• Ca2+–ATPase;
  • IR spectroscopy;
  • phosphoenzyme;
  • recombinant protein;
  • Saccharomyces cerevisiae


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Recombinant Ca2+–ATPase was expressed in Saccharomyces cerevisiae with a biotin-acceptor domain linked to its C–terminus by a thrombin cleavage site. We obtained 200 μg of ~ 70% pure recombinant sarcoendoplasmic reticulum Ca2+–ATPase isoform 1a (SERCA1a) from a 6–L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca2E1P and Ca2E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca2E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.




sarcoendoplasmic reticulum Ca2+–ATPase isoform 1a


sarcoplasmic reticulum


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Sarcoendoplasmic reticulum Ca2+–ATPase isoform 1a (SERCA1a) is the major protein component in the sarcoplasmic reticulum (SR) membrane with a molecular mass of ~ 110 kDa [1]. It plays an important role in muscle relaxation by pumping Ca2+, which has been released into the cytosol during muscle contraction, against its gradient back into the SR lumen using the energy of ATP hydrolysis [2, 3]. SERCA1a belongs to the P–type ATPases family for which an Asp residue (here Asp351) becomes transiently phosphorylated during the catalytic cycle. The phosphorylation and dephosphorylation cycle is coupled to Ca2+/H+ exchange. Two Ca2+ bind to the E1 form of SERCA1a (Ca2E1) from the cytoplasmic side before the enzyme becomes phosphorylated from ATP (Ca2E1P). Using the energy released by ATP hydrolysis, Ca2+ ions are subsequently transferred to the SR lumen. Upon Ca2+ release, the enzyme undergoes a conformational change to the E2P state, and reorients to the E1 state after dephosphorylation. The catalytic cycle is shown in Fig. 1 [2]. 3D structures reveal that SERCA1a is composed of a nucleotide-binding domain (N domain), a phosphorylation domain (P domain) and an actuator domain (A domain) in the cytosolic part, as well as a 10-helix transmembrane domain (M), and that the enzyme undergoes large conformational changes both in the cytoplasmic domains and the transmembrane domain in the catalytic cycle [3-7]. Despite all the detailed functional and structural information, some key steps of the catalytic cycle remain unclear, for example, the calcium translocation processes or the final reorientation from the E2 to E1 conformation. Mutagenesis and the development of new biophysical techniques are useful for shedding some light on these steps.


Figure 1. Simplified scheme of the SERCA1a catalytic cycle.

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Point mutants are used to investigate the role of some important amino acid residues [8-10]. To obtain mutants, it is important to apply an efficient expression system. For large-scale production of recombinant SERCA1a, a yeast-based expression system has been developed to express active protein. Recombinant SERCA1a was expressed with a biotin-acceptor domain at its C–terminus connected to the SERCA1a moiety via a thrombin cleavage site. It was endogenously biotinylated in the yeast host during expression and then purified with avidin affinity chromatography and released from the column by thrombin cleavage [11, 12]. This system has several advantages: yeast has similar Ca2+ homeostasis and signaling as higher eukaryotes, rapid and inexpensive conditions for culture, the biotin–avidin interaction is strong and specific, naturally biotinylated proteins are rare in nature and SERCA1a is not coded in yeast genome [11, 13, 14]. From a functional point of view, the purified recombinant protein behaves in a manner similar to the native rabbit SERCA1a regarding the intrinsic fluorescence changes [10, 11, 15] and its ability to transport Ca2+ and hydrolyse ATP [16]. Moreover, the purified recombinant SERCA1a was crystallized and the structure obtained was identical to the one previously obtained from rabbit SERCA1a [16].

IR spectroscopy has been applied to investigate the structure and mechanism of SERCA1a by several groups [17-25]. Different intermediate states of rabbit SERCA1a can be accumulated and IR bands have been assigned to most of them [25, 26]. The enzymatic reaction is triggered by releasing ATP or Ca2+ from caged compounds, which are designed not to react with enzymes. Upon illumination in the UV range, caged compounds release substrates like ATP, and the reaction can be recorded using FTIR spectroscopy with a time resolution of 60 ms [23].

Here, we present the first IR spectroscopic study of recombinant SERCA1a. By contrast to previous studies, we characterize the conformational changes associated with ATP-induced formation of the two phosphoenzymes and find that the recombinant protein has similar catalytic properties to rabbit SERCA1a. This finding is a prerequisite for using biophysical techniques such as IR spectroscopy in the future to investigate the role of individual amino acids.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Expression and purification of recombinant SERCA1a

Approximately 100 g of cell pellets and ~ 900 mg of light membrane fraction were obtained from a 6–L culture. The cell pellet amount was one third of that obtained by Jidenko et al., in spite of the similar expression conditions [11]. Although the expression level was relatively lower in our case, it was sufficient to perform solubilization and avidin affinity purification.

Electrophoresis analysis was performed on various fractions during purification, but because Ca2+–ATPase only accounts for 1% of the light membrane fraction, it is difficult to detect on the gel. Therefore, only the Coomassie Brilliant Blue-stained SDS/PAGE result of the purified protein fraction is shown in Fig. 2. Densitometric analysis indicated that the purity of the recombinant protein and of the rabbit enzyme was ~ 70%. Although this is a typical value for sarcoplasmic reticulum preparations from rabbit muscle, the purity is probably over-estimated for the recombinant protein because this preparation contains impurities just below the threshold of detection by Coomassie Brilliant Blue (Montigny & le Maire, unpublished). Our aim is to use the recombinant protein for reaction-induced IR difference spectroscopy, this method produces signals only from the ‘active’ protein fraction in the reaction, and therefore the obtained purity was considered sufficient.


Figure 2. SDS/PAGE analysis of the purified Ca2+–ATPase followed by Coomassie Brilliant Blue staining. M, 7 μL molecular mass standards (masses in kDa are indicated on the scale, on the left); 1, ~ 2 μg of purified recombinant SERCA1a; 2, ~ 2 μg of rabbit SERCA1a was loaded as a positive control.

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FTIR spectra of formation of Ca2E1P

Figure 3 shows difference spectra of the reaction from Ca2E1 to Ca2E1P obtained upon the release of ATP from caged ATP at 10 mm Ca2+. This high Ca2+ concentration inhibits enzyme activity and accumulates protein in the Ca2E1P state upon ATP release [27]. Bands below 1300 cm−1 are dominated by changes in phosphate absorption due to the photolysis of caged ATP. The negative bands at 1527 and 1345 cm−1 are assigned to the antisymmetric and symmetric stretching vibrations of the disappearing NO2 group in caged ATP [28, 29].


Figure 3. Difference spectra of formation of the Ca2E1P state from Ca2E1 of rabbit SERCA1a (dotted line) and recombinant SERCA1a (solid line).

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The amide I region between 1610 and 1700 cm−1 is best for observing the structural changes in the protein. The band at 1653 cm−1 has been assigned to an α–helix structural change, bands at 1695, 1641, ~1628 cm−1 have been assigned to β–sheet changes and the band at 1665 cm−1 has been assigned to changes in the turn structure [26, 30]. The signals in the amide I region reflect the net change in backbone structure, comprising changes in secondary structure as well as structural changes within existing secondary structures [23], e.g. the bending of helices or the distortions of β sheets. The band at 1628 cm−1, for example, was tentatively assigned to the joining of two parts of a β sheet in the P domain upon ATP binding [31]. This change persists upon Ca2E1P formation from Ca2E1ATP.

Figure 3 shows that recombinant and rabbit SERCA1a shared similar bands, in not only the amide I region, but also the amide II region around 1550 cm−1, and in the region between the photolysis bands at 1527 and 1345 cm−1, where several small bands were assigned to protein bands [26]. Furthermore, the band at 1718 cm−1 assigned to the phosphorylated Asp351 [23] provides direct evidence for phosphorylation of the recombinant protein and the band position similar to that of rabbit SERCA1a indicates a similar environment for its carbonyl group.

The band amplitudes in the amide I region in the difference spectra are related to the amount of active protein in the sample. In the following we relate this amount to the total protein content. The protein amount is not exactly known a priori because of our sample preparation, which is optimized for the detection of difference signals with small amounts of protein. A consequence is that the precise protein concentration in the IR beam was not known. Instead, the amide II absorbance from the absorption spectrum, which is proportional to total protein concentration, is used as an indicator of the total protein amount.

The band amplitudes from the difference spectra are similar for both samples, although there is twice the amount of protein in the recombinant SERCA1a sample compared with the rabbit SERCA1a sample according to the amide II absorption (Table 1). This indicates that only ~ 50% of the protein in the recombinant sample is active SERCA1a in comparison with the rabbit sample.

Table 1. Approximate concentrations of chemicals and proteins in IR sample
Sample nameProtein amide II absorbanceBufferpHK+ (mm)Ca2+ (mm)Mg2+ (mm)Dithiothreitol (mm)Caged ATP (mm)Me2SO
Rabbit Ca2E1P0.045100 mm Mops7.010010.21010
Rabbit E2P0.072100 mm Imidazole7.00.25101010%
Rabbit hydrolysis0.046100 mm Mops7.01000.231515
Recombinant Ca2E1P0.098100 mm Mops7.010010.21010
Recombinant E2P0.106100 mm Imidazole7.00.25101010%
Recombinant hydrolysis0.086100 mm Mops7.01000.231515

FTIR spectra of formation of E2P

Figure 4 shows difference spectra for the reaction from Ca2E1 to E2P in the presence of 10% Me2SO and absence of K+ [27]. These conditions accumulate protein in the E2P state after ATP release, which is indicated by the amplitude of the bands at 1689 and 1607 cm−1 [23, 26, 32, 33]. Another marker band for the E2P state is found near 1193 cm−1, which has been assigned to the phosphate group by isotopic labeling experiments [33].


Figure 4. Difference spectra of formation of the E2P state from Ca2E1 of rabbit SERCA1a (dotted line) and recombinant SERCA1a (solid line).

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The recombinant protein showed similar band positions as rabbit SERCA1a in the amide I region (1610–1700 cm−1). Moreover, they share the same phosphate group band at 1193 cm−1 (Fig. 4). This is evidence for a very similar phosphate environment in both samples. Recombinant and rabbit SERCA1a also have similar positions for the bands near 1750 and 1710 cm−1, which indicate protonation of the Ca2+ ligands [34]. Their similar position in both samples indicates a similar strength of hydrogen bonding to the carbonyl group of the protonated carboxyl ligands.

The band amplitudes in the difference spectra for rabbit SERCA1a are approximately twice as large as those for the recombinant protein, although similar amounts of protein were detected in the absorption spectra (Table 1). This is consistent with the previous finding that only ~ 50% of the protein is active SERCA1a in the recombinant protein sample as compared with the rabbit SERCA1a sample.

Measurement of enzyme activity with FTIR difference spectroscopy

Figure 5 shows difference spectra obtained upon ATP release in the presence of 3 mm Mg2+ and 100 mm KCl. Under these conditions, hydrolysis of ATP is fast, both Ca2E1P and E2P are present and Ca2E1P dominates. This is evident in the spectra because they resemble those for the formation of Ca2E1P in Fig. 3. However, E2P absorption also contributes to the spectra, and the presence of E2P state is clearly shown through the marker band at 1193 cm−1 and the negative band near 1689 cm−1 (Fig. 5).


Figure 5. IR spectra of ATP hydrolysis (1 °C, pH 7.0). The four spectra shown in the figure from top to bottom at 1244 cm−1 are the average from time intervals 0.70–7.5, 7.5–35, 35–103 and 103–172 s, respectively. (A) Rabbit SERCA1a, (B) recombinant SERCA1a.

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The negative band near 1245 cm−1 has been assigned to the antisymmetric stretching vibration of PO2 groups [35]. Its negativity reflects the conversion of a PO2 group to a PO32− group in the hydrolysis reaction. It is thus proportional to the disappearing ATP and was used before measuring the hydrolytic activity [36]. The kinetics of this band is shown in Fig. 6. The spectroscopic-specific enzyme activity was calculated as the initial change in the 1245 cm−1 band area with time divided by the amide II absorbance as a measure of the protein amount. The specific activity was 62 × 10−5 and 145 × 10−5 cm−1·s−1 for recombinant and rabbit SERCA1a, respectively. Thus the rabbit SERCA1a showed twice the activity of the recombinant protein, in line with the above findings regarding the band amplitudes.


Figure 6. Kinetics of ATP hydrolysis using the band near 1245 cm−1 as a marker for the progress of the reaction. Rabbit SERCA1a (dotted line); recombinant SERCA1a (solid line).

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The spectroscopic activity values can be converted to absolute activity values using a calibration between protein mass and amide II absorbance and a calibration between the photolysis bands at 1344 and 1527 cm−1 and the amount of released ATP. The activity determined by IR spectroscopy compares favorably with values determined using the coupled enzyme assay [36]. Activities at 1 °C for the rabbit and recombinant ATPase were estimated to 0.06 and 0.03 μmol·min−1·mg−1, respectively. These are in line with our previously determined activities for the native ATPase of 0.25 μmol·min−1·mg−1 at 5 °C [37] and 4.3 μmol·min−1·mg−1 at 37 °C [36]. The activity values can further be compared with studies of the temperature dependence of ATPase activity [38, 39]. The activity values in these two studies were in the range of 4–5 μmol·min−1·mg−1 at 37 °C and the activity values extrapolated from near 5 to 1 °C were between 0.04 and 0.09 μmol·min−1·mg−1, in line with our values.

The lower activity of the recombinant protein indicates that the content of active ATPase in this sample is ~ 50% that of the SR preparation, which is in line with the above estimates from the band amplitudes in the difference spectra. It is therefore probable that the recombinant protein sample contains a fraction of inactive ATPases. The band at 1628 cm−1 can be used as an indicator of the concentration of active enzyme, and the spectroscopic-specific enzyme activity for the active enzyme fraction was 0.17 and 0.16 cm−1·s−1 for rabbit SERCA1a and recombinant SERCA1a when normalizing the enzyme activity to the absorbance of the 1628 cm−1 band. This indicates that the enzyme activity for active SERCA1a in both samples was similar.

A further conclusion can be drawn from the fact that the 1628 cm−1 band develops when ATP binds to Ca2E1 [23] and the enzyme adopts a closed conformation [31, 40]. Because the inactive fraction does not seem to contribute to this band, ATP binding must be impaired, either directly or because the inactive form is not in the Ca2E1 state due to a defect in high-affinity Ca2+ binding.

Current state of the structural and functional characterization of recombinant SERCA1a

Because of the requirement for relatively large amounts of purified protein, the study of recombinant SERCA1a using biophysical techniques is only just beginning. An essential prerequisite for this work is that the recombinant SERCA1a behaves like the native enzyme in all aspects. This refers to the protein itself and to the membrane system used for reconstitution because, for example, the length [41] and mobility [42] of the lipid acyl chains affect activity. Previous work on recombinant SERCA1a used solubilized [15] and reconstituted [11, 16] enzyme, the latter was used also in this study.

The SERCA1a construct used here (C–terminal biotin-acceptor domain that is removed by thrombin during purification) has been found to have a crystal structure that is very similar to that of the rabbit enzyme [16]. Activity studies demonstrated Ca2+ transport [16] and ATP hydrolysis [11], although this specific activity is still 30% lower than that of the rabbit enzyme. As we demonstrate here, the active fraction has the same hydrolytic activity as the native enzyme. Therefore, the reduced activity in our preparation is not due to a slower turnover of the recombinant protein but probably reflects the presence of a fraction of inactive ATPase.

Here, recombinant SERCA1a was only purified via a single affinity chromatography step which allowed us to obtain sufficient amounts of active protein for biophysical characterization. However, in Jidenko et al. [16], to obtain a sample appropriate for crystallization, affinity chromatography is followed by exchange of n-dodecyl-β-d-maltopyranoside (DDM) for C12E8 with a size-exclusion chromatography that provides an additional purification step in which most of the aggregated proteins are removed. A final ultracentrifugation at high speed after lipid addition is then performed to remove the remaining aggregates. All the steps are likely to eliminate inactive proteins and in this way contribute to a better specific activity.

To date, conformational changes in the recombinant protein have been investigated for Ca2+ binding [11, 15], ATP binding to the Ca2+-free protein and adenosine 5'-(β,γ-methylene)triphosphate binding to Ca2E1 [10]. We investigated the conformational changes that are associated with ATP-induced formation of the two phosphoenzymes, making this the first study to scrutinize the structure of transient enzyme intermediates of recombinant SERCA1a.

Returning to the characterization of conformational changes, previous studies found that the changes in fluorescence were similar to those of the rabbit enzyme [10, 11, 15]. Nevertheless, intrinsic fluorescence provides very limited structural information with only two parameters, sign and amplitude, which makes it difficult to judge whether the recombinant enzyme performs exactly the same conformational changes as the rabbit enzyme. In this respect, IR spectroscopy offers a more stringent control. More than 20 spectral features can be discriminated in the spectra presented here, which are caused by structural changes in the protein backbone and by changes in the environment around functionally important groups. Thus much more molecular information is available than previously. Examples discussed above include several types of secondary structural elements that are perturbed by the conformational changes, the strength of hydrogen bonding to the phosphorylated Asp351 and to the protonated Ca2+ ligands, as well as the environment of the E2P phosphate group. In all these aspects, the recombinant enzyme behaves as the rabbit enzyme.

In conclusion, the evidence accumulated from this and previous studies shows that the active fraction of recombinant SERCA1a after cleavage of the biotin-acceptor domain behaves in a way that is very similar to the rabbit enzyme regarding its structure and conformational changes in all the main partial reactions of the pump cycle. The conclusion is valid for both the DDM-solubilized and the reconstituted enzyme.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Biochemical products were from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. Bactocasamino and yeast nitrogen base were purchased from Difco (Detroit, MI, USA). Glass beads (diameter 0.5 mm) were also from Sigma. Complete EDTA-free protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Mannheim, Germany). Bovine thrombin was from Calbiochem (La Jolla, CA, USA). Softlink™ Soft Release Avidin resin was purchased from Promega (Madison, WI, USA). 50K centricon micro-concentrators were from Millipore (Cork, Ireland). Purified egg-yolk phosphatidylcholine and egg-yolk phosphatidic acid were from Avanti Polar Lipids (Alabaster, AL, USA). Pure DDM was purchased from Anatrace (Maumee, OH, USA). Poly-Prep® Chromatography columns (0.8 × 4 cm), Biobeads SM-2 absorbent (20–50 mesh), Mini-PROTEAN® TGX™ Gels, protein standards as well as Coomassie Brilliant Blue stain were all from Bio-Rad (Hercules, CA, USA).

Expression and purification of recombinant SERCA1a

Plasmid and strain

The construction of expression plasmid pYeDP60–SERCA1a was as described previously [11, 12]. The C–terminus of the rabbit SERCA1a gene was linked to a sequence coding a biotin-acceptor domain via a thrombin cleavage site. Competent Saccharomyces cerevisiae yeast W3031.b/Gal4 (a, leu2, his3, trp1: TRP1-GAL10-GAL4, ura3, ade2-1, canr, cir+) was transformed with this plasmid and selected on minimal medium [12].

Culture and expression

The conditions for large-scale expression were as described previously [12, 13, 17]. First, a preculture was obtained by incubating the selected clone in 10 mL of minimal medium (0.1% w/v Bacto™ Casamino acids, 0.7% w/v yeast nitrogen base, 2% w/v glucose, 20 μg·mL−1 of adenine) for 24 h at 28 °C with vigorous shaking. This preculture was then used to inoculate another 100 mL of minimal medium to obtain a second preculture. Large-scale culture was initiated by adding 20 mL of the second preculture to 1 L of YPGE2X-rich medium (2% w/v Bacto™ Peptone, 2% w/v yeast extract, 1% w/v glucose, 2.7% v/v ethanol) and incubated at 28 °C with shaking. A total of 6 L of yeast culture was prepared in this way. After 36 h, the temperature was lowered to 18 °C, and expression was induced with 20 g·L−1 galactose. A second galactose addition was carried out after 13 h, and the culture was kept growing for another 6 h before harvest.

Preparation of membrane fractions

All preparation steps were performed at 4 °C unless noted otherwise. Cell pellets were collected by centrifugation at 6238 g, for 10 min in a JLA8.1000 rotor. The pellets were then resuspended in cold sterile water and centrifuged at 6238 g for another 10 min. The weight of the pellets (wp) was measured and ~ 100 g of cell pellets were collected from 6 L culture. Pellets were suspended in TEKS buffer (50 mm Tris/Cl, 1 mm EDTA, 0.1 m KCl, 0.6 m sorbitol, pH 7.5) to a final volume equivalent to 2 wp (~200 mL). The suspension was incubated for 5 min before centrifugation at 4424 g, for 10 min in a JA10 rotor. Pellets were then resuspended in TES buffer (50 mm Tris/Cl, 1 mm EDTA, 0.6 m sorbitol, 1 mm phenylmethanesulfonyl fluoride, 2 protease inhibitor tablets, pH 7.5) to a final volume equivalent to 2 wp (~ 200 mL). Note that phenylmethanesulfonyl fluoride and protease inhibitor tablets were added to TES buffer just before use. The suspension was then fully covered with glass beads (diameter 0.5 mm) in a sealed bottle, and cells were broken by mechanical agitation of the horizontally placed bottle in an incubator with 350 rpm shaking for 20 min at 15 °C. Cells and glass beads were subsequently transferred to a filter flask and a crude extract was recovered by connecting a vacuum pump to the filter flask. Glass beads were washed with TES buffer until the color from the crude extract disappeared. All liquid collected was then centrifuged at 1387 g in a JA10 rotor for 10 min to give pellet P1 and supernatant S1. Supernatant S1 was centrifuged at 22 095 g for 15 min in a JA14 rotor to give pellet P2a and supernatant S2a. P2a, corresponding to the heavy membrane fraction, was resuspended in a small amount of TES buffer (~ 15 mL) and the suspension was then centrifuged at 22 095 g to give supernatant S2b and pellet P2b. S2b together with S2a was centrifuged at 186 010 g for 1 h at 8 °C in a Ti45 rotor, to give pellet P3 and supernatant S3. P3, corresponding to the light membrane fraction, was resuspended in Hepes buffer (20 mm Hepes/Tris, 0.3 m sucrose, 0.1 mm CaCl2, pH 7.4) to a final volume equivalent to 0.5 wp (~ 50 mL), 1 protease inhibitor tablet was added just before use. The suspension was homogenized and stored at −80 °C. Protein concentration from the light membrane fraction was determined by using bicinchoninic acid, with BSA as a standard [43].

P3 was then diluted into solubilization buffer (50 mm Tris/Cl 0.1 m KCl, 20% v/v glycerol, 1 mm CaCl2, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, pH 7.0) to a final protein concentration of 2 mg·mL−1 and with a DDM to protein ratio of 3 : 1 (w/w). The suspension was stirred gently for 1 h at room temperature before centrifugation at 142 414 g for 45 min at 10 °C to give a supernatant S4 and pellet P4.

Purification of recombinant SERCA1a by affinity chromatography

Unless specified otherwise, purification was carried out at 4 °C. Six milliliters of Softlink™ Soft Release Avidin resin was mixed with solubilized membrane fraction S4 and stirred gently overnight. The suspension together with the resin was then loaded into a 0.8 × 4 cm column. The resin was first washed with 10 resin volumes (~ 60 mL) of high-salt buffer (50 mm Tris/Cl, 1 m KCl, 20% v/v glycerol, 1 mm CaCl2, 1 mm dithiothreitol, 0.05% w/v DDM, pH 7.0), and then with 10 resin volumes (~ 60 mL) of low-salt buffer (50 mm Tris/Cl, 100 mm KCl, 20% v/v glycerol, 2.5 mm CaCl2, 1 mm dithiothreitol, 0.05% w/v DDM, pH 7.0) at a flow rate of 2 mL·min−1. The resin was subsequently transferred to a measuring cup and 25 U thrombin·g resin−1 were added. The mixture was stirred gently at room temperature for 30 min before adding another 25 U thrombin·g resin−1. After further 30 min room temperature incubation, 1 mm phenylmethanesulfonyl fluoride and half of a protease inhibitor tablet were added to stop thrombin activity. Solutions were collected into a 50K filter tube and thrombin was subsequently removed by centrifuging the tube at 2465 g for 30 min. Purity was checked by electrophoresis on SDS/polyacrylamide gradient gel (4–15%) and the gel was stained with Coomassie Brlliant Blue.

Densitometric analysis

The purity of SERCA1a was evaluated in the following way. Density profiles of each lane on the electrophoresis gel were produced with imagej. They were evaluated with the spectrometer software opus (Bruker). Densities were first converted to absorbance values according to the Beer–Lambert law using as the I0 value (intensity in the absence of absorption) the highest intensity in each profile. Finally, distinct protein bands were integrated with respect to a baseline drawn between two points just outside the band in question and the ATPase band area was related to the total area of all bands. The obtained values were 73% for recombinant ATPase and 75% for rabbit ATPase.

Lipid preparation

Fifty milligrams of egg-yolk phosphatidycholine and 5 mg of egg-yolk phosphatidic acid were dissolved in 2 mL chloroform and connected to vacuum overnight to completely remove liquid. A 1.84 mL aliquot of degassed buffer (50 mm Tris/Cl, 20% v/v glycerol, pH 7.0) was added and the final concentration of egg-yolk phosphatidycholine/egg-yolk phosphatidic acid phospholipids was 30 mg·mL−1 (9/1, w/w). The resulting suspension was vortexed at maximum speed for 15 min. It was then frozen in liquid nitrogen and thawed in water, this freeze and thaw step was repeated 15 times before storing the lipids at −80 °C.

Detergent removal and membrane reconstitution

Phospholipids were added to the purified protein solution at a ratio of mlipid/mprotein = 3:1, and biobeads were added at a ratio of mbiobeads/mDDM = 200:1. The removal of DDM and the membrane reconstitution process were carried out by incubating the mixture at 4 °C overnight. Protein solution together with biobeads was then loaded onto an empty 0.8 × 4 cm column and protein solution was collected upon washing with small amounts of low-salt buffer without detergent. The protein sample was frozen in liquid nitrogen and stored at −80 °C before use.

IR measurements

Sample preparation

Rabbit SERCA1a was a generous gift from W. Hasselbach (Max-Planck-Institut, Heidelberg, Germany) and was prepared as described previously [44]. Both rabbit and recombinant SERCA1a were centrifuged at 154 000 g for 30 min at 4 °C in a TLA55 rotor, the supernatant was removed and the protein pellet was subsequently resuspended in either Mops buffer (10 mm Mops-KOH, 10 mm KCl, 20 μm CaCl2, pH 7.0) or imidazole buffer (10 mm Imidazole-HCl, 500 μm MgCl2, 20 μm CaCl2, pH 7.0) according to the type of experiments (Table 1).

Reaction-induced IR difference spectroscopy measurements

Dithiothreitol and caged ATP solution were dried on a CaF2 window with a 5 μm trough, and then 10 μL of diluted rabbit SERCA1a (~ 1 mg·mL−1) or 10 μL recombinant SERCA1a (~ 1 mg·mL−1) suspensions were added in the middle of the window. The protein samples were dried and subsequently rehydrated without mixing with 1 μL 3 mm MgCl2, 10 mm CaCl2 or 10% Me2SO, according to the type of experiment. We tested whether the drying process had an effect on the enzyme activity. For these experiments, we dried all ingredients on the CaF2 window and rehydrated with 1 μL recombinant SERCA1a sample, or dried all ingredients together with 1 μL recombinant SERCA1a sample and rehydrated with 1 μL water. The final results showed that the enzyme that was dried had ~ 1.5× higher activity and was ~ 1.4× more concentrated than the undried protein sample. This indicates that the enzyme does not lose activity during the drying process.

The conditions were designed for observation of ATP hydrolysis, to accumulate the Ca2E1P state and E2P state, respectively [23]. The sample preparation was completed by closing the window with a second flat window. Approximate concentrations of each sample preparation are shown in Table 1.

FTIR difference measurements were performed at 1 °C with a Bruker IFS 66 rapid scan FTIR spectrometer equipped with an MCT detector. First, a reference spectrum, which represented the Ca2E1 state of the sample before the enzymatic reaction, was recorded and the time was set to zero. ATP was then released photolytically from caged ATP by a UV flash. Following the flash, 10 spectra of 1 scan, 10 spectra of 10 scans, 10 spectra of 40 scans and 10 spectra of 200 scans were recorded. Difference spectra were calculated with respect to the reference spectrum and they showed only the absorbance changes caused by the photolysis of caged ATP, conformational changes of the enzyme and the catalytic reaction. Six flashes were applied in total to each sample and the voltage of the power supply was 800 V for the first three flashes and increased to 850 V for the fourth flash and to 900 V for the last two flashes. Sample equilibrating time was 1 h between each flash.

The enzyme activity was calculated as described previously and data from six flashes were averaged [36]. Data were evaluated with opus software, the first 10 spectra of one scan were averaged, together with the other 30 spectra gave data at 0.35, 1.03, 1.71, 2.4, 3.08, 3.76, 4.45, 5.13, 5.8, 6.5, 7.2, 8.9, 11.6, 14.4, 17.1, 19.8, 22.6, 25.3, 28, 30.8, 33.5, 41.7, 55.4, 69.1, 82.7, 96.4, 110, 124, 137, 151, 165 s. The band area between 1240 and 1250 cm−1 was plotted against time and the initial rate of the reaction was obtained by calculating the slope of a fitted curve. The slope was divided by the amide II absorbance as a measure of the protein amount in order to obtain a spectroscopic-specific enzyme activity. These values were then converted to absolute activity values using calibrations between protein mass and amide II absorbance and between the photolysis bands at 1345 and 1527 cm−1 and the amount of released ATP: an amide II absorbance of 1 is produced by 0.69 mg SR protein·cm−2 (± 17%) [26, 45] and 1345 and 1527 cm−1 photolysis bands with an absorbance of 0.001 are produced by the release of 14.6 and 5.2 nmol ATP·cm−2 (±8%), respectively [45].


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

The authors thank W. Hasselbach (Max-Planck-Institut, Heidelberg, Germany) for the rabbit Ca2+–ATPase and J.E.T. Corrie and F. von Germar for helping with the preparation of the caged compounds. This work was supported by the Swedish Research Council and Knut och Alice Wallenbergs Stiftelse.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References
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