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* Laboratory of Biomolecular Dynamics; and Laboratory of Gene Technology; Catholic University of Leuven, Leuven, Belgium
Correspondence: Address reprint requests to Yves Engelborghs, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium. Tel.: 321-632-7160, Fax: 321-632-7982; E-mail: yves.engelborghs{at}fys.kuleuven.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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) and a relative molecular mass of 19,485. The three-dimensional x-ray structure (at 2 Å resolution) of Ca2+-bound NSCP (Fig. 1) is described by Vijay-Kumar and Cook (1992) and the nuclear magnetic resonance (NMR) structure is described by Craescu et al. (1998). NSCP has four domains with the typical EF-hand (Kretsinger and Nockolds, 1973) Ca2+-binding sequence, but only sites I, III, and IV can bind Ca2+ or Mg2+. The protein has a compact structure with a central hydrophobic core of 20 amino acids, most of which are aromatic. The domains I and II form a pair by a back-to-back ß-sheet just like the pair of III and IV. NSCP is mainly 
-helical (58%) with eight helices (A–H). Initial Ca2+-binding studies were analyzed in terms of three identical intrinsic binding constants of 1.7 x 108 M-1 at pH 7.5 and 25°C, whereas Mg2+ binding was found to be cooperative with intrinsic binding constants of 0.83, 2.6, and 15 x 104 M-1, respectively (Cox and Stein, 1981). Further conformational and microcalorimetry studies on the protein (Luan-Rilliet et al., 1992) and on isolated tryptic fragments lead to the proposal of a new sequential binding model starting at site I (Durussel et al., 1993). In this study we use site-directed mutagenesis instead of protein fragments to analyze the binding mechanism.
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), e.g., the sarcoplasmic calcium-binding proteins from shrimp, crayfish, and streptomyces. In proteins, tryptophan fluorescence displays a multiexponential decay originating from the different possible microconformations of tryptophan (Dahms et al., 1995; Sillen et al., 2000). Other models are also suggested to explain the existence of a multiexponential decay (Alcala et al., 1987; Van Gilst et al., 1994; Lakowicz, 2000). NSCP is also an interesting protein to gain more insight into protein fluorescence. |
EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Mutagenesis
The NSCP gene was transferred from the plasmid pNDner06 (Dekeyzer et al., 1994) into the plasmid pET22b(+) (Studier et al., 1990) resulting in pETNSCP (van Riel, 1997). In this plasmid the NSCP gene is under control of the strong T7 promoter. For expression of NSCP mutants, the plasmid pETNSCP was used, into which the NSCP mutant genes were constructed either by polymerase chain reaction or by cassette mutagenesis. Site-directed mutagenesis was performed according to Sambrook et al. (1989). The following oligonucleotides were used:
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The tryptophan mutations and R25D and D58R are described in Sillen et al. (2000).
Mutants were identified by restriction analysis and the mutated genes were entirely sequenced using the ABI PrismTM Big Dye Terminator Cycle Sequencing Ready Reaction system (PerkinElmer, Boston, MA).
Expression and purification
The Escherichia coli strain Bli5 (Bl21(DE3) + pDIA17) (Chang and Cohen, 1978; Studier and Moffat, 1986; Raleigh et al., 1988; Munier et al., 1991) was transformed by electroporation with the pETNSCP plasmids. Cells were then treated as described by Dekeyzer et al. (1994). The difference in negative charges between the Ca2+ and the apo states of the protein was used to purify NSCP. In a first step, NSCP, in the presence of Ca2+, was isolated from the other cell proteins by anion exchange chromatography (DEAE, Fast-Flow, Pharmacia, Uppsala, Sweden). This was repeated, but with adjusting to 5 mM EGTA (apo conformation). In a last step, NSCP was purified on a Hiload Superdex 75 prepgrade 16/60 column (Pharmacia). All NSCP variants were 
99% pure as judged by Coomassie-stained SDS gels. All the variants where stored with 2 mM CaCl2. The apo form was made by adding EGTA and EDTA to a 10-mM concentration each. The Mg2+ form was made by adjusting the apo form solution to 20 mM MgCl2.
Steady-state fluorescence
Steady-state fluorescence was measured with a SPEX spectrofluorometer (Fluorolog 1691, Spex Industries, Edison, NJ) with excitation and emission slits providing a bandpass of 7.2 and 3.6 nm, respectively. Spectra are corrected for the wavelength dependence of the emission monochromator and the photomultiplier and also by subtracting background intensities of the buffer solution. The cuvette holder was thermostated at 22°C. The excitation wavelength was 295 nm to ensure that the measured signal is only due to tryptophan fluorescence.
Ultraviolet absorption
Ultraviolet absorption was measured on a UVIKON Kontron 940 spectrophotometer (Kontron AG, Eching, Germany). The molar absorption coefficients at 295 nm are calculated by taking the ratio of the absorbance at 295 nm and at 280 nm, multiplied by the molar absorption coefficients at 280 nm, which was calculated according to the method of Mach et al. (1992).
Fluorescence lifetime data
Fluorescence lifetime data were determined as described previously (Sillen et al., 2000) using an automated multifrequency phase fluorometer. The instrument is similar to that described by Lakowicz et al. (1985), except for the use of a high-gain photomultiplier (Model H5023, Hamamatsu, Bridgewater, NJ) instead of a microchannel plate. The detection part is described by Vos et al. (1997). The excitation source consists of a mode-locked titanium-doped sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA) pumped by a Beamlok 2080 Ar+-ion laser (Model 2080, Spectra-Physics). After frequency-tripling (GWU, Spectra-Physics) the excitation wavelength is 295 nm. The fluorescence lifetime measurements are performed by measuring the phase-shift of the modulated emission at 50 frequencies ranging from 1.6 MHz to 
1 GHz. N-Acetyltryptophanamide (in water filtered by the Milli-Q system, Millipore) with a fluorescence lifetime of 3.059 ns or p-terphenyl in cyclohexane with a lifetime of 1.04 ns (Desie et al., 1986) both at 22°C was used as a reference fluorophore. The measured phase shifts, 
, at a modulation frequency, 
, of the exciting light are related to the fluorescence decay in the time domain I(t),
 | (1) |
i, as described by Weber (1981).
Time-resolved anisotropy
Measuring the frequency-dependent phase difference between the parallel and perpendicular components of the modulated fluorescence revealed the rotational correlation times i with their relative amplitudes (
gi = 1), together with the initial anisotropy r0 (anisotropy in the absence of depolarizing processes) by fitting the frequency transform of a multiexponential anisotropy decay law (Weber, 1977),
 | (2) |
i the rotational correlation times. The analysis was performed assuming that each fluorescing species with lifetime i has the same anisotropy function (Lakowicz et al., 1985). No indication for associative decay was found. The rotation angle of tryptophan was calculated according to Lipari and Szabo (1980) for free diffusion within a cone angle 
.
 | (3) |
). Several statistical techniques were used in the fitting procedure, as described by Clays et al. (1989). Measurements performed at different emission wavelengths were analyzed simultaneously with global analysis (GLOBALS Unlimited, University of Illinois, Urbana, IL) to increase the resolution of the lifetimes and the corresponding amplitudes (Beechem et al., 1983). Phase data were fitted using the modified Levenberg-Marquardt algorithm (More and Sorensen, 1983) assuming fluorescence lifetimes that are independent of the emission wavelength and a variable amplitude ratio. The lifetimes did not change across the different wavelengths.
Quantum yields were determined relative to tryptophan in water according to the method of Parker and Rees (1960), where the intensity is integrated over the wavelength region 300–450 nm, with the absorbance at 295 nm, and the quantum yield QTrp for tryptophan in water is taken as 0.14 (Kirby and Steiner, 1970).
Decay-associated spectra
Decay-associated spectra are constructed by multiplying the intensity fraction with the intensity of the emission spectra at the respective wavelength (Ross et al., 1981). A log-normal function (Burstein and Emelyanenko, 1996) is fitted to the associated intensities to obtain the decay-associated spectra.
The average radiative rate constant is calculated by dividing the quantum yield by a wavelength-independent amplitude average lifetime (Willis and Szabo, 1992; Sillen and Engelborghs, 1998). Each lifetime is integrated over the wavelength region 300–450 nm and then normalized (Sillen et al., 2000).
Calculation of the change in fluorescence intensity due to static and dynamic quenching and by a change in the population of microconformations
We suggested splitting the ratio of the quantum yield of different variants relative to a reference protein, e.g., Wt, into a factor (fkr) representing the change in kr, or homogenous static quenching, a factor (fPR) reflecting heterogeneous static quenching or population reshuffling, and a factor (fDQ) representing pure dynamic quenching (Sillen and Engelborghs, 1998; Sillen et al., 1999). The factor fPR is affected by static quenching only if the static quenching is heterogeneous. If there is static quenching and an increase of the fluorescence due to population reshuffling, then fPR is the minimum increasing factor of fluorescence intensity due to a change in the balance between microconformations.
EGTA titration
NSCP Wt and variants are excited at 295 nm, and the emission is measured at maximum emission wavelength (Ca2+ state). We use a quartz cuvette with stirrer (1-cm optical pathway, Hellma Benelux, Aartselaar, Belgium) that contains 1.2 ml of the protein solution. The protein concentration is 13 µM. This solution contains 0.2 mM CaCl2. The correct concentration of Ca2+ in the solution is determined by an atomic absorption spectrophotometer (Model 372, PerkinElmer).
First, the fluorescence is measured 60x during 1 min. Then a certain volume (2–10 µl) of a 250 or 500 mM EGTA solution is added, depending on the current free calcium concentration. After addition of EGTA, the solution is equilibrated for 3 min before the next measurement.
To calculate the concentration of Ca2+ present in the solution, we use the software program Chelator (Schoenmakers, 1992) that takes the concentration of EGTA, buffer, temperature, and ionic strength into account. During the titration the ionic strength increases, due to the addition of Na2-H2·EGTA, from the original value of 0.1 M to 0.106 M. Between Ca2+ concentrations it decreases from 0.2 mM to 10-8M and the ionic strength increases to 0.16 M when Ca2+ is further decreased to 10-9 M. Calcium Green-1 indicator (Molecular Probes, Eugene, OR) is a fluorescent indicator used to check the titration procedure and the calculations made by Chelator. Calcium Green-1 indicator has a single binding site for Ca2+. The indicator is excited at 488 nm and the fluorescence is measured at 533 nm. Upon binding of Ca2+ the fluorescence increases. The indicator is titrated the same way as the protein samples and the correct binding constant of 5.2 x 106 M-1 was obtained.
Quin 2 titration
Quin 2 titrations are done according to Linse et al. (1991). Quin 2 separately and Quin 2 (25 µM), together with NSCP (10 µM), are titrated with Ca2+ by monitoring the absorption of Quin 2 at 263 nm. First, the exact concentration of Quin 2 is determined by titration with Ca2+ and the dissociation constant is 60 nM (Tsien and Pozzan, 1989) at the ionic strength of 0.10 M. During the titration the ionic strength increases to 0.11 M due to the addition of Ca2+. The absorption in the absence of Ca2+ (ODmax) is determined by measuring Quin 2 with 10 mM EGTA. Then Quin 2, together with NSCP, are titrated with Ca2+. The exact concentration of NSCP is determined by measuring the absorption at 280 nm. The absorption of the Ca2+ free system (ODmax) is determined by measuring Quin 2, NSCP, and 10 mM EGTA. The amount of bound Ca2+ is calculated from the ODmax and the absorption at excess Ca2+. From this it is possible to calculate the fractional saturation of NSCP with Ca2+.
Fitting
Curve fitting of the fluorescence titration curves is done with a nonlinear least-squares fitting program (SigmaPlot-5, SPSS, Chicago, IL). Attempts were made to fit the whole titration curve to the full Adair equation
 | (4) |
I is the Adair coefficient, and Fi is the relative fluorescence of the species NSCP). Cai was not acceptable, because the parameters obtained displayed a strong dependency (parameter d), indicating that too many parameters are being extracted from the data set. Therefore, a simplified equation assuming variable cooperativity was used,
 | (5) |
This equation, however, cannot be used to fit the titration curve of the Wt because in Wt the fluorescence change is biphasic, i.e., a fluorescence decrease is followed by an increase. The biphasic curve could be fitted with the following equation:
 | (6) |
We also used Quin titrations in this case and analyzed them with the Hill equation. Our results and numerical simulations show that the Hill coefficient obtained from direct binding studies can be substantially lower (1.8x) than those obtained from protein fluorescence titrations, due to the nonlinear relation between protein fluorescence and calcium binding.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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).
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R2 and a significant nonrandomness in the autocorrelation function of the weighted residuals as a function of the frequency. Best fit with lowest R2 and no systematic deviations in the autocorrelation function of the weighted residuals is obtained with a triple- or quadruple-exponential function. To improve the recovery of the decay parameters, a global analysis of all the phase measurements at the different wavelengths was performed. The results of this global fit are summarized in Table 2. The decay-associated spectra are shown in Figs. 3 and 4.
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R2 near unity and no systematic deviations in the weighted residuals is obtained with a double-exponential fit resulting in two rotational correlation times. The results of the fit are summarized in Table 3. The long rotational correlation time can be associated with the overall rotation of NSCP, whereas the short rotation correlation time can be associated with the local motion of the tryptophan (Lakowicz, 1999). The apparent time zero anisotropy r0 for tryptophan and tryptophan derivatives is 0.19 at 295 nm (Valeur and Weber, 1977; Lakowicz et al., 1983), usually interpreted as due to the presence of 1La and 1Lb absorption bands (Callis, 1997; Ruggiero et al., 1990). This is also the case for most of the NSCP variants.
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However, titration of Ca2+ Wt with EGTA gives rise to a biphasic fluorescence change (Fig. 5). Starting from the high Ca2+ concentrations, first a decrease in fluorescence, and subsequently an increase, is observed. Such a biphasic curve cannot be fitted with a simple Hill equation. We performed Quin titrations as an alternative method to monitor the binding of Ca2+ to NSCP Wt by measuring the absorption change of Quin 2 in the presence of NSCP Wt with Ca2+. From this the association constants was determined to be (9.8 ± 0.4) x 107 (M-1) and the Hill coefficients was 1.6 ± 0.2. It should be noted that this Hill coefficient is an underestimation of the one determined by protein fluorescence changes by as much as a factor of 1.8 (shown by numerical simulations) due to the nonlinear relation between saturation and conformation.
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1 = (1.3 ± 0.5) x 109 M-1, and 
3
1/3 = (2.9 ± 0.2) x 108 M-1. However, we do not trust the first binding constant, because at calcium concentrations <10-9 M the ionic strength of the solution increases too much. Fitting the titrations of W57 and W170 (Fig. 6 and Table 4) and comparing with Wt reveals that mutating two tryptophans into Phe changes the association constant of Ca2+ to NSCP to somewhat lower values. The association constants and Hill coefficients are summarized in Table 4. But the mutations have no effect on the cooperativity of the binding because the Hill coefficient of Wt and W4F/W57F are approximately the same, and is only slightly lower for the W4F/W170F variant (n = 2.6) (Table 4), taking into account the effect of the factor 1.8 between the Hill coefficient obtained from fluorescence titrations and from direct binding studies. Mutating calcium ligands in the ion-binding loop of domain I has only little effect on the association constants and Hill coefficients (Table 4). Mutations in domains III and IV, however, lower the association constants and reduce the Hill coefficients to 1. To check whether the mutations of tryptophan have an influence on the fluorescence of the remaining tryptophan residues, we calculated the fluorescence intensity of the different tryptophans and compared the addition of the intensities with the fluorescence intensity of Wt (Table 5). In all ion-binding states of NSCP, the sum of the fluorescence intensity approximates the intensity of Wt very closely.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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). Changing the calcium binding residue (z-position) in domain I has only a small influence on Ca2+ binding, although changing the residues (z-positions) in domains III and IV, on the other hand, has a large influence on the Ca2+ binding, in that the average affinity is not much influenced but the cooperativity is totally lost. Mutation of the z-position in site I (D20N) has very little effect on the calcium-binding properties (data not shown). Mutation at the z-position (D108N) in site III has a similar effect as the mutation at z in site III: loss of cooperativity, but little effect on the overall binding constant (data not shown). A sequential binding mechanism was observed for recoverin (Permyakov et al., 2000) and calmodulin (Haiech and Kilhoffer, 2000). But in the case of recoverin the binding was totally lost by a mutation in the first binding site. Here it is clear that binding at sites III or IV both cause a cooperative transition, and that the ligands at z-position are crucial probably by pulling the -helices together to form the EF-hand.
Fluorescence properties of individual tryptophan residues
W4
Trp4 is positioned at the beginning of the helix A and is not involved in a EF-hand, but is close to helix D which, together with helix E, makes the connection between domains II and III. Quantum yield, average lifetime, decay-associated spectra, lifetimes, anisotropy data. and quenching analysis reveal that there is only a minor change in the environment of W4 upon addition of Ca2+ or Mg2+ (Table 6). The high quantum yield is the result of the high population (80%) of the long lifetime (5.2 ns). This mutant protein was not further studied.
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max) of 338 nm in the Ca2+ state. This is considered to be typical for tryptophan residues in an environment that is highly hydrophobic but also with water molecules with low mobility bound to the protein matrix (Burstein, 1983; Eftink, 1990). In the apo state, max shifts to 350 nm, which is characteristic for solvent-accessible tryptophans. This indicates that, although W170 is in the core of NSCP in the Ca2+ state, upon changing to the apo state the core opens and water can penetrate resulting in a partially denatured structure. This is confirmed by FTIR measurements: the percentage of -helix drops in the apo state by 14% compared to the Ca2+ state, whereas the unordered structure rises from 26% to 38% (van Riel, 1997) and this is also confirmed by NMR and circular dichroism measurements (Christova et al., 2000; Prêcheur et al., 1996; Cox and Stein, 1981). Also the rotational correlation time associated with the rotation of the whole protein and the rotation angle of W170 and W57 increases in the apo state (Table 3). The hydrophobic core of NSCP consists of mainly Phe and Tyr residues. Three of these hydrophobic core residues (F28, Y116, and F150) are in direct contact with the EF-hand structure and they are always the immediate neighbor of the amino acid responsible for the complexation of the ions at z-position in all three sites. These amino acids are conserved in most sarcoplasmic calcium-binding proteins (Collins et al., 1988). Consequently, removal of Ca2+ or Mg2+ destabilizes the hydrophobic core of NSCP.
W57
Trp57 is positioned at the second inactive domain with the indole group close to the binding site of the first domain. This makes W57 ideally located for observing the changes at the interdomain region of domains I and II upon binding of Ca2+ or Mg2+. Upon binding of Ca2+ or Mg2+, there is a fluorescence intensity decrease with a factor of 0.76 and 0.77, respectively. This decrease is largely caused by the higher population of the two lowest lifetimes—together, 89%—and is only to a limited extent due to direct dynamic quenching (Table 6). The different lifetimes of W57 have been explained on the basis of microstates of tryptophan (Sillen et al., 2000). The rotational correlation time of the whole protein and the rotation angle of W57 increases in the apo form, whereas the rotational correlation time of W57 itself decreases, indicating a more flexible environment of W57 in the apo form.
The max of W57 in the Ca2+ state is very blue-shifted (317 nm), a phenomenon that could be due to the electromagnetic field over the tryptophan (Vivian and Callis, 2001).
Role of arginine R25
Although arginine is not found to be among the solution quenchers (Chen and Barkley, 1998), it is often observed as being a dynamic quencher of protein fluorescence (Clark et al., 1996; Christiaens et al., 2002) when arginine is very close to the aromatic ring of Trp. Studying the x-ray structure of NSCP, however, shows that the guanidinium moiety of R25 is not in contact with the aromatic rings of W57. Moreover, a detailed analysis of the quenching parameters (Table 6) indicates that the fluorescence change upon mutation R25D is largely due to population reshuffling (fPR).
Influence of the salt bridge R25-D58 on the fluorescence properties of W57
Trp57 is located near the salt bridge R25-D58. This salt bridge connects the binding site of domain I with domain II. R25 is located adjacent to the amino acid responsible for the x-binding coordinate of the metal, and D58 is located in helix C. A similar situation exists in the two other domains of III and IV, where R113 is located just next to the amino acid responsible for the x-binding coordinate of the metal and forms a salt bridge with D135 located in helix G, and is in close contact with binding site IV (9A from the Ca2+). To investigate the influence of the salt bridge R25-D58 on the fluorescence of W57 and to investigate its function in the conformational change upon binding of the ions, two additional mutations were made—i.e., R25D, which breaks the salt bridge; and R25D/D58R, which potentially switches the orientation of the salt bridge.
W4F/W170F/R25D
Upon breaking of the salt bridge, the two lower lifetimes of the Ca2+ state converge to their arithmetic mean while the two longer lifetimes remain identical. The fluorescence lifetimes of the Ca2+, Mg2+, or apo forms are the same; only the amplitude fractions depend upon the binding of an ion. The apo state has a higher fluorescence intensity due to a higher population of the two longer lifetimes. The max shifts 10 nm to the red in changing from the Ca2+ state to the apo state in both W4F/W170F and W4F/W170F/R25D. This indicates that W57 is more solvent-accessible in the apo state.
There is a strong red-shift in all forms upon removing the salt bridge, indicating the existence of the salt bridge even in the apo form. Additionally, there is still a red-shift upon removing the Ca2+ ions from the binding site. The calcium binding site on its own is not able to create the fully shielded state of the W57. The intact salt bridge is partially responsible for this.
W4F/W170F/R25D/D58R
This mutation can potentially restore the salt bridge (but with opposite orientation, although we have no experimental evidence that it actually does so). This mutation has the effect that Ca2+ binding changes the amplitude fractions but keeps the lifetimes similar to those of the Wt apo state. Thus the lifetimes of W57 are Wt-apo-like in this mutant and the binding site is only affecting the population of the different micro conformations. The max shifts only 3 nm to the red upon removal of Ca2+, indicating a more similar solvent accessibility in the vicinity of W57 in comparison with W4F/W170F. Also in this variant all the lifetimes of the apo state are longer. Arginine is thus necessary for the longer fluorescence lifetimes and this elongation of the lifetimes is stronger in the apo state (Table 6). In the variant W4F/W170F, R25 interacts differently in the metal-bound state than in the metal-free state, resulting in a different conformation around W57 between the two states.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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EGTA titration of the Wt and mutants and the Quin 2 titration of Wt reveals that cooperative binding occurs at domains III and IV probably due to the formation of the ß-sheet that connects the two domains and that domain I behaves more as an independent site. The Hill coefficient, which is a measure for cooperativity, is 2, indicating that there is cooperativity (although there is no real full cooperativity; n = 3). Both W57 and W170 are sensitive to the full conformational change due to ion binding (Fig. 5).
The behavior of the individual tryptophan residues (W57 and W170) is in agreement with the studies on tryptic fragments of NSCP done by Durussel et al. (1993). The N-terminal peptide (1–80) showed a single binding site with a Ka = 3.1 x 105 M-1 and a fluorescence decrease upon calcium binding; the C-terminal peptide (90–174) showed two binding sites with Ka = 3.2 x 104 M-1 and a fluorescence increase upon calcium binding. Combining this information with the biphasic behavior of the Wt protein it is tempting to assume a sequential-binding mechanism with initial binding at site I and then with strong cooperativity at sites III and IV. This sequence also explains that lowering the affinity at sites III and IV shows up as a decreased cooperativity, although the same mutation at site I does not influence the overall transition. This cooperativity questions the possibility to limit the saturation of NSCP to one and the same site at stoichiometric amounts of calcium and protein as was done in an NMR study of NSCP (Prêcheur et al., 1996). The biological relevance of this binding mechanism is not clear as long as the exact biological role of these proteins is not known or limited to the concept of a calcium buffer.
The lifetime data on the R25D and R25D/D58R variants indicate that the salt bridge is present also in the apo conformation of W57 and that orientation of the salt bridge is important to create the Ca2+-like environment of W57. Recently it has been shown that the local electric field around the tryptophan residue can have a pronounced influence on the energy level of the low Trp ring-to-backbone charge transfer (Callis and Vivian, 2003) and the effect of Arg on the fluorescence might also be explainable by this effect. Quantum mechanical calculations are, however, necessary to confirm this.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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This project was supported by a research grant from the Fund for Scientific Research, G-00-92-01 (Flanders, Belgium), and a Concerted Research Action grant, GOA/2001/02.
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FOOTNOTES |
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Submitted on December 6, 2002; accepted for publication May 22, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Beechem, J. M., J. R. Knutson, J. B. A. Ross, B. W. Turner, and L. Brand. 1983. Global resolution of heterogeneous decay by phase modulation fluorometry: mixtures and proteins. Biochemistry. 22:6054–6058.
Bevington, P. R. 1969. Gradient-expansion algorithm. In Data Reduction and Error Analyses for the Physical Sciences. McGraw-Hill, New York. pp.235–240.
Burstein, E. A. 1983. The intrinsic luminescence of proteins is a method for studies of the fast structural dynamics. Mol. Biol. 17:455–467.
Burstein, E. A., and V. I. Emelyanenko. 1996. Log-normal description of fluorescence spectra of organic fluorophores. Photochem. Photobiol. 64:316–320.
Callis, P. R. 1997. 1La and 1Lb transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins. Methods Enzymol. 278:113–150.[Medline]
Callis, P. R., and J. T. Vivian. 2003. Understanding the variable florescence quantum yield of tryptophan in proteins using QM-M simulations: quenching by charge transfer to the peptide backbone. Chem. Phys. Lett. 369:409–414.
Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles from the P15A cryptic miniplasmid. J. Bacteriol. 1:1141–1156.
Chen, Y., and M. D. Barkley. 1998. Toward understanding tryptophan fluorescence in proteins. Biochemistry. 37:9976–9982.[Medline]
Christiaens, B., S. Symoens, S. Verheyden, Y. Engelborghs, A. Joliot, A. Prochiantz, J. Vandekerckhove, M. Rosseneu, and B. Vanloo. 2002. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. Eur. J. Biochem. 269:2918–2926.[Medline]
Christova, P., J. A. Cox, and C. T. Craescu. 2000. Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the Apo state is a molten globule. Proteins. 40:177–184.[Medline]
Clays, K., J. Jannes, Y. Engelborghs, and A. Persoons. 1989. Instrumental and analysis improvement in multifrequency phase fluorometry. J. Phys. E Sci. Instrum. 22:297–305.
Clark, P. L., Z. P. Liu, J. Zhang, and L. M. Gierach. 1996. Intrinsic tryptophans of CRABPI as probes of structure and folding. Protein Sci. 5:1108–1117.[Abstract]
Collins, J. H., J. A. Cox, and J. L. Theibert. 1988. Amino acid sequence of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor. J. Biol. Chem. 263:15378–15385.
Cox, J. A., and E. A. Stein. 1981. Characterization of the new sarcoplasmic calcium binding protein with magnesium-induced cooperativity in the binding of calcium. Biochemistry. 20:5430–5436.[Medline]
Craescu, C. T., B. Precheur, A. van Riel, H. Sakamoto, J. A. Cox, and Y. Engelborghs. 1998. H-1 and H-15 resonance assignment of the calcium-bound form of the Nereis diversicolor sarcoplasmic Ca2+-binding protein. J. Biomol. NMR. 12:565–566.[Medline]
Dahms, T. E. S., K. J. Willis, and A. G. Szabo. 1995. Conformational heterogeneity of tryptophan in a protein crystal. J. Am. Chem. Soc. 117:2321–2326.
Dekeyzer, N., Y. Engelborghs, and G. Volckaert. 1994. Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase. Protein Eng. 7:125–130.
Desie, G., N. Boens, and F. C. De Schryver. 1986. Study of the time-resolved tryptophan fluorescence of crystalline a-chymotrypsin. Biochemistry. 25:8301–8308.[Medline]
Durussel, I., Y. Luan-Rilliet, T. Petrova, T. Takagi, and J. A. Cox. 1993. Cation binding and conformation of tryptic fragments of Nereis sarcoplasmic calcium-binding protein: calcium-induced homo- and heterodimerization. Biochemistry. 32:2394–2400.[Medline]
Eftink, M. R. 1990. Fluorescence techniques for studying protein structure. Methods Biochem. Anal. 35:117–129.
Engelborghs, Y., K. Mertens, K. Willaert, Y. Luan-Rilliet, and J. A. Cox. 1990. Kinetics of the conformational changes in Nereis sarcoplasmic calcium binding protein upon binding of divalent ions. J. Biol. Chem. 265:18809–18815.
Haiech, J., and M. C. Kilhoffer. 2000. Tryptophan calmodulin mutants. In Topics in Fluorescence Spectroscopy, Vol. 6: Protein Fluorescence. J. R. Lakowicz, editor. Kluwer Academic/Plenum Publishers, New York. pp.175–209.
Kirby, E. P., and R. F. S. Steiner. 1970. The influence of solvent and temperature upon the fluorescence of indole derivatives. J. Phys. Chem. 74:4480–4490.
Kretsinger, R. H., and C. E. Nockolds. 1973. Carp muscle calcium-binding protein. II. Structure determination and general description. J. Biol. Chem. 248:119–174.
Lakowicz, J. R., B. P. Maliwal, H. Cherek, and A. Balter. 1983. Rotational freedom of tryptophan residues in proteins and peptides. Biochemistry. 22:1741–1752.[Medline]
Lakowicz, J. R., G. Laczko, and I. Gryczinski. 1985. 2-GHz frequency-domain fluorometer. Rev. Sci. Instrum. 57:2499–2506.
Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy, 2nd Ed. Kluwer Academic/Plenum Publishers, New York. pp.329–330.
Lakowicz, J. R. 2000. On spectral relaxation in proteins. Photochem. Photobiol. 72:421–437.[Medline]
Luan-Rilliet, Y., M. Milos, and J. A. Cox. 1992. Thermodynamics of cation binding to Nereis sarcoplasmic calcium-binding protein: direct binding studies, microcalorimetry and conformational changes. Eur. J. Biochem. 208:133–138.[Medline]
Linse, S., C. Johansson, P. Brodin, T. Grundström, T. Drakenberg, and S. Forsén. 1991. Electrostatic contribution to the binding of Ca2+ in calbindin D9k. Biochemistry. 30:154–162.[Medline]
Lipari, G., and A. Szabo. 1980. Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes. Biophys. J. 30:489–506.
Mach, H., C. R. Middaugh, and R. V. Lewis. 1992. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal. Biochem. 200:74–80.[Medline]
More, J. J., and D. C. Sorensen. 1983. Computing a trust region step. SIAM J. Sci. Stat. Comput. 4:553–572.
Munier, H., A.-M. Gilles, P. Glaser, E. Krin, A. Danchin, R. Sarfati, and O. Bârzu. 1991. Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adanylate cyclase. Eur. J. Biochem. 196:469–474.[Medline]
Nakayama, S., and R. H. Kretsinger. 1994. Evolution of the EF-hand family of proteins. Annu. Rev. Biomol. Struct. 23:473–507.[Medline]
Parker, C. A., and W. T. Rees. 1960. Corrections of fluorescence spectra and the measurement of fluorescence quantum efficiency. Analyst. 85:587–600.
Permyakov, S. E., A. M. Cherskaya, I. I. Senin, A. A. Zargarov, S. V. Shulga-Morskoy, A. M. Alekseev, D. V. Zinchenko, V. M. Lipkin, P. P. Philippov, V. N. Uversky, and E. A. Peryakov. 2000. Effects of mutations in the calcium-binding site of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites. Protein Eng. 13:783–790.
Prêcheur, B., J. A. Cox, T. Petrova, J. Mispelter, and C. T. Craescu. 1996. Nereis sarcoplasmic Ca2+ binding protein has a highly unstructured apo state which is switched to the native state upon binding the first Ca2+ ion. FEBS Lett. 395:89–94.[Medline]
Raleigh, E. A., N. E. Murray, H. Revel, R. M. Blumenthal, D. Westaway, A. D. Reith, P. W. Rigby, J. Elhai, and D. Hanahan. 1988. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res. 16:1563–1575.
Ross, J. A., C. J. Schmid, and L. Brand. 1981. Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase. Biochemistry. 20:4369–4377.[Medline]
Ruggiero, A. J., D. C. Todd, and G. R. Fleming. 1990. Subpicosecond fluorescence anisotropy studies of tryptophan in water. J. Am. Chem. Soc. 112:1003–1014.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Schoenmakers, T. J. 1992. CHELATOR: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques. 12:870–879.[Medline]
Sillen, A., and Y. Engelborghs. 1998. The correct use of "average" fluorescence parameters. Photochem. Photobiol. 67:475–486.
Sillen, A., J. Hennecke, D. Roethlisberger, R. Glockshuber, and Y. Engelborghs. 1999. Fluorescence quenching in the DsbA protein from Escherichia coli. The complete picture of the excited state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan. Proteins. 37:253–263.[Medline]
Sillen, A., J. F. Díaz, and Y. Engelborghs. 2000. A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data. Prot. Sci. 9:158–169.[Abstract]
Studier, F. W., and B. A. Moffat. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113–130.[Medline]
Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorf. 1990. Use of T7 polymerase to direct expression of cloned genes. Methods Enzymol. 185:60–89.[Medline]
Tsien, R., and T. Pozzan. 1989. Measurement of cytosolic free Ca2+ with Quin 2. Methods Enzymol. 172:230–262.[Medline]
Valeur, B., and G. Weber. 1977. Resolution of the fluorescence excitation spectrum of indole into the 1La and 1Lb excitation bands. Photochem. Photobiol. 25:441–444.[Medline]
van Gilst, M., C. Tang, A. Roth, and B. Hudson. 1994. Quenching interaction and nonexponential decay: tryptophan 138 of bacteriophage T4 lysozyme. J. Fluoresc. 4:203–207.
van Riel, A. 1997. Karakterisatie van het Ca2+-bindingsmechanisme van Nereis SCP met behulp van plaatsgerichte mutagenese. Katholieke Universiteit Leuven, Leuven, Belgium. (PhD thesis.)
Vijay-Kumar, S., and W. J. Cook. 1992. Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 Å resolution. J. Biol. Chem. 224:413–426.
Vivian, J. T., and P. R. Callis. 2001. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys. J. 80:2093–2109.
Vos, R., R. Strobbe, and Y. Engelborghs. 1997. Gigahertz phase fluorometry using a fast high-gain photomultiplier. J. Fluoresc. 7:33S–35S.
Weber, G. 1977. Theory of differential phase fluorometry: detection of anisotropic molecular rotations. J. Chem. Phys. 66:4081–4091.
Weber, G. 1981. Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements. J. Phys. Chem. 85:949–953.
Willis, K. J., and A. G. Szabo. 1992. Conformation of parathyroid hormone: time-resolved fluorescence studies. Biochemistry. 31:8924–8931.[Medline]
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, longest lifetime; 
, second longest lifetime; •, middle lifetime; 
, smallest lifetime; –, measured emission spectra; and ..., sum of the decay-associated spectra.
) of the tryptophans in NSCP