgCl₂ for Mg²⁺-loaded Ocm. The spectral contribution of the buffer was subtracted from the experimental spectra. Bandwidth was 2 nm, averaging time 2 s, and accumulation 3. Quantitative estimates of the secondary structure fractions were performed using CDPro software [45]. The experimental data in the 200–240 nm range were treated by SELCON3, CDSSTR, and CONTIN algorithms, using SDP48 and SMP56 reference protein sets. The final secondary structure fractions represent averaged values.

2.9. Chemical Cross-Linking Experiments

The metal-depleted rWT/C18S Ocm (1.6–1.7 mg/mL) was cross-linked with 0.02% glutaric aldehyde at 20 °C in 20 mM H₃BO₃-KOH, pH 8.4 buffer, 1.5 mM EDTA or 1 mM CaCl₂/MgCl₂. The reaction proceeded overnight and was terminated with a SDS sample buffer. The samples were subjected to reducing SDS-PAGE (5% concentrating and 15% resolving gels; 5 µg of Ocm per lane) and staining with Coomassie Brilliant Blue R-250. The gel was scanned using Molecular Imager PharosFX Plus System and analyzed by Quantity One software (Bio-Rad Laboratories, Inc.).

2.10. Dynamic Light Scattering Studies (DLS)

DLS measurements were carried out for rWT/C18S Ocm at 20.0 °C using a Zetasizer Nano ZS system (Malvern Instruments Ltd.). The backscattered light from a 4 mW He-Ne laser (632.8 nm) was collected at an angle of 173°. Protein concentration was 1.3–1.7 mg/mL. The buffer conditions: 20 mM H₃BO₃-KOH, pH 8.4, 1.5 mM EDTA or 1 mM CaCl₂/MgCl₂. The protein samples were preliminarily passed through 0.02 µM Whatman Anotop 10 syringe filters. The acquisition time for a single autocorrelation function was 100 s. The resulting autocorrelation function was an average of ten measurements. The intensity-weighted size distributions were calculated using preset parameters for distilled water at 20 °C (refractive index of 1.33 and a viscosity value of 1.0031 cP). The estimates of mean hydrodynamic radii (R_h) of Ocm were derived from these distributions.

2.11. The Scanning Calorimetry Measurements

The differential scanning calorimetry (DSC) studies were carried out on a Nano DSC microcalorimeter (TA Instruments) at a heating rate of 1 K/min and an excess pressure of 4 bar. Ocm concentration was 1.2–2.1 mg/mL. Buffer conditions: 10 mM H₃BO₃-KOH, 1 mM CaCl₂, pH 9.0 (Ca²⁺-loaded Ocm); 20 mM glycine-KOH, 1 mM EDTA, pH 9.2 (apo-Ocm) or 20 mM glycine-KOH, 1 mM MgCl₂, pH 9.2 (Mg²⁺-loaded Ocm). The DSC measurements and calculations of protein specific heat capacity (C_p) were performed as described earlier [46]. The partial molar volume of Ocm and specific heat capacity of the fully unfolded protein were estimated according to the works in [47,48], respectively. The temperature dependence of C_p was analyzed according to the cooperative two-state model:

n∙N ↔ n∙D

[III]

where N and D denote native and denatured protein states, respectively, and n is a number of molecules involved in the transition. The experimental data were fitted using OriginPro v.9.0 software (OriginLab Corp., Northampton, MA, USA), using the equations derived earlier [49]. The heat capacity change accompanying the transition (∆C_p) was supposed to be independent of temperature. ∆C_p, mid-transition temperature (T₀), enthalpy of protein denaturation at temperature T₀ (∆H₀) and n were used as fitting parameters.

2.12. Fluorescence Measurements

Fluorescence studies were carried out at 20 °C on a Cary Eclipse spectrofluorimeter (Varian, Inc.), equipped with a Peltier-controlled cell holder. Quartz cells with pathlength of 10 mm were used. Protein and bis-ANS fluorescence were excited at 275 nm and 385 nm, respectively. Maximum intensities (I_max) and maximum positions (λ_max) of fluorescence emission spectra were obtained from fits of the spectra with log-normal curves [50] using LogNormal software (IBI RAS, Pushchino).

2.13. Fluorimetric Estimation of Ca²⁺ Affinity of Ocm

Ca²⁺ affinity of rWT/C18S Ocm at 20 °C was estimated mainly as described in [6] from spectrofluorimetric titration of the metal-depleted protein with CaCl₂ standard solution, followed by titration of the Ca²⁺-loaded protein with EDTA potassium salt standard solution. Ocm concentration was 20–23 μM. Buffer conditions: 10 mM HEPES-KOH, pH 8.2. Intrinsic Ocm fluorescence intensities were corrected for the dilution effect via division of the experimental values by a factor of (1–10^−Dexc), where D_exc is the protein absorption at the excitation wavelength [51]. The experimental data were described by the sequential metal binding scheme [2]:

K_a1
P + M ↔ P∙M,
K_a2
P∙M + M ↔ P∙M₂,

[IV]

where P and M denote protein and metal ion, respectively, and K_a1 and K_a2 are equilibrium metal association constants for the two active EF-hands of Ocm. The competition between Ocm and EDTA for metal ions was taken into account:

K_EDTA

EDTA + M ↔ EDTA∙M

[V]

The metal association constant for EDTA, K_EDTA, was calculated according to the work in [52], using the thermodynamic constants derived from IUPAC Stability Constants Database, SC-Database v.4.79 (Academic Software, Timble, UK), and corrected for temperature and ionic strength using van’t Hoff and Davies equations, respectively. The data were globally fitted according to the schemes [IV] and [V] using FluoTitr v.1.4 software (IBI RAS, Pushchino, Russia). Log(K_a1), log(K_a2) and specific fluorescence intensities of Ocm states were used as the fitting parameters. K_a1 and K_a2 values were estimated at 295 nm, 306 nm and 315 nm, and averaged.

2.14. Estimation of Kinetic Metal Association/Dissociation Constants

Stopped-flow fluorescence measurements were performed using RX.2000 Rapid Mix Accessory (Applied Photophysics Ltd., Leatherhead, UK) with a dead time of 6 ms. Temperature of all solutions was kept at 20 °C using NESLAB RTE-7 circulator (Thermo Scientific) and Peltier-controlled cell holder. Equal volumes (0.3 mL) of 5–18 µM rWT/C18S Ocm (Ca²⁺ to protein molar ratio of 2:1, or 5:1 for Mg²⁺ and the metal-depleted protein) and 1–2 mM EGTA/EDTA in 10 mM HEPES-KOH buffer, pH 8.2 (Ca²⁺ dissociation experiments) or pH 7.3 (Mg²⁺ dissociation), were mixed using a pneumatic drive. To prevent Ca²⁺ contamination of Ocm solution during the Mg²⁺ dissociation studies, 1 mM EGTA was added. Tyr fluorescence emission was measured at 305 nm. Each experiment was repeated 4 times. The experimental data were fitted within the sequential metal binding scheme [IV] [2] as described earlier [53].

2.15. Estimation of Mg²⁺ Affinity of Ocm by Equilibrium Dialysis

Mg²⁺ binding to rWT/C18S Ocm was studied by equilibrium dialysis method using a 96-well micro-equilibrium dialysis system (HTDialysis, LLC) [54,55]. Each well (500 μL) of the Teflon block is separated by dialysis membrane (regenerated cellulose with MWCO of 3.5 kDa, preliminarily soaked in 20% ethanol). The membrane and the block were cleaned from metal ions according to the procedure described in [56]. One half of the each well was filled with 200 μL of 30–220 µM solution of Ocm in a buffer (30 mM HEPES-KOH, 1 mM EGTA, pH 7.4), whereas the another half contained 200 μL of the same buffer with 5–1500 µM Mg(NO₃)₂ without Ocm. The wells were tightly sealed and equilibrated by continuous shaking (130 rpm) of the block at (20.0 ± 0.5) °C for 17–20 h. Total concentrations of Mg²⁺ in the equilibrated solutions were measured by electrothermal atomization atomic absorption spectrometer iCE 3000 (Thermo Scientific), using argon as an inert gas. The magnesium absorption band at 202.6 nm and Zeeman background correction were used. Ten microliters of diluted sample solution was analyzed in the graphite cuvette from 2 to 3 times. The analytical signal was calibrated using Mg(NO₃)₂ standard solution (0–40 μg/L) in the same buffer. Concentration of Mg²⁺ bound to Ocm ([bM]) was estimated for the each well as a difference between the total Mg²⁺ concentrations measured for both halves of the well, assuming that free Mg²⁺ concentrations ([fM]) do not differ between the two halves of the well. The total protein concentration (P₀) was determined spectrophotometrically for the each well after the equilibrium dialysis. Since at Ocm concentration of 220 μM the Donnan coefficient is close to 1, the error arising due to the Gibbs–Donnan effect is negligible compared to the experimental uncertainties in Mg²⁺ and Ocm concentrations. The experimental data were analyzed using the sequential metal binding scheme [IV]. The number of Mg²⁺ ions bound per Ocm molecule was described by the following equation.

$$\frac{[\mathrm{bM}]}{{\mathrm{P}}_0}=\frac{K_1[\mathrm{fM}]+2\cdot K_1{K}_2{[\mathrm{fM}]}^2}{1+K_1[\mathrm{fM}]+K_1{K}_2{[\mathrm{fM}]}^2}$$

(5)

Log(K₁) and log(K₂) were used as fitting parameters in OriginPro v.9.0 software (OriginLab Corp., Northampton, MA, USA). The weighting factor $w_i=\frac{1}{{\sigma }_i^2}$ was used for the experimental points.

3. Results and Discussion

3.1. Isolation of Recombinant Rat Ocm

Recombinant wild type rat (Rattus norvegicus) oncomodulin (rWT Ocm; Swiss-Prot entry P02631) and its mutant form with homologous substitution of the highly conservative Cys18 in the inactive AB-loop (Figure S2) for Ser (C18S Ocm) were isolated mainly as described in [33]. The protein samples were homogeneous as judged by native and SDS-PAGE. The absence of Trp-containing contaminating proteins in the Ocm samples was confirmed by the lack of Trp contribution in their UV absorption and fluorescence spectra (data not shown). The rWT Ocm sample was stored in the presence of 2-ME to prevent unfavorable oxidation of Cys18 thiol. The 2-ME molecules were removed from the protein by fresh DTT treatment, followed by Ocm cleaning from the reducing agents by gel filtration (see Materials and methods section). The resulting free thiol content was 0.89 thiols per Ocm molecule, as evidenced by Ellman’s test.

Mass spectrometry measurements revealed two major fractions of the reduced rWT Ocm sample. One of them (~45%) had a mass of 12,057 Da, which is consistent with mass of the reduced Swiss-Prot entry P02631 without Met1 and N-terminal acetyl group. The other rWT Ocm fraction (~46%) was 131 Da heavier, in line with the presence of Met1. The minor fractions (~9%) of this sample corresponded to the Ca²⁺-bound and/or oxidized states of the abovementioned forms. The major fraction (~81%) of C18S Ocm had a mass of 12,041 Da, which corresponds to the mass expected for the protein without Met1 and N-terminal acetyl group. The remaining C18S Ocm (~19%) had a mass consistent with the presence of Met1.

3.2. Thiol Group pK_a Value

As the reactivity of a thiol group depends on its pK_a value, we tried to estimate the pK_a value of Cys18 of rWT Ocm. Deprotonated alkyl thiol group displays an absorption band around 240 nm (ε ≈ 4000 M⁻¹cm⁻¹) in the UV absorption spectrum, where absorption of the protonated thiol is negligible [57]. Therefore, the pH-induced change in ionic state (protonation-deprotonation) of the single thiol of rWT Ocm can be monitored by the protein absorbance at 240 nm, after subtraction of spectral contribution of deprotonated Tyr residues [40] of Ocm. Surprisingly, the resulting pH dependence of molar extinction coefficient at 240 nm (ε_{240 nm}) for apo- and Ca²⁺-loaded forms of reduced rWT Ocm showed no changes within pH range from 6.5 to 9.5 (Figure 1). The identical control experiments for C18S Ocm gave analogous results. This means that the the single thiol of rWT Ocm does not change its ionic state within the pH range from 6.5 to 9. The changes in ε_{240 nm} at pH above 9.5 can be related to Cys18 deprotonation and/or alkaline denaturation of the protein. As average pK_a value of Cys in folded proteins is 6.8 ± 2.7 [58], the result obtained show that Cys18 of rWT Ocm is characterized by pK_a value which is either well below the average level or above most of the values reported for Cys residues of folded proteins, regardless of calcium level. In any case, the Cys18 of rWT Ocm is expected to be either fully protonated (pK_a > 9.5) or fully deprotonated (pK_a < 6.5) at physiological pH values, in contrast to the conservative Cys38 of the inactive EF-hand loop of bovine recoverin (pK_a = 7.6 [39]). As Ca²⁺ removal from Ocm increases solvent accessibility of the conservative Cys residue [24,28,30,31,32], the thiol group of apo-Ocm is expected to exhibit a higher pK_a value compared to that for the Ca²⁺-bound protein. Meanwhile, we did not detect this effect.

3.3. Redox Potential of Cys18 of rWT Ocm

The high intracellular concentrations of Ocm (up to 2–3 mM [14]) favor its involvement into thiol-disulfide exchange reactions. One of them is the interaction with a major cell redox buffer, glutathione (GSH). The studies of thiol-disulfide exchange between rWT Ocm and GSH were performed with disulfide dimer of the protein (referred to as “dOcm”), which was prepared by Cu²⁺-induced oxidation of rWT Ocm (see Preparation of disulfide dimer of rWT Ocm section). The equilibrium between metal-free/Ca²⁺-loaded dOcm (1.5 μM) and glutathione redox couple (scheme [I]) at 35 °C has been studied mainly as described in [41], in the presence of 0.2 mM GSSG and 25 μM–1.6 mM GSH, which corresponds to the redox potential of GSSG/2GSH redox pair (scheme [II]), E_h, from −128 mV to −239 mV (Equation (4)). The incubation of this system under anaerobic conditions for 62 h ensures its efficient equilibration [41]. The weight fractions of dOcm, f_D, were estimated using non-reducing SDS-PAGE (Figure 2). Regardless of Ca²⁺ content, the more reducing solvent conditions induce dOcm reduction with half-transition at E_h values about −170 mV. The experimental data are well described by the simplest scheme [I] (Equation (1); Figure 2), which gives the apparent dimer dissociation constant, K, of 0.026 ± 0.004 and 0.048 ± 0.003, for Ca²⁺-loaded and apo-Ocm, respectively. The corresponding x_1/2 values are −3.94 ± 0.06 and −4.20 ± 0.03 (Equation (2)), while the E_h values are (−176.5 ± 1.9) mV and (−168.4 ± 0.9) mV (Equation (4)), for Ca²⁺-loaded and apo-Ocm, respectively. The resulting E_h values are close to those reported for erythrocytes of diabetic patients (−179 mV [59]), apoptotic murine hybridoma and HL-60 cells (−170 mV and −167 mV, respectively), and differentiating HT29 cells (−160 mV) (reviewed in [43]). Therefore, the intracellular conditions accompanied by the elevated redox potential are likely to favor accumulation of disulfide dimer of Ocm. Furthermore, extracellular Ocm [19,20,21] is confronted with even more oxidizing conditions with the E_h values of −140 mV to −131 mV, which drastically increase with age (−93 mV to −84 mV) and under such pathological conditions as diabetes and age-related macular degeneration (up to −65 mV) [59]. Early atherosclerosis [60] and smoking [61] are accompanied by less marked increase in the E_h values. The disulfide dimer of Ocm accumulated under the more oxidizing conditions is expected to possess an altered functional activity, as exemplified by efficient activation by the dimer of two calmodulin-dependent enzymes [29]. As Ocm regulates GSSG and GSH levels via Ca²⁺-dependent inhibition of glutathione reductase [62], the disulfide dimerization of Ocm could affect the intracellular redox potential due to the respective changes in the [GSH]/[GSSG] ratio.

The difference in pK_a values of the conservative thiols located in the inactive EF-hand loops of rat Ocm and bovine recoverin (see above) is probably translated into differences in their E_h values (−152 and −128 mV, for the Ca²⁺-loaded and Ca²⁺-free recoverin [41]). The relatively high E_h values of recoverin are sufficient for manifestation of its disulfide dimerization under in vivo conditions [41,63], thereby indicating such possibility for Ocm. The E_h values decrease upon calcium binding to Ocm and recoverin, implying that calcium promotes accumulation of their disulfide dimers.

3.4. Impact of C18S Substitution on Structural and Functional Properties of Rat Ocm

Aside from disulfide dimerization of Ocm, oxidizing physiological conditions may induce numerous chemical modifications of its Cys18 residue, including formation of mixed disulfides, sulfenic, sulfinic, and sulfonic acids [64], as observed for recoverin [63]. To explore the susceptibility of rWT Ocm properties to minor structural modifications of the Cys18, the latter was replaced by a homologous residue, Ser.

The secondary structure fractions for apo- and Mg²⁺/Ca²⁺-loaded rWT/C18S Ocm were estimated by far-UV CD using CDPro software [45] (

Table 1. The secondary structure fractions of rWT/C18S Ocm (7–13 µM) estimated from far-UV CD spectra [45] and parameters of fluorescence emission spectrum (λ_max, spectrum maximum position; I_max, maximum fluorescence intensity) of hydrophobic probe bis-ANS (1 µM) in presence of the protein (18–23 µM) at 20 °C. Buffer conditions: 10 mM H₃BO₃-KOH, pH 8.8 or 10 mM Tris-HCl, pH 7.3 for CD studies; 10 mM HEPES-KOH, pH 7.3/8.2 for bis-ANS fluorescence measurements. Ca²⁺/Mg²⁺ content was controlled by addition to the metal-depleted protein of 1.5 mM EDTA (apo-Ocm), 1 mM CaCl₂ (Ca²⁺-loaded Ocm) or 1 mM EGTA, 1 mM MgCl₂ (Mg²⁺-loaded Ocm). Standard deviations for estimates of the secondary structure fractions are indicated.

Ocm State	Secondary Structure Fractions				Bis-ANS Fluorescence Emission
	rWT		C18S		rWT		C18S		Free Bis-ANS
	α-helices,%	β-sheets, %	α-helices, %	β-sheets, %	λmax, nm	Imax, a.u.	λmax, nm	Imax, a.u.	λmax, nm	Imax, a.u.
apo	32.4 ± 4.3	17.1 ± 3.0	12.7 ± 1.8	26.3 ± 2.3	496	90	497	115	538	8
Mg2+	53.2 ± 4.3	9.1 ± 3.0	32.6 ± 1.8	15.9 ± 2.3	494	74	496	98	539	8
Ca2+	54.8 ± 4.3	8.0 ± 3.0	53.9 ± 1.8	8.1 ± 2.3	499	34	494	58	536	8

). Although α-helices content of Ca²⁺-loaded rWT Ocm (55%) is in line with the X-ray structure 1OMD (55.6%), α-helicity of the apoprotein (32%) is 22% lower as compared to that derived from the NMR (nuclear magnetic resonance) structure 2NLN (54.6%). The latter is likely due to the Na⁺-binding induced stabilization of Ocm structure during NMR measurements (150 mM NaCl should favor Na⁺ binding to apo-Ocm according to its dissociation constant of 17 mM [65]). While Ca²⁺-bound states of rWT and C18S Ocm exhibit equivalent secondary structures, α-helices content of Mg²⁺-bound and metal-free states of C18S Ocm are lowered compared to rWT Ocm by 20–21%, partly at expense of the increase in β-sheets content (Table 1). Overall, secondary structure of apo-C18S Ocm is severely disorganized, but recovers upon Ca²⁺ binding, while Mg²⁺ binding is insufficient for full recovery of the secondary structure. The disturbance in secondary structure of rat Ocm in response to C18S substitution increases solvent exposure of its hydrophobic residues, as judged from elevated fluorescence intensity of a hydrophobic fluorescent probe 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS), regardless of Mg²⁺/Ca²⁺ content (Table 1).

As the increased solvent exposure of hydrophobic residues of C18S Ocm could promote its multimerization, the quaternary structure of rWT/C18S Ocm was studied by chemical cross-linking and dynamic light scattering (DLS) at 20 °C (

Table 2. Study of quaternary structure of rWT/C18S Ocm at 20 °C according to chemical cross-linking with 0.02% glutaric aldehyde and DLS data (mean hydrodynamic radius, R_h); 1.3–1.7 mg/mL Ocm in 20 mM H₃BO₃, pH 8.4 buffer, 1.5 mM EDTA or 1 mM MgCl₂/CaCl₂ for the apo- and Mg²⁺/Ca²⁺-loaded protein, respectively. Standard deviations for the R_h values are indicated.

Ocm	Protein State	Fractions of Protein Multimers		Rh, nm
		12 kDa Band, %	24 kDa Band, %
rWT	apo	100	0	2.22 ± 0.08
C18S		100	0	2.42 ± 0.04
rWT	Mg2+	100	0	2.01 ± 0.06
C18S		100	0	1.97 ± 0.04
rWT	Ca2+	96	4	1.80 ± 0.03
C18S		95	5	1.81 ± 0.04

). SDS-PAGE of the Ocm forms cross-linked with 0.02% glutaric aldehyde shows that vast majority of the protein is monomeric, regardless of Mg²⁺/Ca²⁺ content. Therefore, the increase in mean hydrodynamic radius (R_h) of rWT/C18S Ocm observed upon the metal depletion and Ca²⁺ replacement by Mg²⁺ reflects expansion of the protein molecule due to disorganization of its structure, in accordance with the CD data (Table 1). While R_h values of the Mg²⁺/Ca²⁺-loaded rWT and C18S Ocm do not differ from each other, apo-state of C18S Ocm is considerably less compact compared to apo-state of rWT Ocm, in line with the drastically lowered α-helicity of the mutant.

As metal-free C18S Ocm exhibits the combination of features specific for the proteins lacking rigid tertiary structure, we have studied the integrity of its structure by differential scanning calorimetry, DSC (Figure 3). In contrast to the distinct heat sorption peak observed for apo-state of rWT Ocm (mid-transition temperature, T₀, estimated within the cooperative two-state model [III], of 30.7 °C–

Table 3. Thermodynamic parameters of thermal denaturation of various states of rWT/C18S Ocm, estimated from the DSC data shown in Figure 3 according to the cooperative two-state model [III]: mid-transition temperature (T₀), enthalpy of protein denaturation at temperature T₀ (∆H₀), heat capacity change accompanying the transition (∆C_p), and number of molecules involved in the transition (n).

Ocm	Protein State	T0, °C	ΔH0, J/g	ΔCp, J/(g·K)	n
rWT	apo	30.7	11.3	0.59	0.83
	Mg2+	60.9	27.2	0.26	0.83
	Ca2+	89.3	52.9	−0.31	0.77
C18S	apo	n.d.	n.d.	n.d.	n.d.
	Mg2+	55.9	16.4	−0.09	1.25
	Ca2+	85.6	31.8	−0.41	0.87

n.d.: not determined.

), metal-free C18S Ocm does not reveal measurable heat sorption peaks characteristic for first-order thermal transitions. Furthermore, its specific heat capacity closely approaches that calculated for the fully unfolded protein [48]. Therefore, DSC data also strongly evidence unordered tertiary structure in apo-state of C18S Ocm. Meanwhile, Mg²⁺/Ca²⁺-loaded states of C18S Ocm demonstrate well-defined heat sorption peaks, shifted relative to the corresponding peaks of rWT Ocm towards lower temperatures by 5 °C and 3.7 °C, respectively (Table 3). Thus, Mg²⁺/Ca²⁺ binding evens out the differences between the properties of rWT and C18S Ocms, as observed for hydrodynamic radius (Table 2) and α-helices content (Table 1).

The drastic changes in structural properties of metal-depleted Ocm induced by C18S substitution could affect kinetic and equilibrium parameters of Ca²⁺/Mg²⁺-binding to the protein, which are crucial for PA functioning (at least as a muscle relaxation factor [10,11]). Ca²⁺-binding to rWT/C18S Ocm was explored using spectrofluorimetric titrations of their apo-forms by Ca²⁺, followed by competitive Ca²⁺ removal with Ca²⁺ chelator, EDTA (Figure 4a). The binding of Ca²⁺ to the proteins results in an increase in Tyr fluorescence intensity (rat Ocm has no Trp residues), while the Ca²⁺ removal causes the opposite effect. Analysis of the data according to the sequential metal-binding scheme [IV] shows that our estimates of Ca²⁺ affinity of rWT Ocm (

Table 4. The values of equilibrium Ca²⁺ association constants for rWT/C18S Ocm at 20 °C estimated from the experimental data shown in Figure 4a (fluorimetric Ca²⁺/EDTA titrations) and 5 (equilibrium dialysis) according to the sequential metal-binding scheme [IV]. Standard deviations for the estimates of equilibrium metal binding constants (K_a1 and K_a2) are indicated.

Ca2+			Mg2+
Ocm	Fluorimetric Ca2+/EDTA Titrations		Equilibrium Dialysis
	log Ka1	log Ka2	log Ka1	log Ka2
rWT	9.1 ± 0.3	7.5 ± 0.3	5.15 ± 0.03	4.16 ± 0.09
C18S	7.6 ± 0.2	7.9 ± 0.2	4.71 ± 0.04	4.00 ± 0.19

) exceed the previous estimates [66] by 1–1.6 orders of magnitude, which is likely due to much lower KCl concentration used in our work. It is clearly seen that Ca²⁺ ions are removed from C18S Ocm by EDTA essentially easier than from rWT Ocm, implying that the latter has higher calcium affinity (Figure 4a). Indeed, Ca²⁺ affinity of the EF-hand of C18S Ocm, which is filled first, is 1.5 orders of magnitude lower than that of rWT Ocm (Table 4). As the CD and EF sites of Ocm are considered as low- and high-affinity sites, respectively [67], the C18S substitution seems to suppress Ca²⁺ affinity of the EF site.

Mg²⁺-binding to rWT/C18S Ocm was studied by equilibrium dialysis method with determination of total Mg²⁺ concentration using electrothermal atomization atomic absorption spectrometry (Figure 5). Scatchard plots (Figure 5b) reveal two linear regions corresponding to filling by Mg²⁺ of the two metal-binding sites. Again, analysis of the data within the sequential metal-binding scheme [IV] evidences that our estimates of Mg²⁺ affinity of rWT Ocm (Table 4) are 0.9–1.6 orders of magnitude higher compared to those reported earlier [66]. Similar to the results for Ca²⁺-binding to Ocm, the C18S substitution suppresses Mg²⁺ binding to the site, which is occupied first, by 0.4 orders of magnitude (Table 4). Importantly, the ratio of total free energies of Mg²⁺ and Ca²⁺ binding to C18S Ocm (calculated as ΔG_∑ = −RT∙(ln K_a1 + ln K_a2)) equals to the same ratio calculated for rWT Ocm (0.64; Table S1). Therefore, the C18S substitution equally compresses the energetic scales of Mg²⁺ and Ca²⁺ binding to Ocm, thereby confirming self-consistency of our estimates of the metal-binding constants.

In accordance with the lowered Ca²⁺ affinity of C18S Ocm, fluorimetric stopped-flow measurements of EGTA-induced Ca²⁺ removal from the protein (Figure 4b) demonstrate accelerated compared to rWT Ocm Ca²⁺ dissociation from the mutant. Analysis of the kinetic data according to the sequential metal-binding scheme [IV] (

Table 5. Equilibrium and kinetic parameters of Ca²⁺ binding to rWT/C18S Ocm at 20 °C, estimated from the fluorimetric experiments. The values of equilibrium metal association constants, K_a1 and K_a2, and rate constants of metal dissociation, k_off1 and k_off2, were estimated according to the sequential metal-binding scheme [IV] from equilibrium and stopped-flow experiments shown in Figure 4a,b, respectively. The kinetic constants of Ca²⁺ binding to Ocm, k_on1 and k_on2, were derived from these estimates. Standard deviations for the parameters of Ca²⁺ binding to Ocm are indicated.

Ocm	Ka1, M−1	Ka2, M−1	kon1, M−1s−1	kon2, M−1s−1	koff1, s−1	koff2, s−1
rWT	(1.3 ± 0.9) × 109	(4 ± 2) × 107	(3 ± 2) × 109	(5 ± 4) × 108	2.6 ± 0.3	14.4 ± 5.0
C18S	(4 ± 2) × 107	(8 ± 4) × 107	(1.5 ± 0.8) × 108 *	n.d. *	3.6 ± 0.4 *	n.d. *

* Ca²⁺ dissociation from the second site occurs within dead time of the instrument (6 ms).

) shows that Ca²⁺ dissociation from the site of C18S Ocm, which is occupied first, occurs 38% faster than from that of rWT Ocm. Ca²⁺ dissociation from the second site of C18S Ocm occurs within the dead time of the instrument (6 ms). In contrast to the dissociation stage, Ca²⁺ association with the first site of the C18S mutant is 20-fold slower in comparison to rWT Ocm (Table 5). Thus, Ca²⁺ affinity of C18S Ocm is decreased mostly due to the drop in Ca²⁺ association rate. Mg²⁺ dissociation from rWT/C18S Ocm occurs within the dead time of the instrument (data not shown), in accordance with their relatively low Mg²⁺ affinities (Table 4).

Overall, the structural and functional consequences of the homologous C18S substitution are unexpectedly pronounced. For this reason, the numerous possible oxidative modifications of the C18 thiol could induce noticeable changes in functioning of Ocm. Similarly, the oxidation mimicking substitution of the conservative C39 residue of recoverin markedly changes its structural properties and affinity for photoreceptor membranes [68].

4. Conclusions

We have shown that the highly conservative C18 thiol of rat Ocm exhibits physiologically relevant redox potential values, which suggests its disulfide dimerization under oxidizing conditions, inherent to numerous disorders. Another possibility in this case is an accumulation of the thiol-oxidized derivatives. As minor structural modification of the C18 residue (C18S substitution) significantly affects structural and functional status of Ocm, its thiol derivatives are expected to possess altered functional activity. Thus, oxidizing conditions are likely to favor accumulation of the oxidized forms of Ocm with modified activity, thereby supporting the redox sensory function of its C18 thiol. The effects described in the present work closely resemble those reported for the conservative among neuronal calcium sensor proteins C39 residue of recoverin, located in the loop of its inactive EF-hand 1. Light-induced oxidation of this thiol results in appearance under in vivo conditions of disulfide dimer of recoverin and its oxidized monomer, which demonstrate altered structural and functional properties [41,63]. Ocm and recoverin are both descendants of calmodulin [69,70], but their conservative thiols located in the inactive EF-loops corresponding to EF-hands 2 and 1 of calmodulin, respectively. Therefore, the selected EF-hands of Ocm and recoverin independently lost affinity to calcium in the course of their evolution, at the expense of the gain in redox sensitivity. The appearance of redox sensitivity in calcium-binding proteins gives them a unique ability to serve as a hub for calcium signaling and redox signaling pathways. The role of redox-sensitive EF-hand proteins in the well-studied crosstalk between calcium and reactive oxygen species [71,72] needs further clarification.