Use of Creatine Kinase To Induce Multistep Reactions in Infrared

Nov 14, 2013 - To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanis...
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Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments Nadejda Eremina and Andreas Barth* Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden ABSTRACT: An extension of current approaches to trigger enzymatic reactions in reaction-induced infrared difference spectroscopy experiments is described. A common procedure is to add a compound that induces a reaction in the protein of interest. To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanisms. The enzymatic reaction of CK could be followed using bands at 1614 and 979 cm−1 for creatine phosphate consumption, at 944 cm−1 for ADP consumption, and at 1243, 992, and 917 cm−1 for ATP formation. The potential of CK to induce multistep reactions in infrared spectroscopic experiments was demonstrated using the sarcoplasmic reticulum Ca2+ATPase (SERCA1a) as the protein of interest. ADP binding to the ATPase was triggered by photolytic release of ADP from P3-1-(2-nitro)phenylethyl ADP (caged ADP). CK added in small amounts converted the released ADP to ATP on the time scale of minutes. This phosphorylated the ATPase and led to the formation of the first phosphoenzyme intermediate Ca2E1P. Thus a difference spectrum could be obtained that reflected the reaction from the ADP ATPase complex to the first phosphoenzyme intermediate. Comparison with a phosphorylation spectrum obtained when the initial state was the ATP ATPase complex revealed the contribution of ATP’s γ-phosphate to the conformational change of the ATPase upon nucleotide binding: γ-phosphate binding modifies the structure of a β-sheet, likely in the phosphorylation domain, and shifts its spectral position from ∼1640 to ∼1630 cm−1. Upon phosphorylation of the ATPase, the β-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state.

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sites of the ATPase in state E1, which releases H+ into the cytoplasm. This is followed by ATP binding and phosphorylation, Ca2E1 → Ca2E1ATP → Ca2E1P. The next step is phosphoenzyme conversion, Ca2E1P → E2P. Dephosphorylation to E2 and conversion of the Ca2+ free enzyme to E1 complete the reaction cycle. Most of these major partial reactions have been investigated with infrared difference spectroscopy.17−21 Further partial reactions can be induced by employing helper enzymes. In our previous helper enzyme experiment, ATP was initially released from caged ATP and phosphorylated the ATPase. When the produced ADP dissociated from the phosphoenzyme, the helper enzyme apyrase slowly converted it to AMP, which has a low affinity for the ATPase and therefore did not rebind. The experiment revealed that bound ADP stabilized a closed conformation of the phosphoenzyme, which relaxed to a more open conformation when ADP dissociates. In this work we characterize creatine kinase (CK) for its use as helper enzyme. CK’s substrate, creatine phosphate (CP), serves as an energy reservoir for the rapid buffering and regeneration of ATP, which is catalyzed by CK according to the following reaction: ADP + CP → ATP + C. CK transfers the phosphate group of the creatine phosphate (CP) to the ADP

INTRODUCTION Infrared spectroscopy is a versatile bioanalytical technique that provides information on a wide variety of biological systems: from tissues1,2 to individual bonds in large biomolecules.3−9 In studies of protein reactions, it combines two of its advantages: high time resolution and high information content. Reactions usually have to be induced directly in the infrared cuvette to achieve the necessary sensitivity. This approach is called reaction-induced difference spectroscopy. Several methods to trigger reactions are available,4,6,9−11 one of which10,12 employs so-called caged compounds13 to release a compound of interest upon light excitation. A considerable extension of the existing approaches for reaction-induced infrared difference spectroscopy would be the possibility to induce a second reaction after initiation of the first reaction. In this context we have suggested the use of helper enzymes to modify the compound that is initially present in the sample. Adding only a small amount of helper enzyme ensures that it does not contribute to the signals of the protein of interest and that the reaction catalyzed by the helper enzyme is significantly slower than the initial reaction. We have used this approach in studies of the Ca2+-ATPase,14,15for example, to remove its enzymatic product ADP by conversion to AMP using the helper enzyme apyrase.14 The Ca2+-ATPase (SERCA1a) pumps Ca2+ ions from the muscle cells into the sarcoplasmic reticulum. According to a model established by de Meis and Vianna16 the cycle proceeds as follows: two Ca2+ ions bind to the high affinity Ca2+ binding © 2013 American Chemical Society

Received: September 26, 2013 Revised: November 11, 2013 Published: November 14, 2013 14967

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molecule, producing an ATP molecule and unphosphorylated creatine (C). The present study has a second methodological implication, the direct measurement of enzyme activity with infrared spectroscopy. This is one of the largely overlooked applications of infrared spectroscopy in spite of its high potential for biotechnology applications. Virtually any product of any enzyme reaction has a different infrared spectrum than the respective substrate because the chemical structures are sufficiently different. Therefore, enzyme activity can be followed directly in time-resolved experiments without employing modified substrates or further enzymes, needed for example in other spectroscopic assays to convert the product into a molecule with a spectral signature in the UV/visible spectral range. Furthermore, restrictions regarding, e.g., pH or temperature that are imposed on the system from the additional or artificial components needed for other methods are irrelevant for infrared spectroscopy. Nevertheless, the number of applications is limited, as summarized recently.22 Here, we characterize the spectral changes that are associated with the catalytic reaction of creatine kinase (CK). They have been partially described before.23

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RESULTS Infrared Spectra of CK’s Enzymatic Reaction: Band Assignment. In the following section the infrared spectra of the components of the CK enzymatic reaction are discussed. Figure 1A shows the absorption of ATP (full line) and ADP (dotted line). The bands in the 1700−1300 cm−1 region are assigned to the ring and NH bending vibrations of the adenine and ribose rings.24 The broad band at 1229 cm−1 is due to the antisymmetric stretching vibrations of the α- PO2− and β-PO2− groups of ATP. The second broad band between 1120 and 1070 cm−1 arises from the stretching vibrations of γ-PO32− as well as from the overlapping symmetric stretching vibration of the PO2− group(s). The 994 cm−1 band is assigned to the stretching vibration of P−O in the Pα−O−Pβ and PO32− groups of ATP, whereas the 920 cm−1 band comes from the P−O stretching vibration in Pβ−O−Pγ. The main difference between the ATP and ADP absorption spectrum is in the phosphate region. For ADP the signature bands are observed at 1211 and 940 cm−1 and are assigned to the antisymmetric stretching vibration of the PO2− and the symmetric stretching vibration of P−O in the Pα−O−Pβ.25−29 Figure 1B presents the absorption spectra of C (full line) and CP (dotted line). CP has several typical bands in its spectrum. A strong band at 1612 cm−1 is assigned to the antisymmetric stretching vibration of its carboxyl group; a medium strong band at 1527 cm−1 stems from a bending vibration of the NH2 group; two medium bands at 1395 and 1308 cm−1 are associated with the symmetric vibration of the carboxyl group and two distinct bands at 1117 and 978 cm−1 are typical of the stretching vibrations of the PO32− group.24 The absorption spectrum of C resembles that of CP; however, it lacks the phosphate bands and has instead three bands at 1670, 1628, and 1594 cm−1 that originate from the stretching vibrations of CN and the scissoring vibration of NH230 and the antisymmetric stretching vibration of the carboxyl group. All our absorption spectra are in agreement with those published previously, some of which (CP, C) were shown in a more limited spectral range. Figure 1C shows a model difference spectrum of the CK catalyzed reaction from reactant (ATP + C) to product (ADP +

Figure 1. Infrared spectra associated with the catalytic reaction of CK. Infrared absorbance spectra (A) of 50 mM ATP (black line), 50 mM ADP (gray line) and (B) of 50 mM C (black line), 50 mM CP (gray line). (C) A calculated spectrum of ATP with C minus ADP with CP. The spectra have been shifted for a clearer presentation. (D) Reactioninduced double difference spectra of the enzymatic reaction ATP + C → ADP + CP of CK generated by subtracting a spectrum recorded at ∼2.5 s from the spectra recorded at three different times: ∼5 s (black line), ∼50 s (dark gray line), and ∼500 s (gray line).

CP) obtained by subtraction of the respective absorbance spectra. The model spectrum shows the changes expected in the observation of the enzymatic reaction. The negative bands indicate the transfer of the phosphate from CP to ADP and the positive bands show the formation of ATP and C. In the following, this spectrum will be compared to the reactioninduced difference spectrum directly obtained during the CK reaction. In the reaction-induced experiment, the phosphorylation of ADP in the presence of CK was initiated by the photolysis of caged ADP and the following formation of ATP was monitored with time-resolved FTIR spectroscopy. Figure 1D presents spectra from the CK enzymatic reaction recorded 5 s (bold line), 50 s (dashed line), and 500 s (dotted line) after the flash. Comparing the model difference spectrum to the reactioninduced difference spectrum, we assign the positive bands at 917, 992, and 1243 cm−1 to the formation of ATP, the negative band at 979 cm−1 to the consumption of CP, and the negative band at 944 cm−1 to the consumption of ADP. The negative band at 1614 cm−1 was previously assigned to CO of the peptide backbone of CK accessible to water.23 However, in the 14968

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which indicates that Ca2+ also causes an inhibitory effect at mM concentrations. CK as a Helper Enzyme To Induce Multistep Reactions in the Ca2+-ATPase Reaction Cycle. In the following section, we evaluate the use of CK to study multistep reactions with infrared spectroscopy. Our test case and protein of interest is the Ca2+-ATPase, but the approach is generally applicable to phosphate transfer reactions involving ATP. Here, we were interested in the effects of the γ-phosphate of ATP on the conformation of the ATPase with bound nucleotide. In our multistep experiment, ADP is released from caged ADP and the released ADP binds to the ATPase Ca2E1 → Ca2E1ADP. Subsequently, CK converts ADP to ATP, which binds to the Ca2+-ATPase and phosphorylates the enzyme Ca2E1ADP → Ca2E1P. The absorbance changes observed upon the release of ADP from the caged compound, ADP binding to Ca2+-ATPase, ADP conversion to ATP by CK, and phosphorylation of the ATPase by the produced ATP are presented in Figure 3 (top panel). Note that in these

model spectrum (Figure 1C) there is a similar band at 1612 cm−1, which stems from the dephosphorylation of CP. Because the concentration of CK in the reaction-induced experiment is low, 2 mg/mL, it is not expected to contribute to the difference spectrum. In addition, all bands in the reaction-induced difference spectrum appear also in the model difference spectrum, which does not have contributions from CK. Therefore, we assign the 1614 cm−1 band in the reactioninduced difference spectrum (Figure 1D) to the disappearing absorption of CP. The two positive bands above 1620 cm−1 can be assigned to the formation of C. Thus, all molecules participating in the reaction can be followed with infrared spectroscopy. Kinetics of the CK Enzymatic Reaction. Kinetics for some marker bands of the enzymatic reaction are shown in Figure 2. For evaluating the kinetics of the enzymatic reaction,

Figure 3. (A) Difference spectra showing the absorbance changes observed upon the release of ADP from caged ADP, ADP binding to Ca2+-ATPase, ADP conversion to ATP by CK, and phosphorylation of the ATPase by the produced ATP. Spectra were recorded 0−7 s (black line), 70 s (dark gray line), and 690 s (gray line) after photolysis of caged ADP. The reference spectrum was recorded immediately before the photolysis flash. (B) Absorbance change in the early (black line) and late (gray line) phases after ADP release calculated by subtraction of difference spectra. The early spectrum (spectrum at 70 s minus spectrum at 0−7 s) is dominated by protein absorbance changes due to the reaction Ca2E1ADP → Ca2E1P, the late spectrum (spectrum at 690 s minus spectrum at 70 s) by the CK enzymatic reaction. The spectra were shifted for a clearer presentation.

Figure 2. Kinetics of the enzymatic reaction of CK, monitored by integrating difference bands at (A) 1243 cm−1 arising from ATP formation and at (B) 1614 cm−1 caused by CP consumption.

the two most prominent bands were chosen: (A) the increasing 1243 cm−1 band of ATP (product) and (B) the decreasing 1614 cm−1 band of CP (reactant). These were integrated and averaged because the rates were the same. The plots show that the activity of CK can easily be determined with infrared spectroscopy, using only 2 μg of enzyme. The relative rates for the reaction were calculated to be 0.072 ± 0.013 s−1 in the presence of 6 mM Ca2+ and 3 mM Mg2+, 0.026 ± 0.002 s−1 in the presence of 6 mM Ca2+ alone, 0.125 ± 0.023 s−1 in the presence of only 3 mM Mg2+, and 0.058 ± 0.011 s−1 without ions added. It has been seen in earlier studies that Mg2+, Ca2+, Mn2+ ,and Co2+ can be used as activators of CK. Optimal activity was obtained when Mg2+ was present in access. It is also known that other metals such as Ni2+, Cr2+, and Cd2+ can cause inhibitory effects.31,32 Our results show that the enzymatic activity drops drastically in presence of 6 mM Ca2+,

experiments, the reference spectrum was recorded before the photolysis flash so that the spectra reveal also absorbance changed due to the caged ADP photolysis. This is in contrast to the reaction-induced difference spectrum shown in Figure 1 where difference spectra recorded late after the flash were subtracted from an early difference spectrum and therefore only show processes after ADP release. The earliest spectrum (bold line, recorded within the first 7 s) shows absorbance changes due to the photolysis of caged ADP and the binding of ADP to the Ca2+-ATPase. The large negative bands at 1525 and 1347 cm−1 arise from the 14969

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that is characteristic of phosphoenzyme formation. Other bands differ in the two spectra, reflecting differences between ADP and ATP binding. The negative bands at 1689 and 1627 cm−1 in the Ca2E1ATP → Ca2E1P spectrum are replaced by positive bands at 1693, 1678, and 1631 cm−1 and a positive band at 1639 cm−1 is replaced by a negative band at 1643 cm−1 in the spectra of the present work. All these changes are in regions of β-sheet absorption, indicating that γ-phosphate binding affects the structure of a β-sheet. Upon Ca2E1P formation and when the ADP bound state is the initial state, the main β-sheet band shifts down from 1643 cm−1 (negative band) to 1631 cm−1 (positive band) but shifts up from 1627 cm−1 (negative band) to 1639 cm−1 (positive band) when the initial state is the ATP bound state. This indicates that the position of this band in Ca2E1P is intermediate between that in Ca2E1ADP and in Ca2E1ATP and that the band is most downshifted in Ca2E1ATP. Thus, the combination of negative and positive bands suggests that γ-phosphate binding induces a downshift of a β-sheet band from ∼1640 to ∼1630 cm−1, which is partially reversed upon formation of Ca2E1P. The downshift has been observed before for nucleotide binding35,36 and tentatively assigned to the β-sheet in the phosphorylation domain, which becomes more planar when nucleotides bind.33,37 The new finding of this work is that binding of the γ-phosphate considerably contributes to this conformational change. Upon phosphorylation of the ATPase, the β-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state. In the amide II region, a negative band is observed at 1534 cm−1 in the Ca2E1ADP → Ca2E1P spectrum (Figure 3 bottom part, full line) that is not present in the Ca2E1ATP → Ca2E1P spectrum (previous work). Bands in the Ca2E1ADP → Ca2E1P spectrum that are not observed in the Ca2E1ATP → Ca2E1P spectrum are due to conformational changes that are necessary to reach the Ca2E1P state when the starting state is the ADP bound state but that are not executed when the ATP bound state is the starting state. Therefore, these changes are already induced by ATP binding. The band at 1534 cm−1 is close to a strong photolysis band but has been identified before upon subtraction of a photolysis spectrum and assigned to an amide II vibration because it is reduced in D2O.35 This band and the procedure of subtracting the photolysis spectrum are confirmed here by an independent and more direct approach, and the band can be attributed to a conformational change induced by γ-phosphate binding.

antisymmetric and symmetric vibrations of the disappearing nitro group of caged ADP. Bands below 1300 cm−1 are assigned to alterations in phosphate absorption caused by photolysis18 in analogy to the absorbance changes observed for caged ATP.29 The bands observed in the amide I region (1700−1600 cm−1) indicate the conformational changes of the Ca2+-ATPase upon nucleotide binding. The positive band at 1628 cm−1 is indicative of the binding event and has been tentatively assigned to the conformational changes in the β-sheet of the phosphorylation domain along with the 1693 cm−1 band.33 The positive shoulder at 1619 cm−1 can also be considered a marker band for the binding of ADP to Ca2+-ATPase.34 In the two later spectra after 70 and 690 s, new negative bands are observed at 1607 and at 977 cm−1, indicating consumption of CP due to the CK reaction. We do not see positive bands due to ATP production by CK because the ATP is consumed by the ATPase. The final products when all CP is consumed are C, ADP, and phosphate. The production of phosphate can be seen at the positive band near 1070 cm−1. In addition to bands due to the enzymatic reactions, absorbance changes due to the phosphorylation of the ATPase are observed. The positive band at 1717 cm−1 is characteristic of the accumulation of the Ca2E1P state and is assigned to the formation of the CO group of the phosphorylated Asp351. Two positive bands at 1550 and 1391 cm−1 are also indicative of Ca2E1P formation.25 Two double difference spectra are shown in the bottom part of Figure 3 obtained by subtracting the early spectrum of the top part from the middle spectrum and the middle spectrum from the late spectrum. The spectrum showing the late absorbance changes (Figure 3 bottom part, dotted line) is due to the catalytic reactions of the ATPase and of CK. It is different from the spectrum of the CK reaction (Figure 1) because the ATP produced by CK is immediately consumed by the ATPase as mentioned above. The ATP band at 1243 cm−1 is therefore not observed in the late spectrum of Figure 3B. Instead, a band at 1070 cm−1 is present, which is due to phosphate, one of the products of ATP hydrolysis. The reaction reflected in the late spectrum can be summarized in the following way: ADP + CP → ATP + C → ADP + Pi + C

The early absorbance changes reveal the conformational transition from Ca2E1ADP to Ca2E1P, i.e., the absorbance of Ca2E1P minus that of Ca2E1ADP. Previously, we have published the spectrum of the phosphorylation reaction,25 i.e., the absorbance of Ca2E1P minus that of Ca2E1ATP. These reactions have the same end state but different starting states. Therefore, the differences between these two spectra reflect the different starting states, i.e., are due to the effects of binding of the γ-phosphate of ATP. When the present spectrum of Ca2E1ADP → Ca2E1P is compared with our published spectrum of Ca2E1ATP → Ca2E1P,25 it becomes clear that the positive bands at 1717, 1653, and 1550 cm−1 are found at similar positions in both spectra. These can therefore be assigned to Ca2E1P specific structural features that are induced by neither ATP nor ADP binding. The 1717 cm−1 band has been attributed to the phosphorylation of Asp351,25 the 1653 cm−1 band to a conformational change of an α-helix, and the 1550 cm−1 band to a conformational change reflected by the amide II absorption. Also a negative band is common for both spectra, the band near 1600 cm−1. This indicates a structural change

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DISCUSSION This work evaluated the use of CK as a helper enzyme to induce multistep reactions in infrared spectroscopic experiments. In this context we characterized the absorbance changes that accompany the catalytic reaction of this enzyme. We demonstrate that the catalytic activity of CK can be directly followed by infrared spectroscopy using the absorbance of any of the involved molecules. As compared to enzyme assays that couple a catalytic reaction of interest to further enzymatic reactions that eventually produce a signal in the UV/visible spectral range, the direct infrared spectroscopic approach is not limited by the requirements of the secondary enzymatic reactions. Any condition (temperature, chemical composition of the medium) on the activity of the assayed enzyme can be tested. In contrast to the enzymes that we previously employed as helper enzymes, adenylate kinase and apyrase,15 the CK 14970

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reaction produces a distinct band at 1614 cm−1 outside the region of phosphate absorption. This is an advantage when the helper enzyme is employed to study a protein of interest that also catalyzes phosphate transfer reactions, which are then difficult to distinguish from those catalyzed by the helper enzyme. In the case of CK, the absorbance changes in the phosphate region in the presence of the protein of interest Ca2+-ATPase were different from those in the presence of the helper enzyme CK alone because the ATPase transformed the produced ATP back to ADP. However, the band at 1614 cm−1 provided a clear measure of the extent of the CK reaction. One advantage of helper enzymes in reaction-induced difference spectroscopy experiments is that a particular reaction can be delayed and its rate determined by the concentration of the helper enzyme. In the present work, formation of the Ca2E1P phosphoenzyme was controlled by the helper enzyme CK. Slowing down particular reactions brings the advantage of longer recording times for the spectra and thus of increased signal-to-noise ratio. In addition, a transition between enzyme states can be induced that does not normally take place in the catalytic cycle. In the case of the Ca2+-ATPase, the ADP bound complex is a dead end complex. Nevertheless, using CK as a helper enzyme allowed us to observe the transition from the ADP bound state to the first phosphoenzyme Ca2E1P, which provided detailed information on the conformational effects of the binding of ATP’s γ-phosphate. Generating “unphysiological” transitions between enzyme states with helper enzymes follows the philosophy of reaction-induced difference spectroscopy, which states that it is more reliable to generate a difference spectrum directly in the infrared cuvette than to compare spectra obtained with different samples.

Corp., Woburn, MA) with the release yield of ∼20%. Spectra were recorded in the following way: (1) a reference spectrum was recorded, and (2) after the photolysis flash was applied, time-resolved infrared spectra with 65 ms time resolution were recorded. Difference spectra were obtained by subtracting the reference spectrum from the spectra recorded after photolytic release of ATP or ADP. Kinetic plots of the enzymatic reaction were obtained by integrating the spectrum using method E of the Bruker OPUS software. For evaluation of ATP formation, the peak integrations limits were set to 1235−1250 cm−1 and calculated with respect to a baseline drawn between two baseline points on either side of the peak. These points were obtained by averaging data points between 1267 and 1280 cm−1 and between 1213 and 1224 cm−1. For evaluation of CP dephosphorylation the peak integration limits were set to 1605−1621 cm−1. The baseline points were obtained by averaging the data points between 1623−1632 and 1591−1600 cm−1. The curve fitting was performed using the program Simfit using the following formula y = Ae(−kt) − C. The rate constants were determined from the fitting to the averaged data from three to four experiments, whereas its error was obtained by calculating the standard deviation of the rate constants calculated from individual experiments.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank W. Hasselbach (Max-Planck-Institut, Heidelberg, Germany) for the rabbit Ca2+-ATPase and J. E. T. Corrie for the preparation of the caged compounds. This work was supported by the Swedish research council and Knut och Alice Wallenbergs Stiftelse.

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EXPERIMENTAL PROCEDURES Materials. CK (C3755) from rabbit muscle, creatine phosphate (P7939), MgCl2 and MOPS (3-[N-morpholino]propanesulfonic acid) were purchased from Sigma. Tris−HCl was obtained from Angus. KCl and CaCl2 and were obtained from Scharlau. Ca2+-ATPase from rabbit hind leg and back muscle was prepared in the laboratory of W. Hasselbach by the method of Hasselbach and Makinose and stored at −20 °C.38 Caged compounds were synthesized by J. E. T. Corrie at the National Institute of Medical Research, London. Sample Preparation. Infrared samples for reactioninduced difference spectroscopy were prepared by drying the sample solution on a BaF2 window with a trough of 5 μm depth and 8 mm diameter. The samples were immediately rehydrated with 0.8 μL of 2 mg/mL CK and sealed with a second flat BaF2 window. The approximate composition of samples without the Ca2+-ATPase is 6 mM CaCl2, 3 mM MgCl2, 5 mM CP, 5 mM DTT, 2 mg/mL CK, 7 mM caged ADP, and 150 mM KCl. For some samples, addition of either Mg2+ or Ca2+ or both was omitted. In the samples with Ca2+-ATPase, there is also 1.2 mM Ca2+-ATPase. Before infrared sample preparation, the SR Ca2+ATPase was dialyzed for 90 min in a buffer containing 10 mM MOPS (pH 7 adjusted by KOH, at 1 °C), 20 μM CaCl2, 10 mM KCl, and distilled H2O before 10 μL was dried on the BaF2 window. The samples were placed in the spectrometer and left for equilibration for 45 min at 1 °C before measuring. Fourier Transform Infrared Spectroscopy. Timeresolved Fourier transform infrared measurements were performed at 1 °C with a Bruker IFS 66/S spectrometer as described previously.19,35 Photolytic release of ADP from caged ADP was triggered by a Xenon flash tube (N-185C; Xenon

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dx.doi.org/10.1021/jp409599p | J. Phys. Chem. B 2013, 117, 14967−14972