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* Department of Biophysical Chemistry, Lund University, Chemical Centre, Lund, Sweden; Laboratory of Bioorganic Chemistry, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia; and Department of Theoretical Chemistry, Lund University, Chemical Centre, Lund, Sweden
Correspondence: Address reprint requests to Sara Linse, Dept. of Biophysical Chemistry, Lund University, Chemical Centre, P.O. Box 124, Lund, Sweden. Tel.: 46-46-222-8238; Fax: 46-46-222-4543; E-mail: sara.linse{at}bpc.lu.se.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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,2), and the inositol tris 1,4,5-phosphate receptor (3). A number of different "structural modes of interaction" with target protein have been identified (4), usually involving a helical segment in the target protein.
In complexes with kinases, (Ca2+)4-CaM binds to a segment of the partner protein rich in positively charged and hydrophobic residues. An isolated 20-residue target sequence corresponding to residues 796–815 (1–20 in this work) in chicken gizzard smooth-muscle myosin light-chain kinase (smMLCKp or P7) retains its affinity for CaM and represents an adequate model for a complex of CaM with intact smMLCK protein (5). In the globular (Ca2+)4-CaM-P7 complex the protein is wrapped around the 
-helical peptide. All the lysine and arginine charges of P7 are in close contact with negatively charged side chains of acidic residues of CaM (6). In addition to paired ionic interactions in the complex, the net charges of unbound CaM and peptide are large and opposite. Accordingly, electrostatic interactions are expected to be importantly involved in formation of CaM complexes. However, they do not seem to be significantly manifested in binding affinity, which is highly surprising. For example, replacement of any positively charged residue by alanine in the CaM-binding sequence of MLCK from skeletal muscle does not result in decreased affinity, but rather an increase was observed (7). This unexpected observation was recently confirmed in another study (8). Even more intriguing results were obtained for complex formation between smMLCK peptides and CaM (9). The binding affinity was found to be essentially independent of changes in the net charge of peptide between +4 and +8, and of CaM from –6 to –18 (9). This observation was made while monitoring the binding equilibria in low-ionic-strength buffer where electrostatic interactions are supposed to be close to maximum. An analysis of the interaction of a protein with an oppositely charged surface shows that electrostatic interactions become "saturated" provided the surface is highly charged (10). The same type of "saturation" appears to take place in the case of CaM-target complex formation.
In this study we find an increase in binding affinity with added salt for highly and oppositely charged CaM and MLCK peptides. (See Table 1 for peptide nomenclature.) In many cases, the binding affinity for a charged ligand to an oppositely charged protein is reduced on salt addition or charge reduction, although the effect can be complicated by desolvation penalties. To shed some light on the unusual manifestation of electrostatic interactions in CaM action we investigated the combined effect of salt and pH on complex formation between CaM and MLCK peptides of variable charge number. Monte Carlo simulations of electrostatic interactions in the complex formation are performed along with experimental studies. We show that the complex formation at physiological ionic strength and pH still occurs in conditions of saturated electrostatic interactions. We find that despite the opposite charges of CaM and MLCK peptides the complex formation is facilitated by reduction of ionic interactions with added salt. We suggest that at low ionic strength the collapse of CaM into a globular complex with smMLCK peptides might be disfavored by the repulsion between the negatively charged domains. The repulsion between CaM domains apparently becomes overridden by increasing ionic strength to near-physiological values, where the formation of a wrapped complex is facilitated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). P7 was also produced recombinantly in the IMPACT system (New England Biolabs, Beverly, MA) and purified according to the instructions by the manufacturer. N- and C-terminal domains of CaM (TR1C and TR2C, respectively, corresponding to residues 1–75 and 78–148, respectively, of CaM) were purified as previously described (11,12). Purity was confirmed by agarose and sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as 1H NMR. The concentrations were determined by absorbance at 280 nm (or 265 nm for TR1C) using an extinction coefficient of 3200 M–1 cm–1 for Ca2+-CaM (13), 2950 M–1 cm–1 for TR1C, 760 M–1 cm–1 for TR2C, and 5550 M–1 cm–1 for each peptide (14), as confirmed by amino acid analysis after acid hydrolysis.
Fluorescence spectroscopy
Binding constants were obtained from titrations of peptide with protein in 5 mM buffer containing 1 mM CaCl2 and NaCl concentrations ranging from 0 to 3 M. Sodium acetate, MES, Bis-Tris, Tris, Bis-Tris propane, Tricine, and CAPS were used as buffers to cover the pH range from 4 to 11. Overlapping buffering areas were used to test for specific buffer effects on the binding, but none were found. The pH readings taken before and after titration agreed within ±0.1 units. Below pH 5, the Ca2+ concentration was increased to 3–5 mM to saturate CaM. The peptide concentration was between 0.3 and 2 µM, and calmodulin aliquots were added from a concentrated stock solution. Fluorescence data were obtained with excitation at 295 nm and emission at 335 nm, as previously described (9). The reported binding constants are averages of at least two independent measurements, except for the CaM mutants, where an average is reported for all mutants binding to the same peptide and the error in binding constants is <±0.2 log units. Fluorescence titration data were analyzed according to a 1:1 binding model, as previously described (9). In cases of weak binding, a large number of CaM titration points had to be collected and a linear part proportional to the concentration of unbound CaM was added to the fitting function. For binding between tryptic fragments and peptides, the stoichiometry of the formed complexes was an extra adjustable parameter in the fits. Binding constants above 108 M–1 are difficult to determine, prohibiting a comparison of different CaM charge mutants at high salt.
CD spectroscopy
CD spectra (180–250 nm, response time 16 s, resolution 1 nm, scan rate 20 nm/min, four scans averaged) were obtained at 5°C using a Jasco J-720 spectropolarimeter with a Jasco PTC-343 Peltier type thermostatic cell holder and a quartz cuvette with a path length of 1 mm. The peptide concentration was 46–74 µM at pH 7.2–7.3. smMLCKp adopts a random coil in solution at room temperature as shown by circular dichroism (CD) spectroscopy. To be able to compare the helix propensity and its salt dependence for the smMLCKp charge variants, the helical state needs to be favored. This was done by lowering the temperature to 5°C and by addition of trifluoroethanol (TFE) that shifts the coil to helix equilibrium toward the helical state. The helix propensity of different charge variants of smMLCKp was estimated from far-ultraviolet CD spectra in 20% TFE at 5°C, taking a CD signal at 221 nm of –35,300 deg cm2 dmol–1 (15) to represent a fully formed helix. The CD signal at 221 nm was converted into fraction helix for the different peptides in 20% TFE and the salt dependence of the helix formation was studied by adding 100 mM NaF. Difference spectra for complexes of P4 or P7 with CaM were generated by digitally subtracting the spectra of free CaM and free peptide from the spectra recorded for 1:1 complex of 10 µM peptide and 10 µM CaM in 5 mM Tris pH 7.5, as well as in 5 mM NaAc at pH 5.0. Data for substoichiometric mixtures were obtained by titrating P4 or P7 into 30 µM CaM in 5 mM Tris pH 7.5 and 5 mM NaAc at pH 5.0.
Isothermal calorimetry
The enthalpy associated with CaM-smMLCKp binding was determined by isothermal titration microcalorimetry using a VP-ITC instrument manufactured by Microcal (Northhampton, MA). Peptide stock (120–280 µM) was injected (5–11 µl per injection) into the 1.42-ml reaction cell containing 6–14 µM CaM or TR1C in 5 mM MOPS, pH 7.5, with 2 mM CaCl2 and 0 or 100 mM NaCl. Controls included injection of peptide after complete saturation of CaM and injection of peptides into buffer. Wintrode and Privalov previously reported weak buffer dependence of smMLCKp binding to CaM at pH 7.0, so the measured heats should represent the intrinsic heat of binding. The data obtained from titration experiments were analyzed using the Origin software package for isothermal titration calorimetry (ITC) analysis from Microcal and Mathlab (Mathworks, Natick, MA). For most cases a 1:1 binding model was used to fit the data but for two peptides in low salt (P7 and P4) a second lower-affinity binding step was observed and then a sequential binding model was used to fit the data, and the reported enthalpy value is for the first binding step. The binding is too strong to allow determination of binding constants by ITC except for P4. For this peptide log K values of 7.5 and 7.0 were found in 5 mM buffer and in 100 mM NaCl, respectively. These values are in excellent agreement with those measured by fluorescence spectroscopy.
NMR relaxation measurements
The backbone dynamics of the complex of CaM and P7am-ac was studied by 15N relaxation measurements in 0 and 100 mM KCl using 15N-labeled protein. R1, R2, and {1H}-15N nuclear Overhauser effect (NOE) values were determined using previously published assignments for the complex (16). No peaks associated with free protein could be found, indicating that CaM was fully saturated with Ca2+ and peptide. Both Ca2+ and peptide dissociate from the complex slowly on the NMR timescale, so the dynamics of the components in the complex could be studied.
CaM and P7am-ac were dissolved in 1.4 ml ultrapure H2O (Millipore, Bedford, MA, 0.22 µm) and the sample was concentrated and buffer exchanged using a Vivaspin concentrator. The final sample volume was 300 µl, containing 1 mM CaM, 1.2 mM peptide, 5 mM CaCl2, 0.2 mM NaN3, 0.1 mM DSS, 10% D2O, and 0 or 100 mM KCl. The final pH values were 6.7 and 7.6, respectively, for the 0- and 100-mM samples. Spin-lattice (R1) and spin-spin (R2) relaxation rate constants, as well as {1H}-15N steady-state hetero-NOEs were measured using two-dimensional pulse field gradient-enhanced experiments as described (17) using a 600-MHz Varian UNITY PLUS spectrometer operating at a 1H frequency of 599.89 MHz. Spectra were acquired with 2048 complex points in 
2 (1H), spectral width 7500 Hz, and 128 complex points in 1 (15N), spectral width 1800 Hz. The recovery times between experiments were 1.5 s, 2.0 s, and 10.0 s for R1, R2, and {1H}-15N, respectively. R1 values were determined at 25°C with the following spectral delays: 2 (*2), 170, 320, 470 (*2), 620, 770 (*2), 920, 1220, 1370 (*2), and 1520 ms. R2 values were determined at 25°C with the following spectral delays: 2 (*2), 4, 6 (*2), 8 (*2), 10, 120, 140, 180, 250, and 300 (*2) ms. (*2) indicates experiment duplicates. In the NOE experiment, two spectra were recorded interleaved, one with NOE and one without. All spectra were processed using the nmrpipe program suite with Lorentzian-to-Gaussian transformation in 2 and a shifted sinebell in 1. Relaxation rates were estimated by nonlinear fitting of an exponential function to the experimental data.
Computational details
The thermodynamic binding constant, KTH, for a process where a protein (P) binds a ligand (L) and forms a complex (PL) can be formally written as
 | (1) |
are activities, concentrations, and activity factors, respectively, for the molecules indicated by subscripts. In Eq. 1 we have also made use of the relation a = C. The first ratio on the right-hand side of Eq. 1 is the stoichiometric binding constant, Ks = CPL/CPCL. This is the quantity measured in the experiments. Thus, since KTH is a true constant, any measured change in Ks reflects a change in the activity factors. The activity factor is related to the excess chemical potential,
 | (2) |
 | (3) |
The notation "I" and "II" could, for example, correspond to the binding at two different pH values and 
The indices "B" and "L" stand for bound and free peptide, respectively. The excess chemical potentials in Eq. (3) are averaged over all protonation states of the protein.
The above equations are formally exact, but in the following text, the focus will be on electrostatic interactions only and we will assume that the change in binding constant is solely due to electrostatics. Note also that it is the shift in binding constant that is calculated. This means, for example, that structural changes upon binding do not affect the result as long as they are the same at different conditions. It also means that any "self-energy terms" cancel when the salt effect is studied; not necessarily because the self-energy of the bound and unbound form are the same but because the difference in self-energy between bound and unbound forms at two different salt levels are similar. The self-energy itself can be a large term.
We use a dielectric continuum model for the description of the protein solution. Thus, the atomic details of the solvent (water) are assumed to be of secondary importance and the water is characterized only by its dielectric permittivity, 
r = 78.3, at room temperature. However, the protein atoms and the salt particles are treated explicitly as independent particles. Negatively charged amino acids, Glu, Asp, and the C-terminus, are given a charge of –e divided equally between the two carboxylic oxygens. A positive unit charge is assigned to the appropriate nitrogen atoms of basic amino acid residues including Lys, Arg, His, and the N-terminus. All other protein atoms are treated as hard spheres with a radius of 2 Å—the same hard-core radius is assigned to charged protein atoms and any added positive and negative salt ions. With this model, the protein has a nonuniform charge distribution and the detailed form of the protein is taken into account using an experimentally determined structure for the protein-peptide complex (6).
We have assumed a uniform dielectric constant throughout the solution that is equal to the value for pure water. The interaction energy between any two particles can be formally described by
 | (4) |
 | (5) |
0 are the elementary charge and electric permittivity for vacuum, respectively, and 
= 4 Å. Hence, the total energy is a sum over all charged particles 
We use the standard Metropolis algorithm (18) and the protein atoms are kept fixed at the experimental x-ray coordinates, whereas counterions and salt particles are subject to moves in the Monte Carlo (MC) algorithm. In addition to the interactions described above, we have also introduced a confining sphere for the protein and the ions whose radius defines the protein concentration, which was set to 100 µM except for high ion strengths where a concentration of 800 µM was used. The ionization status of acidic and basic amino acid residues is in principle unknown and varies with pH, salt concentration, and protein concentration, as well as the binding of any ligand. This has been taken into account in the simulations by extending the canonical Metropolis algorithm to a semicanonical approach. Thus, the MC procedure consists of two types of moves: 1), random displacement of mobile salt particles; and 2), random change of the ionization status of titrating groups.
The acceptance of the second type of move is controlled by a change in electrostatic interactions plus the cost for ionizing/neutralizing the randomly chosen amino acid. The appropriate Boltzmann factor reads
 | (6) |
,20). Both the excess chemical potential of the bound and free peptide are obtained from the same simulation. In the first case, the peptide is inserted in the binding site, whereas in the latter the peptide is inserted at random in the MC sphere. The excess chemical potential is then obtained as a canonical average,
 | (7) |
) is the interaction energy between a peptide inserted at position and all other particles. The brackets denote an ensemble average over the unperturbed system. The accuracy of the calculated µex for an octavalent peptide is of the order of a few tenths of a kT. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Because of the long-range nature of electrostatic interactions, changes in the charge state of the protein affect the peptide as much in its free as in its bound state, leading to a virtual pH independence of binding over a wide range of pH values. At pH values close to the isoelectric point, an additional cause of the diminished response of binding affinity to changes in pH is a charge regulation mechanism. In particular, highly charged biopolymers change their net charge in response to one another by releasing or binding protons at ionizable residues (21). One might expect that increasing the salt concentration makes the binding even less sensitive to changes in pH. In contrast, we find that the pH dependence of CaM-peptide complex formation in 1 M NaCl is more pronounced than in the absence of NaCl (Fig. 2). In 1 M NaCl, the log K values show a bell-shaped pH dependence with a maximum around pH 8. Between pH 4.5 and pH 8 we see an increase in binding affinity by 1.5 log K units. A further increase in pH leads to a decrease in binding constant, which could be due to structural changes in CaM and peptide.
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The simulated pH dependence of binding in 761 mM salt is shown in Fig. 2. Here the addition of salt has no effect on the binding in the pH range between 5 and 9, and this behavior is a consequence of the high charge of the system. Instead of the log K optimum around pH 7.5 as found by experiment in 1 M salt, the simulations yield a plateau from pH 5 to pH 9. The reason for this discrepancy may be that the conformational changes that take place upon binding are dependent on pH and salt.
Salt dependence of peptide binding
Complex formation between oppositely charged molecules is usually facilitated by attractive electrostatic forces. Addition of electrolytes reduces the binding affinity due to screening of electrostatic interactions, e.g., as observed for the calcium binding to EF-hand sites of calbindin D9k (22). An increase in binding affinity with added salt is expected when complex formation includes interaction between charges of the same sign. For the oppositely charged CaM and P7, it seems reasonable to expect that the addition of salt reduces the binding constant (in the neutral pH region). The bell-shaped pH dependence at high salt (Fig. 2), however, shows that between pH 5.5 and pH 10 the calmodulin-target peptide interaction is enhanced at high salt. This calls for more detailed investigation of the salt dependence. The affinity between charge variants of CaM and the smMLCK peptide was previously determined at pH 7.5 in low-ionic-strength conditions. Here we have measured the binding constants in 100 mM KCl at pH 7.5 for all combinations of five CaM (wt, E84Q, E83Q, E14Q, and D78N) and four peptide (P6G, P6Q, P5, and P4) charge variants. The binding of wt CaM to P7am-ac was also studied. The results (Table 2) clearly show that the affinity for CaM increases with increasing ionic strength from 0 to 100 mM for all peptides except P4. It is also clear that for all CaM mutants, binding of the highly charged peptides (P5–P7) is tighter at 100 mM KCl than at low salt.
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For P7, P7am-ac, and P4, the binding to wt CaM was followed as a function of salt (Figs. 3 a and 4). P7 and P7am-ac both show an increased binding constant up to 
100 mM NaCl, after which a plateau is reached that extends up to 3 M NaCl for P7. Thus, in conflict with the naive picture, the affinity of the highly charged peptides for the oppositely charged protein increases with increasing salt concentration in the range 0–100 mM NaCl, after which no further increase is seen.
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The results of difference CD spectroscopy show that the peptide is bound in the same helical conformation, regardless of net charge or pH. Difference CD spectra of P4 or P7 bound to CaM compared to free components show that the same amount of helicity is induced in P4 and P7 upon binding at pH 7.5, indicating that P4 is also fully helical in the complex. Also both peptides show the same capacity to adopt a helix on complex formation at pH 5.0 and 7.5. 1H-15N heteronuclear single quantum coherence spectra of P7am-ac-CaM complex in low and 100 mM KCl does not provide any evidence of large conformational changes with the addition of salt (data not shown).
Although the simulations capture many essential features of the experimental results (larger pH sensitivity at high compared to low salt, and more "normal" salt dependence in a system with lower charge), the agreement is not perfect and the increase in affinity with added salt for the highly charged system is not seen in simulations. However, the agreement is much better than what is achieved using the popular Debye-Hückel approximation. A traditional and often useful way to calculate the same quantities as we have done here in the Monte Carlo simulations is to use the so-called Tanford-Kirkwood model. This takes into account the detailed charge distribution of both peptide and protein, and the complex. The electrostatic interactions are, however, calculated only within the Debye-Hückel approximation, which is not a valid approach considering the high charge on both calmodulin and the peptides (23). The Tanford-Kirkwood model predicts that the binding constant increases as a function of salt at low pH and that it decreases as a function of salt at high pH. It also predicts a much too large shift upon change of pH, both at low and high salt concentration.
The failure of the Tanford-Kirkwood theory at low pH is due to the neglect of charge regulation. That is, when a positively charged peptide binds to a protein there will be a release of protons from acidic residues. This mechanism is included in the simulations, whereas the Tanford-Kirkwood model assumes a fixed charge distribution equal to that of the unperturbed protein. At high pH, Tanford-Kirkwood overestimates the salt response due to the underlying linearization approximation, and the simulated curve is closer to experiment. However, both theoretical approaches neglect structural changes in CaM, and this is probably the main reason for the difference between Monte Carlo simulation results and experiments.
Salt effect on the binding of P7am-ac to tryptic fragments of CaM, TR1C, and TR2C shows a decrease in binding affinity with increasing salt concentration, as usually observed for complex formation between oppositely charged molecules (Fig. 5). For TR2C, the obtained stoichiometry is close to one fragment per peptide up to 100 mM NaCl but increases to 1.6 at 1 M NaCl. For TR1C, we observe a change in stoichiometry from one fragment per peptide at low salt, between one and two at 100 mM NaCl, and up to around four TR1C per peptide at 1 M salt. NMR self-diffusion measurements of 1:1 mixtures of TR1C and P7am-ac in 0–200 mM NaCl also indicate a complex salt dependence for the stoichiometry (data not shown). Despite these complications, it is clear that both TR1C and TR2C show the expected salt dependence with decreased peptide-binding affinity up to 1 M NaCl. At low salt, the binding affinity of P7am-ac for TR1C and TR2C is similar to the binding affinity of P7am-ac for intact CaM (log K = 7.5 for TR1C, 7.9 for TR2C, and 7.6 for CaM). Hence, at low salt the presence of a second domain as in intact CaM does not contribute much to the binding affinity. However, around physiological salt, CaM is a much more potent peptide binder than any one of the isolated domains. For example, at 100 mM NaCl, the peptide-binding affinity is 40- to 70-fold higher for calmodulin compared to TR1C and TR2C. This shows that salt screening allows the two domains to cooperate in binding and chelate the peptide more tightly.
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Helix propensity
The process of binding a peptide to CaM may be divided into coil-to-helix transition and binding of the helical peptide to CaM. Addition of salt expected to reduce the electrostatic penalty of forming a helix with a large number of like charges. Indeed, our CD measurements show an increased helical signal for all peptides upon addition of 100 mM NaF (Table 4). This corresponds to a decrease in the free energy of helix formation of 0.3–1.1 kJ/mol, accounting for 5–20% of the salt-induced decrease in free energy of binding.
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Calorimetric data
For all measured peptides the enthalpy of binding to CaM increases with the addition of 100 mM NaCl (Table 3). P4 has a significantly larger increase in binding than the other peptides. Also, the enthalpy of binding P7 to TR1C is higher at 100 mM NaCl (–34.4 kJ/mol) than at 0 mM NaCl (–43.2 kJ/mol), and the apparent stoichiometry of TR1C to peptide increases with the addition of salt. The increase in enthalpy with salt is opposite to what should be expected from Debye-Hückel-type theory.
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). An increase in log K with salt means that the free energy of binding decreases, and since the enthalpy of binding increases, the free energy decrease must be a consequence of entropic factors. This is true for P4 also, where the increase in enthalpy with salt is significantly larger than the increase in free energy. The coil-to-helix transition of the peptide upon binding reduces the entropy of the system, and salt effects on the free peptides may account for a maximum of 20% of the observed free energy change. Other entropic factors involve the N- and C-terminal domains, which are moving independently in free CaM, and experience a loss in the number of available conformations upon peptide binding. Side-chains and backbone can have salt-dependent conformational entropy. The NMR relaxation data give no evidence for changes in backbone flexibility of CaM on the picosecond to nanosecond timescales. Finally, interactions between opposite charges and burial of hydrophobic surfaces in the complex increase the entropy of water.
Interdomain repulsion as a conceivable rationalization of CaM electrostatics
The anomalous salt dependence of the CaM-peptide interaction may be due to electrostatic repulsion between the two domains of CaM, both of which are highly negatively charged. At neutral pH, CaM has a formal net charge of –24 that is reduced to –16 upon Ca2+ binding, with the charges distributed between the N- and C-terminal domains. Several structures of CaM-target-peptide complexes show a peptide bound to the C-terminal lobe of extended CaM, leaving the N-terminal domain unbound. This is consistent with a higher net charge of the C-terminal than the N-terminal domain (–14 for TR2C and –10 for TR1C), indicating that basic peptides preferentially bind to the C-terminal domain. Salt dependence of peptide binding is more pronounced for TR2C than for TR1C, indicating a larger electrostatic component to the binding of peptide to the C-terminal domain. Even with peptide bound to the C-terminal domain, there remains a net negative charge on the domains. One may therefore imagine that the two domains are on average found at larger separation at low salt. Upon addition of salt the repulsive electrostatic interactions are screened and the two domains come closer together with a resulting gain in attractive van der Waal's interactions within the complex (Fig. 6). The model presented here relies on a conformational freedom of the two domains. If the peptide would bind to a static structure, the screening of intraprotein electrostatic interactions by salt would not affect the binding in the manner found in this work.
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CONCLUSIONS |
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ACKNOWLEDGEMENTS |
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Submitted on June 15, 2005; accepted for publication December 22, 2005.
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