ABBS 2008,40(11): Thermodynamic study of the binding of calcium and magnesium ions with myelin basic protein using the extended solvation theory

Received: August 25, 2008       

Accepted: September 29, 2008

*Corresponding author: Tel,
008989126820758; Fax, 00982813780040; E-mail, [email protected]

The interaction of myelin basic protein
(MBP) from the bovine central nervous system with Ca2+ and Mg2+ ions, named as M2+, was studied
by isothermal titration calorimetry at 27 篊 in aqueous solution. The extended
solvation model was used to reproduce the enthalpies of MBP+M2+ interactions. The solvation parameters
recovered from the extended solvation model were attributed to the structural
change of MBP due to the metal ion interaction. It was found that there is a
set of two identical and noninteracting binding sites for Ca2+ and Mg2+ ions.

Keywords        myelin basic protein; isothermal
titration calorimetry; binding parameter

The energy of biochemical reactions or molecular interactions at
constant temperature is measured by isothermal titration calorimetry (ITC)
[1,2]. ITC gives invaluable information about biomacromolecule杔igand interaction. During
the last two years, we attempted to study the metal ion binding on different
proteins [3–5]. Metal ions change the conformational
stability and formation of aggregates. The importance of metal ions such as Ca2+ and Mg2+ in regulating
protein stability and function has been widely reported [5–10].

We have previously developed a theory to account for the solvation
of solutes in mixed solvent systems. Studies within our group are aimed at
developing an understanding of how the binding proteins of the metal ions and
other ligands affect the stability of the biomolecules. One of the unique
aspects of our approach is studying the stability of proteins by using the
extended solvation model. Myelin basic protein (MBP) is one of the most
abundant proteins of the myelin sheath
of the central nervous system (CNS), and its primary role is generally considered to be maintenance of the stability of the sheath by holding together
the apposing cytoplasmic leaflets of the oligodendrocyte membrane [11].
Previous investigations have revealed that the efficiency of MBP in preserving
the compactness and the stability of the myelin membrane appeared to be
enhanced in the presence of zinc (Zn) ions. There is also evidence that Zn
stabilizes the in vitro self association of MBP dissolved in a phosphate
buffer [12]. Since MBP addition to its associations with lipids, it has been
shown to interact with calmodulin and cytoskeletal proteins such as actin and
tubulin. Thus, functional roles for MBP in phosphoinositide-mediated signal
transduction and other processes have been postulated [13]. The MBP gene
encodes two families of paired formation of myelin sheets in primary cultures
of cortical proteins: the classic MBPs and the Golli proteins, the function of
which is less well understood. Previous work has suggested that Golli proteins
may play a role in Ca homeostasis in oligodendrocytes and in T-cells [14].

Ca and Mg are of great physiologic importance by their intervention in many enzymatic systems and their function in neural excitability, muscle contraction blood coagulation, bone formation, hormone secretion, and cell adhesion [15]. The human body is equipped with an efficient negative feedback system that counteracts variations of Ca and Mg balance [16]. The maintenance of the Ca and Mg balance is controlled by the concerted action of intestinal absorption, renal excretion, and exchange with bone. After years of research, rapid progress has been made recently in identification and characterization of the Ca and Mg transport proteins that contribute to the delicate balance of divalent cations [15]. The interactions of MBP with Ca2+-calmodulin have been previously investigated by fluorescence spectroscopy [17]. The apparent associated equilibrium binding constant for MBP interaction with Ca2+-calmodulin was previously reported to be 2.10.1 mM-1 [18]. MBP is an 搃ntrinsically unstructured or 搉atively unfolded protein, therefore its three-dimensional structure might only be determined in its interaction with another protein [19-22]. As a clear understanding of operational stability constitutes an important goal in protein technology, our efforts aimed at elucidation of the structure-stability using the extended solvation model. This model is able to correlate the solvation parameters to the effect of metals on the stability of a protein in a very simple way. The present paper reports some interesting experimental data for the heats of interaction of Ca2+ and Mg2+ ions with MBP, and analyzes these using the extended solvation theory for the first time. With regard to the importance of the presence of balance between Ca and Mg values in humans, and the role of MBP in Ca homeostasis in oligodendrocytes and T-cells, it seems that the present study has significant applications in pharmacology, neuroscience and drug delivery.

Materials and Methods

MBP from bovine CNS was obtained from Sigma (The isothermal titration microcalorimetric experiments were
performed with the four-channel commercial microcalorimetric system, Thermal Activity
Monitor 2277 (Thermometric,

Results

The enthalpies interaction of myelin basic protein with Ca2+ and Mg2+ ions, were
calculated in kJ穖ol–1 and are listed in Table 1
and 2 respectively.

Discussion

We have shown previously that the enthalpies of the solute-solvent
(M2++MBP+water in this case)
interactions in the aqueous solvent (M2++water in the present case) system, can be accounted for
quantitatively in terms of three factors: preferential solvation by the
components of a mixed solvent, weakening or strengthening of solvent-solvent
bonds by the solute and the change in the enthalpy of the solute-solvent
interactions [23–31]. This treatment leads to:

                  1                         

The parameters  and  are the indexes of MBP stability in the low M2+ ion
concentrations and in the maximum concentration of the M2+ upon saturation
of all MBP respectively. Cooperative binding requires that the macromolecules
have more than one binding site, since cooperativity results from the
interactions between binding sites with ligands. If the binding of a ligand at
one site increases the affinity for the ligand at another site, the
macromolecule exhibits positive cooperativity. Conversely, if the binding of a
ligand at one site lowers the affinity for a ligand at another site, the
protein exhibits negative cooperativity. If the ligand binds at each site
independently, the binding is non-cooperative: p<1 or p>1
indicate positive or negative cooperativity of the macromolecule for binding
with the ligand, respectively; p=1 indicates that the binding is
non-cooperative. can be expressed as follow:

    2

xB is the fraction of the metal ion needed for
saturation of the binding sites, and xA = 1杧B is
the fraction of unbounded M2+ ions. Now the model is a simple mass action treatment,
with metal ions replacing water molecules, at the binding sites in the present
case. We can express xB fractions, as the total M2+ concentrations divided by the
maximum concentration of the M2+ upon saturation of all MBP as follows:

; 3

 is the total concentration of
metal ions and  is the maximum
concentration of the M2+ upon saturation of all MBP. In general, there
will be 揼
sites for binding of M2+ per MBP molecule and v is defined as
the average moles of bound M2+ per mole of total MBP. LA and LB are
the relative contributions of unbounded and bounded metal ions to the
enthalpies of dilution in the absence of MBP and can be calculated from the
enthalpies of dilution of M2+ in buffer, DHdilut, as follow:

;

    4

The enthalpies of M2++MBP interactions, DH, were fitted to Equation 1 over the whole M2+ compositions. In the procedure the only
adjustable parameter (p) was changed until the best agreement between
the experimental and calculated data was approached (Figs. 1–3). 
and  parameters have been also
optimized to fit the data. The optimized 
and  values are recovered from the
coefficients of the second and third terms of Equation 1. The small
relative standard coefficient errors and the high r2 values (0.99999) support the method. The agreement between the
calculated and experimental results (Fig. 1) is striking, and gives
considerable support to the use of Equation 1.

F is the fraction
of the MBP molecule undergoing complexation with M2+ ions
which can be expressed as follows:

    5

where DHdilut represents the heat value upon saturation of
all MBP. The appearance association equilibrium constant values, Ka, as a function
of M2+ concentration can be calculated
as follows:

    6

 is the
unbounded or the free M2+ ion
concentrations. The variable F represents the fraction of binding sites that are
occupied on the peptide molecule. Therefore, 1朏 represents the fraction of binding sites that are not
occupied. The appearance association equilibrium constants, Ka, for successive
replacement of water molecules by M2+ ions
are as follow:

    7

where the Ki is the macroscopic association equilibrium
constants which are the equilibrium constants for every successive replacement of
water molecules by M2+ ions in the
equilibria:

     
E1

Ka values obtained from Equation 6, have
been fitted to Equation 7 using a computer program for nonlinear
least-square fitting. Therefore, we can approach the 揼 value and the macroscopic association equilibrium
constant in the first, K1, and the second, K2, binding site (Table 3), which
correspond to n = 1, n = 2 and n = 3 respectively.      values can be calculated at any
concentration of M2+ via Equation 2. The binding
parameters obtained from this method are listed in Table 3. The Gibbs
energies as a function of M2+ concentrations
can be obtained as follow:

    8

Gibbs energies, DG, calculated from Equation 8 are shown graphically in Fig.
2. TDS values were calculated
using DG values and
have shown in Fig. 3. The less negative Gibbs free energies in the low M2+ concentrations (Fig. 2) indicate the
lower affinity in this region.

A nonpolar residue dissolved in water induces
a solvation shell in which water molecules are highly ordered. When two
nonpolar groups come together on the folding of a polypeptide chain, the
surface area exposed to the solvent is reduced, and part of the highly ordered
water in the solvation shell is released to the bulk solvent, which results in
an increase in the entropy. It is possible to introduce a correlation between
change in  and increase in the stability
of proteins. The  value reflects the
hydrophobic property of MBP, leading to the enhancement of water structure. The
greater the extent of this enhancement, the greater the stabilization of the
MBP structure and the greater the value of . value (Table 3) for MBP+M2+interactions are
small and positive (Table 3), indicating that in the low concentration
of M2+ the MBP structure is stabilized,
resulting in an increase in its biological activity.  value in high M2+ concentration are big and negative, indicating
that the MBP structure is destabilized by these cations in this region.

The p values are very close to one (p=1.04),
indicating that there are a set of two identical non-operative binding sites
for MBP+Ca2+and MBP+Mg2+. In practice it turns out that even for good
data it is usually very difficult to distinguish between heterogeneity and
cooperativity, if more than two sites are present. Furthermore, it is almost
impossible to derive accurate binding parameters from real data, if binding is
cooperative and the binding sites are not equivalent.

A value of p = 1 would mean that K1 = K2, indicates that the binding of the second
site occurs non-cooperatively as compared to binding of the first site, whereas
a value of p>1 would indicate that binding of the second site is
facilitated and a value of p<1 indicates that binding of the second site is inhibited (anti-cooperativity or negative cooperativity).

The same conclusion is reached via Equation 7 because the
macroscopic association equilibrium constants recovered from this equation are
roughly the same (K1籏Using
fluorescence studies, Barylko et al have represented that there are
two binding sites on MBP for Ca2+-calmodulin,
but they could not determine the exact values of dissociation constants of every site [17]. Whereas, we can
calculate all thermodynamic functions, cooperativity parameters, equilibrium
constants and stability prediction as a result of ligand interaction with a
biopolymer, just using Equations 1 and 7, and it is the most
useful method in the ligand+macromolecule interactions.

References

 1   Saboury AA. A review on the ligand binding
studies by isothermal titration calorimetry. Journal of the Iranian Chemical
Society 2006, 3: 1–21

 2   Saboury AA. New methods for data analysis of
isothermal titration calorimetry. J Therm Anal Cal 2003, 72: 93–103

 3   Saboury AA. A simple method for determination
of binding isotherm by isothermal titration calorimetry and its application to
the interaction between Cu2+ and myelin basic
protein. J Therm Anal Cal 2004, 77: 997–1004

 4   Saboury AA. Atri MS, Sanati MH, Sadeghi M.
Application of a simple calorimetric data analysis on the binding study of
calcium ions by human growth hormone. J Therm Ana Cal 2006, 83: 175–179

 5   Saboury AA, Atri MS, Sanati MH,
Moosavi-Movahedi AA, Hakimelahi GH, Sadeghi M. A thermodynamic study on the
interaction between magnesium ion and human growth hormone. Biopolymers 2006,
81: 120–126

 6   Saboury AA, Atri MS, Sanati MH,
Moosavi-Movahedi AA, Haghbeen K. Effects of calcium binding on the structure
and stability of human growth hormone Int J Biol Macromol 2005, 36: 305–309

 7   Saboury AA, Kordbacheh M, Sanati MH, Mizani
F, Shamsipur M, Yakhchali MB, Moosavi-Movahedi AA. Thermodynamics of binding
copper ion by human growth hormone. Asian J Chem 2005, 17: 2773–2782

 8   Saboury AA, Ghourchaei H, Sanati MH, Atri MS,
Rezaei-Tawirani M, Hakimelahi GH. Application of a simple calorimetric data
analysis on the binding study of calcium ions by human growth hormone. J Therm
Ana Cal 2006, 83: 175–179

 9   Osawa M, Dace A, Tong KI, Valiveti A, Ikura M,
Ames JB. Mg2+ and Ca2+ differentially regulate DNA binding and
dimerization of DREAM. J Biol Chem 2005, 280: 18008–18014

10  Wingard JN, Chan J, Bosanac I, Haeseleer F,
Palczewski K, Ikura M, Ames JB. Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator
of calcium channels. J Biol Chem 2005, 280: 37461–37470

11  Moscarello MA, Wood DD, Ackerley C, Boulias C.
Myelin in multiple sclerosis is developmentally immature. J Clin Invest 1994,
94: 146–154

12  Benfatto M, Della Long S, Qin Y, Li Q, Pan G,
Wu Z, Morante S. The role of Zn in the interplay among Langmuir-Blodgett
multilayer and myelin basic protein: a quantitative analysis of XANES spectra.
Biophys Chem 2004, 110: 191–201

13  Libich DS, Hill MDC, Bates IR, Hallett FR,
Armstrong S, Siemiarczuk A, Harauz G. Interaction of the 18.5-kDa isoform of
myelin basic protein with cacalmodulin: effects of deimination assessed by
intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular
Dichroism. Protein Sci 2003, 12: 1507–1521

14  Paez PM, Spreuer V, Handley V, Feng JM,
Campagnoni C, Campagnoni AT. Increased expression of Golli myelin basic
proteins enhances calcium influx into oligodendroglial cells. J Neurosci 2007,
27: 12690–12699

15  Hoenderop JGJ, Bindels RJM. Epithelial Ca2+ and Mg2+ channels in health and disease. J Am Soc
Nephrol 2005, 16: 15–26

16  Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole
DE, Quamme GA. Magnesium transport in the renal distal convoluted tubule.
Physiol Rev 2001, 81: 51–84

17  Baryłko B, Dobrowolski Z. Ca2+-calmodulin-dependent
regulation of F-actin-myelin basic protein interaction. Eur J Cell Biol, 1984,
35: 327–335

18  Libich DS, Harauz G. Interactions of the 18.5
kDa isoform of myelin basic protein with Ca2+-calmodulin: in vitro studies using
fluorescence microscopy and spectroscopy. Biochem Cell Biol 2002, 80: 395–406

19  Dyer CA, Phillbotte T, Wolf MK,
Billing-gagliardi S. Regulation of cytoskeleton by myelin components: studies
on shiverer oligodendrocytes carrying an MBP transgene. Dev Neurosci 1997, 19:
395–409

20  Doyle HA, Mamula MJ. Post-translational
protein modifications in antigen recognition and autoimmunity. Trends Immunol
2001, 22: 443–449

21  Boggs JM, Rangaraj G. Interaction of
lipid-bound myelin basic protein with actin filaments and calmodulin.
Biochemistry 2000, 39: 7799–7806

22  Hill CMD, Bates IR, White GF, Hallett FR,
Harauz G. Effects of the osmolyte trimethylamine-N-oxide on
conformation, self-association, and two-dimensional crystallization of myelin
basic protein. J Struct Biol 2002, 139: 13–26

23  Rezaei Behbehani G, Saboury AA. Using a new
solvation model for thermodynamic study on the interaction of nickel with human
growth hormone. Thermochimica Acta 2007, 452: 76–79

24 Rezaei Behbehani G, Ghamamy S, Waghorne WE.
Enthalpies of transfer of acetonitrile from water to aqueous methanol, ethanol
and dimethylsulphoxide mixtures at 298.15 K. J Thermochimica Acta 2006, 448: 37–42

25  Rezaei Behbehani G, Tazikeh E, Saboury AA.
Using the extension coordination model (ECM) to reproduce the enthalpies of
transfer of tetraethylurea from water to aqueous ethanol, propan-1-ol and
acetonitrile at 298 K. Acta Chimica Slov 2006, 53: 363–369

26 Rezaei Behbahani G, Saboury AA, Fallah
Baghery A, Abedini A. Application of an extended solvation theory to study on
the binding of magnesium ion with myelin basic protein. J Therm Anal Calorim
2008, 93: 479–483

27  Rezaei Behbahani G,  Saboury AA,
Fallah Baghery A. A thermodynamic study on the binding of calcium ion with myelin
basic protein. J Solution Chem 2007, 36: 1311–1320

28  Rezaei Behbahani G,  Saboury AA,
Taleshi E. A comparative study of the direct calorimetric determination of the
denaturation enthalpy for lysozyme in sodium dodecyl sulfate and
dodecyltrimethylammonium bromide solutions. J Solution Chem 2008, 37: 619–629

29  Rezaei Behbehani G, Saboury AA. A
thermodynamic study on the binding of magnesium with human growth hormone.
Consideration of the new extended coordination model solvation parameters. J
Therm Anal Calorim 2007, 89: 859–863

30  Rezaei Behbehani G, Saboury AA, Takeshi E.
Determination of partial unfolding enthalpy for lysozyme upon interaction with
dodecyl trimethylammonium bromide using an extended solvation model. J Mol
Recognit 2008, 21: 132–135

31  Rezaei Behbehani G, Saboury AA. A
directcalorimetric determination of denaturation enthalpy for lysozyme in
sodiumdodecylsulfate. J Coll Surf B: Biointerfaces 2007, 61: 224–228