HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 19, 16662-16672, May 10, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/19/16662    most recent M200415200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berggård, T. Articles by Linse, S. Search for Related Content PubMed PubMed Citation Articles by Berggård, T. Articles by Linse, S. Social Bookmarking                      What's this?

Calbindin D_28k Exhibits Properties Characteristic of a Ca²⁺ Sensor^*

Tord Berggård^§, Simona Miron, Patrik Önnerfjord^¶, Eva Thulin, Karin S. Åkerfeldt, Jan J. Enghild^**, Mikael Akke, and Sara Linse

From the  Department of Biophysical Chemistry, Lund University, SE-221 00 Lund, Sweden, the  Department of Chemistry, Haverford College, Haverford, Pennsylvania 19041-1392, the ^** Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, and ^¶ Section for Connective Tissue Biology, BMC, C12, Lund University, SE-221 84 Lund, Sweden

Received for publication, January 15, 2002, and in revised form, February 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calbindin D_28k is a member of the calmodulin superfamily of Ca²⁺-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca²⁺ buffer, but the studies presented in this work indicate that it may also act as a Ca²⁺ sensor. The results show that Mg²⁺ binds to the same sites as Ca²⁺ with an association constant of ~1.4·10³ M¹ in 0.15 M KCl. The four high affinity sites in calbindin D_28k bind Ca²⁺ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg²⁺, the Ca²⁺ affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca²⁺ concentration in a resting cell calbindin D_28k is saturated to 40-75% with Mg²⁺ but to less than 9% with Ca²⁺. In contrast, the protein is expected to be nearly fully saturated with Ca²⁺ at the Ca²⁺ level of an activated cell. A substantial conformational change is observed upon Ca²⁺ binding, but only minor structural changes take place upon Mg²⁺ binding. This suggests that calbindin D_28k undergoes Ca²⁺-induced structural changes upon Ca²⁺ activation of a cell. Thus, calbindin D_28k displays several properties that would be expected for a protein involved in Ca²⁺-induced signal transmission and hence may function not only as a Ca²⁺ buffer but also as a Ca²⁺ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca²⁺, becomes exposed upon Ca²⁺ binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calbindin D_28k is a Ca²⁺-binding protein expressed in brain as well as in kidney, bone, pancreas, and other tissues (1). In some tissues it is exceptionally abundant. For example, calbindin D_28k constitutes between 0.1 and 1.5% of the total soluble protein in brain, and protein levels in auditory neurons are estimated to reach concentrations of up to 2 mM (2). Calbindin D_28k contains 261 amino acid residues forming six EF-hands, which are organized in a single globular domain (Fig. 1) (3, 4). The protein is a member of the calmodulin superfamily (5). Some members of this superfamily are Ca²⁺ sensors that undergo Ca²⁺-induced conformational changes resulting in the exposure of a hydrophobic surface. The hydrophobic patch typically serves as a binding surface for target molecules, which become activated or attenuated upon complex formation (6). The targets include many membrane transport proteins and enzymes (7). In this way, intracellular Ca²⁺ influx triggers the regulation of cellular processes, such as muscle contraction and the phosphoinositide cascade.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of human calbindin D28k. The schematic on top outlines secondary structure elements, including the helix-loop-helix motif. Potential trypsin cleavage sites are shaded.

Another class of Ca²⁺-binding proteins are the Ca²⁺ buffers or signal modulators. Parvalbumin and calbindin D_9k are examples of Ca²⁺-buffer proteins. Unlike Ca²⁺ sensors, the Ca²⁺-buffer proteins do not expose hydrophobic surfaces upon Ca²⁺ binding. In fact, the exposure of hydrophobic surfaces would be unfavorable for these proteins because it may limit their stability and Ca²⁺ affinity. Ca²⁺ buffer proteins are thought to be involved in deactivation of signal transducers and/or quenching of Ca²⁺ signals, thus protecting the cell against toxic effects of Ca²⁺, such as the formation of insoluble calcium phosphates (for a review see Ref. 8). The Ca²⁺ affinity is generally higher for buffer proteins than for sensor proteins, and the rates of Ca²⁺ binding and release are lower in the buffer group. Calbindin D_28k is thought to prevent sustained elevations of Ca²⁺ by acting as an intracellular Ca²⁺ buffer. There is evidence that calbindin D_28k has a Ca²⁺-buffering function in neurons. For example, increased intracellular levels of calbindin D_28k causes blunted intracellular Ca²⁺ elevations (9), and Ca²⁺ transients are increased in calbindin D_28k null mutant mice (10). A number of examples where Ca²⁺ buffering by calbindin D_28k has an effect on the electrophysiological behavior of cells have also been reported (11-13). The notion that calbindin D_28k acts as a Ca²⁺-buffering system in the cytoplasm has led to the hypothesis that calbindin D_28k may protect neurons against large fluctuations in free intracellular Ca²⁺ and, hence, prevent cell death (14-17). However, experiments using calbindin D_28k-null mutant mice subjected to cerebral ischemia did not support a cytoprotective effect of the protein (18). Although most reports deal with the Ca²⁺-buffering function of calbindin D_28k, several lines of evidence suggest that the protein also acts as a Ca²⁺ sensor. Spectroscopic investigations (19) and in vitro studies using antibodies (20) have shown that calbindin D_28k undergoes a conformational shift upon Ca²⁺ binding. Additionally, a number of putative Ca²⁺-dependent interactions with target proteins or brain membrane fractions have been reported (20-23), and erythrocyte membrane Ca²⁺-ATPase and 3',5'-cyclic nucleotide phosphodiesterase have been shown to be stimulated in a dose-dependent, saturable manner with calbindin D_28k (24). Moreover, the finding that a fraction of calbindin D_28k (9-55%) (20, 25-27) is specifically associated with particulate structures in the cell indicates that the protein plays other roles in addition to its function as a mobile Ca²⁺ buffer.

In our previous study (28), we showed that although a conformational change occurs upon Ca²⁺ binding, both the Ca²⁺-free and Ca²⁺-loaded forms of calbindin D_28k have exposed hydrophobic surfaces. Thus, the protein behaves neither like a classical Ca²⁺ sensor nor like a Ca²⁺ buffer. The aim of the present study was to further characterize the Ca²⁺-induced conformational change and to determine whether calbindin D_28k is likely to respond structurally to changes in the intracellular concentration of Ca²⁺ in the presence of physiological levels of Mg²⁺ and salt.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins-- Human recombinant calbindin D_28k was expressed in Escherichia coli and purified to homogeneity as described (29). Bovine brain calbindin D_28k was purified from brain homogenate as described (28). F123 is a fragment containing EF-hands 1-3 (residues 1-132) of human calbindin D_28k, whereas F456 contains EF-hands 4-6 (residues 133-261). F123 and F456 were produced by cloning techniques and expressed in E. coli (4).

Chemicals-- CaCl₂ and MgCl₂ of Pro Analysi quality were from Merck; quin 2 was from Fluka Buchs, Switzerland, and 5,5'-Br₂-BAPTA¹ was from Molecular Probes, Eugene, OR. All chemicals were of highest grade commercially available. Buffers were made Ca²⁺-free by incubation with a dialysis tube filled with Chelex 100 (Bio-Rad) for 2 weeks.

Preparation of Apoprotein-- All apo solutions were made from Ca²⁺-depleted protein, which was produced as follows. Purified protein was dissolved in 1 ml of doubly distilled water, containing an excess of EGTA (10-20 eq) at pH 8. It was then applied to a 3.4 × 20-cm Sephadex G-25 superfine gel filtration column. To abolish EGTA binding, the protein was applied to the column after 15 ml of saturated NaCl had been allowed to penetrate the top of the column. The NaCl solution had been depleted from residual Ca²⁺ by dialysis against Chelex 100 resin. During the gel filtration, the protein was passed through the NaCl zone and eluted, now free from EGTA, with doubly distilled Ca²⁺-free water. The final product contained between 0.2 and 0.6 molar eq of Ca²⁺, as determined from Ca²⁺ titrations in the presence of the chelator quin 2 or by high resolution inductively coupled plasma mass spectrometry (analysis performed by SGAB, Luleå, Sweden). ¹H NMR spectroscopy was used to verify that the apo samples were free from EGTA.

Near UV CD Spectroscopy-- Near-UV CD spectra (250-300 nm) were obtained using a Jasco J-720 spectropolarimeter at 25 °C (thermostated) and quartz cuvettes with a path length of 10 mm. A spectrum was first recorded for the apoprotein (25 µM apocalbindin D_28k in 0.15 M KCl, 2 mM Tris, 0.125 mM EGTA, pH 7.3). A second spectrum was recorded after adding 10 mM MgCl₂. Finally, 0.5 mM CaCl₂ was added, and a spectrum for the Ca²⁺ form was recorded.

ANS Fluorescence-- Fluorescence spectra were obtained using a PerkinElmer Life Sciences Luminescence Spectrometer LS 50 B connected to a Julabo F25 water bath. All data were collected at 25.0 °C in a quartz cuvette (10 mm path-length). For the 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments, ANS was added to a final concentration of 120 to 10 µM protein in 10 mM DTT, 0.15 M KCl, 2 mM Tris, pH 7.3, with 0, 1, or 5 mM MgCl₂. Fluorescence spectra were recorded between 400 and 600 nm (bandwidth 5 nm) with excitation at 385 nm (bandwidth 3 nm) and a scan rate of 50 nm per min. Two spectra were obtained for each sample and averaged.

Determination of Macroscopic Ca²⁺ Binding Constants-- The affinity and cooperativity of Ca²⁺ binding to F123, F456, or intact calbindin D_28k were determined by titration with CaCl₂ in the presence of a chromophoric chelator, quin 2, as described previously (30, 31). All experiments were performed in 2 mM Tris/HCl buffer at pH 7.5, either with no added salt, with 0.15 M KCl, or with 0.15 M KCl in the presence of 2 mM MgCl₂. The macroscopic Ca²⁺ binding constants were obtained by least squares fitting of the absorbance at 263 nm versus total Ca²⁺ concentration by minimizing the error square sum (e.s.s.) as shown in Equation 1,

(Eq. 1)

where the sum runs over the n + 1 data points in the titration. The calculated absorbance at each titration point, i, was obtained as shown in Equation 2,

(Eq. 2)

where V_i and V₀ are the total volumes at point i and before adding the first CaCl₂ aliquot, respectively. A_min and A_max are the respective absorbances that the Ca²⁺-chelator complex, and free chelator would have at no dilution. K_D is the dissociation constant of the chelator-Ca²⁺ complex. The free Ca²⁺ concentration, Y_i, was solved using the Newton-Raphson method from Equation 3,

(Eq. 3)

where K₁ through K_N are the N macroscopic binding constants and CQ_i, CP_i, and CCa_i are the total chelator, protein, and Ca²⁺ concentrations, respectively, at point i corrected for the dilutions due to the CaCl₂ additions. The initial chelator concentration, CQ₀, was determined by withdrawing an aliquot of the solution and recording the absorbance at 239.5 nm in the presence of excess Ca²⁺ (using  = 4.2 × 10⁴ liter mol¹ cm¹). The initial Ca²⁺ concentration, CCa₀, was determined by high resolution inductively coupled plasma mass spectrometry (at SGAB, Luleå, Sweden). The initial protein concentration, CP₀, was determined by amino acid analysis after acid hydrolysis on a withdrawn aliquot of the protein/chelator solution that had been lyophilized in a hydrolysis tube (Biomedical Centre, Uppsala, Sweden). The adjustable parameters in the fit were A_min, A_max, and the N macroscopic binding constants. The parameter F was either fixed at 1.0 or could be used as an adjustable parameter to analyze if the assumed stoichiometry (number of macroscopic binding constants used) is correct (F will end up close to 1.0) or not (F will deviate from 1.0). For presentation, the data and fitted curves were normalized according to Equation 4,

(Eq. 4)

The apparent Ca²⁺-binding constants in the presence of Mg²⁺ were derived using the same method as above but with 5,5'-Br₂-BAPTA instead of quin 2. The chelator 5,5'-Br₂-BAPTA has a high level of selectivity against Mg²⁺ (log K_Ca = 5.65; log K_Mg <1, at 0.15 M KCl). Hence, differences in titration curves obtained in the interval 0-10 mM Mg²⁺ are due to Mg²⁺ effects on the protein. The resulting binding constants are apparent Ca²⁺ binding constants for the protein in the presence of Mg²⁺. These constants result from the Ca²⁺ affinity, the Mg²⁺ affinity, and competition or coupling between the two events.

Determination of Ca²⁺ and Mg²⁺ Binding Constants by Fluorescence Spectroscopy-- Mg²⁺ or Ca²⁺ binding constants were determined by monitoring the intrinsic (tryptophan) fluorescence during Ca²⁺ or Mg²⁺ titration in 0.15 M KCl, 2 mM Tris, pH 7.3. Fluorescence spectra were obtained using a PerkinElmer Life Sciences Luminescence Spectrometer LS 50 B connected to a Julabo F25 water bath at 25.0 °C. Quartz cuvettes with a path length of 10 mm were used. The excitation wavelength was 280 nm, and the emission scanned was between 300 and 450 nm. The protein concentration was 25 µM. Ca²⁺-free protein was titrated with increasing amounts of metal ion (Ca²⁺ or Mg²⁺) beyond saturation. The intensity at each point was corrected for dilution. Ca²⁺ and Mg²⁺ titrations were also performed in the presence of 120 µM ANS (excitation at 385 nm, emission scanned between 400 and 600 nm) with 10 µM calbindin D_28k in 10 mM DTT, 0.15 M KCl, 2 mM Tris, pH 7.1. In some experiments, MgCl₂ was added to a final concentration of 1 or 5 mM. The excitation wavelength was 385 nm (bandwidth 3 nm), and the emission was scanned between 400 and 600 nm. The experimental data were fitted according to Equation 5,

(Eq. 5)

where Y is the free Ca²⁺ or Mg²⁺ concentration; K_A is the apparent binding constant, and I₀ and I_p are the intensities for the free and bound state, respectively.

Limited Proteolysis of Calbindin D_28k-- Calbindin D_28k was dissolved in 50 mM Tris, 150 mM KCl, containing either 1 mM CaCl₂, 1 mM EDTA, or 2.5 mM MgCl₂ + 0.5 mM EGTA (EGTA has a very high selectivity for Ca²⁺ over Mg²⁺) at a protein concentration of 0.5 mg/ml. The pH was adjusted to 7.5. Sequencing grade modified trypsin (Promega) was dissolved in the supplied buffer, yielding a stock solution with a concentration of 0.5 mg/ml. Proteolysis was initiated by mixing 100 µl of calbindin D_28k (0.5 mg/ml) with 1 µl of the trypsin stock at room temperature. Aliquots of 5 µl were withdrawn at various time points, and the digestion was blocked by the addition of 1 µl of soybean trypsin inhibitor (1 mg/ml) (Roche Molecular Biochemicals). The digested fragments were then separated by SDS-PAGE (15%). Following electrophoresis, the protein bands were either stained by Coomassie Blue and excised for mass spectrometry analysis (see below) or blotted onto a poly(vinylidene difluoride) membrane (Immobilon, Millipore) and subjected to N-terminal amino acid sequencing (automated Edman degradation using an Applied Biosystems 477 A sequencer with on-line detection of phenylthiohydantoin-derivatives with an Applied Biosystems 120A high pressure liquid chromatography).

Sample Preparation for Mass Spectrometry-- Coomassie-stained protein fragments were excised from the gel and washed with water, followed by 40% acetonitrile in 25 mM NH₄HCO₃, pH 7.8, until the gel piece was transparent. The gel piece was dried in a SpeedVac vacuum centrifuge. Reduction using 10 mM DTT at 48 °C for 30 min was followed by alkylation in 55 mM iodoacetamide for 30 min in darkness at room temperature. The gel piece was washed and dried again before digestion with sequencing-grade trypsin (Promega) in 25 mM NH₄HCO₃ overnight at 37 °C. The digestion was terminated, and the peptides were eluted by adding 10 µl of 2% trifluoroacetic acid. Peptides were purified from buffer using C-18 reversed phase tips (Ziptips, Millipore).

Mass Spectrometry-- Mass spectrometric studies were performed using a Bruker Scout 384 Reflex III matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. The instrument was operated in the positive ion mode with delayed extraction and an acceleration voltage of 25 kV. Peptide samples were analyzed using the reflector detector with 2,5-dihydroxybenzoic acid as matrix, whereas larger fragments and intact protein were measured in the linear mode using ferulic acid as matrix. Improved signal-to-noise ratios were obtained by the accumulation of 50-150 single shot spectra. Autolysis fragments of trypsin were used for internal calibration, whereas an external calibration standard was used for the analysis of intact protein fragments.

NMR Spectroscopy-- NMR spectra were acquired on a 600-MHz Varian Inova spectrometer at 310 K. ¹⁵N HSQC experiments utilizing pulsed field gradient and preservation of equivalent path (32, 33) employing water flip-back (34) were performed on calbindin D_28K at different Mg²⁺ or Ca²⁺ concentrations. The GARP-1 decoupling sequence (35) was used for ¹⁵N decoupling during acquisition. All spectra were acquired with spectral widths of 2000 and 8000 Hz in the F1 and F2 dimensions, respectively, and sampled using 128 and 1024 complex points in the t1 and t2 dimensions, respectively, with 16 transients acquired for each free induction decay. The data were processed and analyzed using the Felix97 Software (Micron Separations, San Diego). The data were multiplied by exponential (F2) and cosine-bell (F1) functions prior to Fourier transformation, and zero-filled to generate matrices of 2048 × 2048 real points. Proton chemical shifts were referenced relative to the water signal, which resonates at 4.64 ppm from the sodium 2,2-dimethyl-2-silapentane sulfonate (DSS) at 310 K. Nitrogen chemical shifts were referenced indirectly relative to DSS, using the nitrogen to proton frequency ratio (36).

The NMR samples contained 0.73 mM apocalbindin D_28k (with less than 0.05 eq of Ca²⁺), 0.1 mM DSS, 10 mM deuterated DTT and were prepared by dissolving the lyophilized protein and deuterated DTT in 620 µl of 93% ¹H₂O, 7% ²H₂O. The pH was adjusted to 6.8 with 0.1 M KOH or HCl. Aliquots from a 73 mM CaCl₂ stock solution was added to one sample directly in the NMR tube using a Hamilton syringe (5 µl for each point of the titration). The protein concentration was determined by amino acid analysis following acid hydrolysis. The concentration of the CaCl₂ stock solution was determined by inductively coupled plasma mass spectrometry. HSQC spectra were also acquired for a 0.8 mM protein sample with 2 or 10 mM MgCl₂ as well as with 10 mM MgCl₂ and 5 mM CaCl₂.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ Binding Determined from Competition with Chromophoric Chelators-- Titration with Ca²⁺ in the presence of a chromophoric chelator was used to determine the macroscopic Ca²⁺ binding constants (30, 37). To minimize experimental errors, care was taken to determine accurately the Ca²⁺ concentrations before and after titration (by high resolution inductively coupled plasma mass spectrometry), as well as the protein concentration before titration (by amino acid analysis after acid hydrolysis). Reliable acid hydrolysis results were obtained only when an aliquot of the sample was freeze-dried in the hydrolysis tube (glass) before shipment to the analysis station. Samples sent as liquid in Eppendorf tubes yielded unreasonably low and inconsistent protein concentrations, probably due to protein adhesion to the plastic.

The stoichiometry of Ca²⁺ binding was analyzed by fitting each titration in a simplified way using Equations 1-3 with n = 2. CP₀ was set to the value obtained by acid hydrolysis, and the parameter F was used as a variable parameter. After fitting, the stoichiometry was calculated as 2F. By using this method, the number of high affinity Ca²⁺-binding sites in the intact protein are 4.03 ± 0.11 (average and S.D. of 9 titrations). Thus the data convincingly show that human calbindin D_28k binds 4 Ca²⁺ ions with high affinity.

To obtain the values of the four macroscopic binding constants, each titration was then fitted using Equations 1-3 with n = 4. CP₀ was set to the value obtained by acid hydrolysis, and F was fixed at 1.0. The intact protein displayed macroscopic binding constants of 1.5·10⁷ to 3.5·10⁸ M¹ at low ionic strength and 8.9·10⁵ to 7.9·10⁶ M¹ in 0.15 M KCl (Table I).

The Ca²⁺ titration curves display a close to linear decrease of the absorbance as a function of total Ca²⁺ concentration. The lack of sigmoidal shape may indicate a low degree of cooperativity of Ca²⁺ binding. More quantitative information on the cooperativity is derived from the relationships between the macroscopic binding constants. For a protein with four Ca²⁺-binding sites, values of lgK₁  lgK₂ < 0.426, lgK₂  lgK₃ < 0.352, lgK₃  lgK₄ < 0.426, and lgK₁  lgK ₄ < 1.204 imply positive cooperativity (38). The results obtained here for the intact protein in the absence of Mg²⁺ yield the following: lgK₁  lgK₂ = 0.393; lgK₂  lgK₃ = 0.275; lgK₃  lgK₄ = 0.281; and lgK₁  lgK₄ = 0.948. Hence there is only a low degree of cooperativity of Ca²⁺ binding. In the presence of 2 mM Mg²⁺, however, the values obtained are as follows: lgK₁  lgK₂ = 0.18; lgK₂  lgK₃ = 0.55; lgK₃  lgK₄ = 0.03; and lgK₁  lgK₄ = 0.80. This indicates that the cooperativity of Ca²⁺ binding is greater in the presence of Mg²⁺, which is the relevant physiological condition. In summary, our results clearly show that calbindin D_28k has four high affinity Ca²⁺-binding sites, which act as a cooperative unit. Hence the situation is very different from calmodulin, in which the four sites are distributed into two groups with different affinity (30).

Ca²⁺ Binding Monitored by Trp Fluorescence-- Calbindin D_28k contains two Trp residues located in the first helix of EF-hands 1 and 3, respectively. Analysis of the Ca²⁺ titration curve monitored by Trp fluorescence for calbindin D_28k yields an apparent binding constant of around 1·10⁶ M¹ in 0.15 M KCl, which is similar to that obtained by Ca²⁺ titrations with chromophoric chelators. Although the titration monitored by Trp fluorescence does not provide the high precision obtained using the competitive chromophoric chelator method, it does confirm that the binding constants obtained by the chelator method are in the correct range.

Ca²⁺ Binding as Monitored by NMR Spectroscopy-- Ca²⁺ binding to calbindin D_28k was monitored by heteronuclear two-dimensional ¹⁵N-¹H HSQC NMR spectroscopy for samples containing 0-6 eq of Ca²⁺. In the absence of Ca²⁺, the signals are broad, and there are few well resolved resonances (Fig. 2A). Addition of Ca²⁺ to the protein sample causes significant changes in the HSQC spectrum. The HSQC spectrum of the Ca²⁺-saturated form shows predominantly sharp resonances and improved spectral resolution (Fig. 2D). In the absence of Ca²⁺, no aggregation or dimerization of the protein is observed by size exclusion chromatography when 100 µl of a 1 mM protein sample is injected on a Superdex 75 column (1.1 × 30 cm; Amersham Biosciences, data not shown). Therefore, the NMR signals of the apo state are probably broadened due to exchange between at least two conformational states with a fast-to-intermediate rate on the NMR chemical shift time scale. During the titration, the cross-peaks initially remain broadened (Fig. 2, B and C) but start to become sharper after the addition of 3 eq of Ca²⁺. Between 3 and 4 eq, very large changes in the spectrum are seen, both in terms of line widths and chemical shift dispersion. No further changes are observed beyond 4 eq of Ca²⁺.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Two-dimensional 1H-15N HSQC spectra of uniformly 15N-labeled calbindin D28k recorded at 310 K and at Ca2+/calbindin D28k ratios of 0 (A), 2.4 (B), 3.2 (C), and 4.0 (D).

The NMR signals of the apo form disappear, and new ones appear during the Ca²⁺ titration. This shows that Ca²⁺ dissociation from calbindin D_28k occurs in the slow exchange regime of the NMR chemical shift time scale. The Ca²⁺ binding process was therefore followed by measuring the intensity of individual cross-peaks, well resolved in the HSQC spectrum as a function of Ca²⁺ concentration. The signals are grouped according to their behavior between 0 and 4 eq of Ca²⁺ in Fig. 3, A-C. A small number of resonances are affected already by the addition of the 1st eq of Ca²⁺; others start to increase during the addition of the 2nd or the 3rd eq of Ca²⁺. However, the majority of the signals do not appear until addition of the 3rd or 4th eq.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Signal intensities of cross-peaks in the two-dimensional 1H-15N HSQC spectra of calbindin D28k as a function of the Ca2+/calbindin D28k ratio. A-C, normalized experimental data grouped according to the shape of the curve. D, fraction of the protein in the apo, Ca1, Ca2, Ca3, and Ca4 forms during the titration calculated using the four macroscopic binding constants.

Ca²⁺ binding to a four-site system may be a multibranched process involving one apo state, four Ca₁ states, six Ca₂ states, four Ca₃ states, and one Ca₄ state. Using the macroscopic calcium binding constants, determined by the chelator method, we have calculated for each point of the HSQC Ca²⁺ titration the fraction of the protein in (i) the apo, (ii) any of the Ca₁ states, (iii) any Ca₂ state, (iv) any Ca₃ state, and (v) the Ca₄ state (Fig. 3D). Comparisons of the calculated curves with the NMR data reveal that a majority of the NMR titration curves monitor the appearance of the Ca₄ state. This shows that most protons have a unique chemical shift in the Ca₄ form. A minority of titration curves follow the disappearance of the apo state, whereas none of the monitored peaks seems to correspond to curves expected for any of the Ca₁, Ca₂ or Ca₃ forms. This suggests that the intermediate states are interconverting rapidly, leading to severe broadening of the resonances. The small number of resonances appearing before addition of the 3rd eq of Ca²⁺ may represent protons having the same chemical shift in one or more intermediate states as in the Ca₄ species. The NMR data hence show that all four sites have similar affinities and are filled in a parallel fashion and that the Ca₄ species has structural features distinct from those of the intermediate states.

Mg²⁺ Binding as Monitored by Trp Fluorescence-- Titration with MgCl₂ to saturation results in a 10% increase of the fluorescence intensity at 333 nm and gives a small but reproducible blue shift of the intensity maximum from 333.5 to 332.5 nm (not shown). Addition of excess Ca²⁺ to the Mg²⁺-saturated sample results in an additional 15% increase in the fluorescence intensity (not shown). This shows that, although Mg²⁺ binds to the protein, it does not invoke the same structural response as Ca²⁺ binding. The Mg²⁺ titration curve in the absence of Ca²⁺ yields an apparent Mg²⁺ binding constant of 1.4 × 10³ M¹ in 0.15 M KCl (average of four independent experiments). The stoichiometry of Mg²⁺ binding cannot be deduced from the experiment, because the Mg²⁺ concentration at half-saturation is almost 2 orders of magnitude higher than the protein concentration. However, the obtained binding constant implies that under physiological intracellular concentrations of Mg²⁺ (0.5-2 mM) and resting levels of Ca²⁺, the Mg²⁺-binding sites in calbindin D_28k are 40-75% occupied by Mg²⁺.

Mg²⁺ Binding as Monitored by NMR Spectroscopy-- Mg²⁺ binding to calbindin D_28k was also monitored by heteronuclear two-dimensional ¹⁵N-¹H HSQC NMR experiments. Fig. 4 shows spectra recorded using 0.8 mM Ca²⁺-free protein samples containing 2 and 10 mM Mg²⁺, corresponding to 2.5 and 12.5 eq of Mg²⁺, respectively. Addition of Mg²⁺ to the apoprotein causes an increase in the chemical shift dispersion with a number of shifted peaks, although the effects are not as pronounced as during addition of Ca²⁺. Even at 10 mM Mg²⁺, most of the peaks are still broadened and clustered between 8.5 and 7 ppm in the proton dimension and between 121 and 115 ppm in the nitrogen dimension. Addition of 3.2 mM Ca²⁺ (4 eq) to the sample with 10 mM Mg²⁺ yields an HSQC spectrum that is identical to the one in Fig. 2D (data not shown). This shows that Ca²⁺ efficiently displaces Mg²⁺, suggesting that Mg²⁺ binds to the same sites as Ca²⁺.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Two-dimensional 1H-15N HSQC spectra of uniformly 15N-labeled calbindin D28k recorded at 310 K with 0.8 mM Ca2+-free protein samples containing 2 and 10 mM Mg2+ (corresponding to 2.5 and 12.5 eq of Mg2+, respectively).

Mg²⁺ Binding to Individual EF-hands as Monitored by CD Spectroscopy-- The far-UV CD spectra were recorded for six synthetic peptides, comprising EF-hands 1-6, at a concentration of 20 µM. The six peptides appear mostly unfolded in the apo form as well as in the presence of 10 or 200 mM MgCl₂ (data not shown). This is in striking contrast to the spectra obtained in the presence of Ca²⁺, which show a high degree of helicity and are consistent with Ca²⁺ binding for EF1, EF3, EF4, EF5, and EF6 (39). The results suggest that although Mg²⁺ binds to the intact protein, the interaction between Mg²⁺ and the individual EF-hands is not strong enough to induce secondary structure in these peptides.

Ca²⁺ Binding in the Presence of Mg²⁺ and a Chromophoric Chelator-- Ca²⁺ binding in the presence of Mg²⁺ was determined using a chromophoric Ca²⁺ chelator (5,5'Br₂-BAPTA), which has a high level of discrimination against Mg²⁺. Fig. 5 shows the experimental data for Ca²⁺ titrations of intact calbindin D_28k in the presence of 0 or 2 mM Mg²⁺, together with the curves of best fit. Already from the appearance of the curves it is evident that the presence of Mg²⁺ reduces the Ca²⁺ affinity for calbindin D_28k. The fitting of the experimental data shows that the Ca²⁺ affinity of calbindin D_28k is reduced approximately 2-fold to yield K = 3.5·10⁶-5.6·10⁵ M¹ in the presence of 2 mM MgCl₂ (Table I). Only very small ionic strength effects are expected for the addition of 2 mM MgCl₂ into 0.15 M KCl. Therefore, the data again suggest that Mg²⁺ competes with Ca²⁺ for the same binding sites.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Titration of 5,5'Br2-BAPTA with Ca2+ in the presence of calbindin D28k with or without Mg2+ in 0.15 M KCl, 2 mM Tris, pH 7.3. The curves of optimal fit to the data points are presented. , titration performed without MgCl2. , titration performed in the presence of 2 mM MgCl2.

Changes in the Tertiary Structure as Monitored by Near UV-CD-- Ca²⁺ binding induces changes in the near-UV CD spectra of calbindin D_28k, which indicates a rearrangement of the tertiary structure (not shown, see also Ref. 28), in agreement with the NMR data. In contrast, addition of 10 mM MgCl₂ to apocalbindin D_28k does not alter the near-UV CD spectrum to any significant degree (not shown).

Limited Proteolysis-- Limited tryptic digestion of human recombinant calbindin D_28k was performed in the presence of EDTA, Ca²⁺, or Mg²⁺. The reaction was quenched at different time points ranging from 1 min to 14 h and was analyzed by SDS-gel electrophoresis (Fig. 6). Both the rate and pattern of tryptic digestion are strongly Ca²⁺-dependent. The Ca²⁺-loaded form of calbindin D_28k is clearly more resistant to proteolysis than the Ca²⁺-free form, although many of the potential trypsin cleavage sites are protected in both forms (Fig. 1). Significant digestion of the apo form is seen already in the 1st min, whereas fragments of the Ca²⁺-loaded form appear at a significantly lower rate. The identities of the tryptic fragments were determined by mass spectrometry and N-terminal amino acid sequencing. The deduced cleavage sites are indicated by the arrows in Fig. 7, and the identity of the major fragments are summarized in Table II. Both apo- and Ca²⁺-bound calbindin are cleaved at Lys-59 and Lys-235 in the N-terminal helices of EF-hands 2 and 6. The apoprotein is cleaved also at Arg-169 in the C-terminal helix of EF-hand 4. In contrast, the Ca²⁺-bound protein is not cleaved at this site but mainly at Lys-72 in the loop of EF-hand 2 and at Lys-98 in the linker between EF-hands 2 and 3. In addition, a minor cleavage site in the Ca²⁺ form is found at Lys-245 in the loop of EF-hand 6. 

View larger version (45K): [in this window] [in a new window]   Fig. 6.   Tryptic digestion of calbindin D28k. Human recombinant calbindin D28k was digested with trypsin as described under "Experimental Procedures." Fragments were generated in the presence of 1 mM CaCl2, in the presence of 1 mM EDTA, or in the presence of or 2.5 mM MgCl2 + 0.5 mM EGTA and separated by SDS-PAGE (15%), transferred to an Immobilon membrane, and stained with Coomassie Brilliant Blue.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Limited proteolysis of calbindin D28k. Schematic of calbindin D28k outlining secondary structure elements, including the helix-loop-helix motifs and major tryptic fragments generated in 1 mM CaCl2 (A) and 1 mM EDTA (B). EF-hand motifs 1-6 are shown by arabic numbers. EF hands 2 and 6, which contain sequence anomalies, are shown by dashed lines. The major cleavage sites are indicated by arrows. The regions that display a difference in the cleavage patterns between the Ca2+ and apo forms are shaded boxes.

At longer incubation times, a number of small fragments appear. These are mainly due to further cleavage of larger fragments and may therefore represent cleavage sites that are inaccessible in the intact protein. It is noteworthy that in EDTA a small fragment containing residues 181-261 (EF-hands 5 and 6) is fairly resistant to further cleavage as its intensity increases all the way up to the last time point (120 min, band A8 in Fig. 6).

Limited proteolysis of calbindin D_28k in the presence of 2 mM Mg²⁺ results in nearly the same digestion pattern as for the apoprotein (Fig. 6). Thus, all identified fragments were equivalent in the EDTA and Mg²⁺ samples, except for a very small amount of a peptide (residues 60-261),² which was not found in the EDTA samples. Mg²⁺ seems to offer some protection to proteolytic digestion, although digestion is much faster than in the presence of Ca²⁺.

Ca²⁺ Binding to F123 and F456 as Monitored by the Chromophoric Chelator Quin 2-- Ca²⁺ binding to F123 and F456, each comprising one-half of calbindin D_28k, was measured with the quin 2 method. The stoichiometry of Ca²⁺ binding to each half was analyzed as described for the intact protein, yielding n = 2.01 ± 0.22 for F123 and 2.14 ± 0.24 for F456. Hence, there are two high affinity sites in each half of calbindin D_28k. One site in each half was found to retain the Ca²⁺ affinity of the intact protein (K₁ 1 × 10⁸ M¹). The second site in each half binds Ca²⁺ with ~50-fold lower affinity (K₂ 2 × 10⁶ M¹).

Ca²⁺-induced Conformational Changes as Monitored by ANS Fluorescence-- The fluorescent probe ANS can be used as a sensitive reporter of solvent-exposed hydrophobic patches in proteins. In an attempt to investigate whether F123 and F456 undergo Ca²⁺-induced conformational changes at the tertiary level, we measured the Ca²⁺ dependence of ANS binding for F123 and F456. In EDTA, both F123 (472 nm) and F456 (485 nm) yield a significant blue shift and intensity increase relative to ANS in buffer (530 nm), indicating binding of ANS. When Ca²⁺ is added, a further blue shift in the fluorescence maximum from 485 to 475 nm is observed for F456 with a 30% increase in intensity (Fig. 8B). In contrast, addition of Ca²⁺ to F123 yields a 43% decrease in intensity, with a shift from 472 to 483 nm. The results clearly indicate that both halves undergo structural changes after binding of Ca²⁺. The response is different in the two halves with the local environment around the bound ANS becoming less polar in F456 but more polar in F123.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   ANS binding to F123 and F456. F123 () or F456 () was dissolved to 6 µM each in 2 ml of 60 µM ANS, 50 µM EDTA, 1 mM DTT, 0.15 M KCl, pH 7.1. The ANS fluorescence emission spectra (excitation at 385 nm) were first recorded at room temperature for the Ca2+-free form (dotted line). CaCl2 (1 µl 1 M) was then added to each solution (solid line), and the measurements were repeated. The dash-dotted line shows the spectrum for 60 µM ANS in buffer without protein.

ANS fluorescence spectra were also recorded for an equimolar mixture of F123 and F456 and for the intact protein. These spectra were compared with one another and to the sum of the ANS spectra recorded individually for F123 and F456. No spectral differences were observed between these three cases, neither in the absence nor in the presence of Ca²⁺. The data suggest that ANS binds to the same exposed hydrophobic patches in the intact protein as in the two halves. Earlier work (4) has shown that each half-fragment has a tendency to form homodimers, hence the contact surface between the two halves in the intact protein would be hidden in the separate fragments and not accessible to ANS binding.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of the present work clearly demonstrate that the tertiary structure of calbindin D_28k is Ca²⁺-dependent. The large Ca²⁺-induced effects observed by two-dimensional (¹H-¹⁵N) HSQC NMR spectroscopy (Fig. 2) indicate that the protein undergoes a conformational change. Whereas the spectrum of the apo form shows limited chemical shift dispersion and broad lines, the Ca²⁺-saturated protein displays shift dispersion and narrow lines typical of a well folded helical protein. Far-UV CD data (28) indicate a significant degree of helicity both for the apo and Ca²⁺-loaded states, suggesting that the limited chemical shift dispersion of the apo form represents a loosely organized tertiary structure, similar to a molten globule. As seen in Figs. 2 and 3, there is not a progressive change in the spectrum throughout the Ca²⁺ titration. Rather, fairly small spectral changes are observed until 4 eq of Ca²⁺ are added, at which point there is a dramatic change with a large number of sharp resonances appearing. For example, four signals appear in a region where the glycine residue in position 6 of the Ca²⁺-bound EF-hand loop is typically found (¹H = 10-10.5 ppm, ¹⁵N = 110-114 ppm). These signals most likely represent the corresponding four glycine residues of the regular EF-hands 1 and 3-5, because synthetic peptides corresponding to these four sites also display ¹H chemical shifts of these glycine residues between 10 and 10.5 ppm in the Ca²⁺-bound form (39). Whereas data on the rat protein have been interpreted as one site having much higher affinity than two or three other sites (40), we do not observe sites differing in affinity. Rather, we have strong evidence from the quin 2 and NMR titrations that all four sites bind Ca²⁺ in a parallel fashion with similar affinity and positive cooperativity.

Limited tryptic proteolysis of the apo- and Ca²⁺ proteins (Figs. 6 and 7 and Table II) provides further details on the Ca²⁺-induced structural changes. In accordance with the HSQC results, the protein appears more loosely folded in the apo state, because it is considerably more sensitive to proteolysis than in the Ca²⁺-loaded state. The cleavage patterns are also distinctly different. Whereas the apoprotein is mainly cleaved at Arg-169, the Ca²⁺ form is cleaved at Lys-72 and Lys-98. Arg-169 is located in the middle of the second helix of EF-hand 4, whereas Lys-72 and Lys-98 are found in the loop of EF-hand 2 and in the N-terminal helix of EF hand 3, respectively. This suggests that the conformational changes upon Ca²⁺ binding involve burial or rotation of the second helix in EF-hand 4 and exposure of the loop of EF-hand 2 and of the N-terminal helix of EF hand 3. EF-hand 2 is a variant site that does not appear to bind Ca²⁺ (39), and its Ca²⁺-induced exposure to solvent is intriguing. One could speculate that EF-hand 2 is part of a target-binding surface that becomes exposed upon Ca²⁺ binding.

It is interesting to compare the limited proteolysis of calbindin D_28k to the digestion patterns of calretinin. Calretinin is a hexa-EF-hand protein that is homologous to calbindin D_28k (58% sequence identity), but in contrast to calbindin, it binds Ca²⁺ to EF-hand 2. In calretinin, regions in EF-hands 2 and 3 are not cleaved in the presence of Ca²⁺, which indicates that this part of the protein is not exposed to solvent in a Ca²⁺-dependent way (41, 42). Instead, cleavage occurs in EF-hand 6 of calretinin, which corresponds to a minor Ca²⁺-induced cleavage point in calbindin D_28k. EF-hand 6 represents a non-canonical sequence in both proteins. The dissimilarities in digestion pattern may reflect functional and evolutionary differences between the two proteins to adapt to different targets.

The heteronuclear two-dimensional ¹⁵N-¹H HSQC NMR experiments show that Ca²⁺ efficiently displaces Mg²⁺, suggesting that Mg²⁺ binds to the same sites as Ca²⁺. The results from limited proteolysis, near-UV CD, and NMR spectroscopy show that the structural changes upon Mg²⁺ binding are much smaller than upon Ca²⁺ binding. This indicates that although Mg²⁺ binds to calbindin D_28k, it coordinates in such a way that is does not drive any substantial conformational change. This is consistent with structural data on other EF-hand proteins, which show that the coordination of Mg²⁺ and Ca²⁺ to the same site generally differs (43). The bidendate Ca²⁺-ligating glutamate at position 12 in the EF-hand loop is important for inducing conformational changes in Ca²⁺ sensor proteins. In the Mg²⁺-loaded form, the ion is often coordinated by none or only by one of the side chain oxygens of this glutamate side chain.

Each of the two isolated halves, comprising EF-hands 1-3 and 4-6, respectively, was found to contain two high affinity Ca²⁺-binding sites. In each of the two halves, one site retains the Ca²⁺ affinity as seen for intact calbindin D_28k, whereas the other site has lost affinity by a factor of 50. Hence, truncation of the protein leads to loss of important interactions between EF-hands at the interface between the two halves. Thus, the Ca²⁺-binding sites of calbindin D_28k are clearly not grouped into two largely independent domains, as in calmodulin. The two halves of calmodulin retain native-like Ca²⁺ binding behavior even when separated (44). The present Ca²⁺ binding data, obtained for F123 and F456, support earlier findings that all six EF-hands in calbindin D_28k are part of one globular domain (3, 4).

Calbindin D_28k appears to have evolved through triplication of an ancestral gene coding for two EF-hands (5). EF-hands often associate in pairs as they occur in the sequence, but in calbindin D_28k there are also extensive interactions beyond the pairwise contacts (3, 4). As isolated synthetic peptides, EF-hands 1 and 3-5 in calbindin D_28k are the main Ca²⁺-binding sites, whereas EF-hand 2 fails to bind Ca²⁺ and EF-hand 6 does so only weakly (39). Hence, it seems reasonable to assume that EF-hands 1 and 3 are active in the Ca²⁺ titrations of the N-terminal half and EF-hands 4 and 5 of the C-terminal half. An attractive model for the intact protein is that EF hands 3 and 4 are paired with one another. A cut in this pair would then have major influences on the Ca²⁺ affinity for both EF-hands 3 and 4, which could explain the reduced Ca²⁺ affinity of one site in each half. In this model, the conformational properties and high Ca²⁺ affinities of EF-hands 1 and 5 depend mostly on interactions within the respective half.

The physiological role of calbindin D_28k as a neuroprotector in conditions related to Ca²⁺ overload, and as a facilitator of neuronal Ca²⁺ signaling and renal Ca²⁺ resorption, has been suggested to be linked to the Ca²⁺-buffering capacity of the protein. However, Ca²⁺-induced conformational changes, reported in an earlier study (28) and further analyzed in the present work, suggest that some of the physiological findings could be due to Ca²⁺ sensor activity of calbindin D_28k. All cells have transport systems for the extrusion of Ca²⁺. The intracellular Ca²⁺ concentration in a resting cell is therefore relatively low, <0.1 µM. The cytosolic Ca²⁺ concentration can be abruptly raised to 1-10 µM when the cell is activated by influx of Ca²⁺. In the activated cell, Ca²⁺ is bound in a highly selective way to Ca²⁺-signaling proteins (Ca²⁺ sensors) and induce conformational changes, which lead to an altered activity of target proteins. As discussed above, calbindin D_28k clearly undergoes a Ca²⁺-induced conformational change; however, to be classified as a Ca²⁺ sensor, several other conditions also need to be fulfilled. The intracellular concentration of Mg²⁺, a potential competitor to Ca²⁺, is kept at a relatively constant level around 0.5-2 mM. Consequently, a Ca²⁺ sensor protein must have ~1000-fold selectivity for Ca²⁺ over Mg²⁺. This condition is met by calbindin D_28k as the protein binds to Mg²⁺ with an affinity constant around 1.4·10³ M¹ in 0.15 M KCl, where the average Ca²⁺ binding constant is 2.5·10⁶ M¹. A Ca²⁺ sensor must further be able to respond structurally within a biologically relevant range of intracellular Ca²⁺ concentrations. This is also true for calbindin as its average Ca²⁺ dissociation constant (K_d,av = 7·10⁷ M) falls within the physiological range of intracellular free Ca²⁺ concentrations. In contrast, a Ca²⁺-buffer protein would be expected to have higher affinity (K_d <10⁷ M), whereas Ca²⁺-binding proteins with a lower affinity (K_d >10⁵ M) cannot act as sensors because they are unable to detect the changes in intracellular free Ca²⁺ concentrations that normally occur in cells. Fig. 9 shows the Ca²⁺ saturation curves for calbindin D_28k, calmodulin, and parvalbumin calculated from the Ca²⁺ binding constants in 0.15 M KCl and 1-2 mM MgCl₂. Parvalbumin is considered to be a Ca²⁺-buffering protein and binds Ca²⁺ with ~20 times higher affinity than calbindin D_28k. It is clear from Fig. 9 that parvalbumin cannot be a Ca²⁺ sensor because it is almost fully saturated with Ca²⁺ at resting concentrations of Ca²⁺. By contrast, calbindin D_28k and calmodulin are only 9 and 3% saturated with Ca²⁺, respectively, at resting concentrations of Ca²⁺ (0.1 µM). At Ca²⁺ concentrations similar to those that follow Ca²⁺ activation of a cell, the saturation levels of calbindin D_28k and calmodulin are changed dramatically, as would be expected for Ca²⁺ sensors.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Ca2+ saturation curves for parvalbumin, calmodulin, and calbindin D28k. The degree of saturation as a function of free Ca2+ concentration was calculated from the macroscopic binding constants in 1-2 mM Mg2+ and 0.15 M KCl, pH 7.3. The Ca2+ concentration intervals of a resting and activated cell are shaded.

If calbindin D_28k is a Ca²⁺ sensor, by which mechanism could it work? As mentioned above, the Ca²⁺-binding sites of calbindin D_28k are clearly not grouped into two largely independent domains, as seen in calmodulin. Therefore, it does not follow the calmodulin paradigm of having two independent domains, which expose hydrophobic surfaces only upon Ca²⁺ binding, and that together clamp around the recognition sequences of target enzymes (45). Instead, the tryptic digestion patterns suggest that the Ca²⁺-induced conformational changes involve the rotation of helices and the reorganization of the interface between EF-hands and the linker loops. The mechanism may resemble the Ca²⁺-induced changes observed for recoverin (46), a protein with 4 EF-hands organized in a single globular domain. Alternatively, calbindin D_28k may function by a unique mechanism, possibly shared with other hexa-EF-hand proteins.

	FOOTNOTES

^* This work was supported by Swedish Natural Science Research Foundation Grants NFR K-Ku.10178 (to S. L.) and NFR 104739 (to M. A.), Inga-Britt och Arne Lundbergs Research Foundation grant (to P. Ö.), a National Science Foundation Career grant (to K. S. Å.), and a postdoctoral fellowship from FEBS (to S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence may be addressed. Tel.: 46-46-2228246; Fax: 46-46-2224543; E-mail: tord.berggard@bpc.lu.se.

To whom correspondence may be addressed. Tel.: 46-46-2228246; Fax: 46-46-2224543; E-mail: Sara.Linse@bpc.lu.se.

Published, JBC Papers in Press, February 28, 2002, DOI 10.1074/jbc.M200415200

² Fragments A3 and Mg3 also start at residue 60. The molecular weight as estimated from SDS-PAGE (25-26 kDa) of fragments A3 and Mg3 is higher than the maximum possible weight of a polypeptide starting at residue 60. It is possible that A3 and Mg3 contain more than one fragment that together form a complex strong enough to resist separation during the conditions of the gel electrophoresis.

	ABBREVIATIONS

The abbreviations used are: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; HSQC, heteronuclear single quantum coherence; ANS, 8-anilinonaphthalene-1-sulfonic acid ammonium salt; DTT, 4-dithiothreitol; DSS, 2,2-dimethyl-2-silapentane sulfonate; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES