1 Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142292, 2 Department of Enzymology, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899 and 3 Branch of M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region 142292, Russia
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Abstract |
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Keywords: metal-binding sites/recoverin/site directed mutagenesis/structure/thermal stability
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Introduction |
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The X-ray crystal structure of recoverin shows that the protein molecule is composed of two domains, each containing two EF-hand motifs (Flaherty et al., 1993). Potential Ca2+-binding sites are distributed relatively uniformly within the recoverin sequence and include amino acids between 36 and 48 (EF1), 73 and 85 (EF2), 109 and 121 (EF3) and 159 and 170 (EF4) (Flaherty et al., 1993
). Of the four potential Ca2+-binding sites, only two (the second and the third) EF-hands are capable of binding Ca2+, whereas the remaining two sites (the first and the fourth) do not possess this ability (Flaherty et al., 1993
; Ames et al., 1995
). The EF-hand structural motif consists of two perpendicularly placed
-helices and a connecting loop, that can be represented as a helixloophelix structure (Ikura, 1996
). In the `working' EF-hands of recoverin, six amino acid residues are involved in Ca2+ binding. Five of them are located in the loop, while glutamate, the sixth residue, is in the second helix of the helixloophelix motif (Ames et al., 1995
).
Calcium binding by myristoylated recoverin has been shown to be a cooperative process with a Hill coefficient of 1.41.75 (Ames et al., 1995; Baldwin and Ames, 1998
). The intermediate value of the Hill coefficient suggests that calcium binding by myristoylated protein cannot be regarded as a totally uncooperative or fully cooperative process, thus raising the question of the exact mechanism of this process (Ames et al., 1995
).
Using site-directed mutagenesis, four mutants of recoverin with amino acid substitutions in the Ca2+-binding sites have been designed (Alekseev et al., 1998). Three of these mutants contained substitutions of glutamate to glutamine at the Z-position of the `working' EF-hands in order to modify the Ca2+-binding properties of these sites. As a result, the myristoylated recoverin mutants EF2 (E85Q), EF3(E121Q) and EF2,3 (E85Q/E121Q) with the modified second, third and (second + third) EF-hands, respectively, were obtained. The fourth mutant, +EF4, had substitutions G160D, K161E, K162N, D165G and K166Q, which gave to the fourth potential Ca2+-binding site of the recoverin the canonical EF-hand sequence. The appearance of enhanced Ca2+ capacity has been expected for this mutant (Alekseev et al., 1998
). In our previous study, the ability of these mutants to bind to photoreceptor membranes and to inhibit the rhodopsin phosphorylation, catalyzed by rhodopsin kinase, was investigated (Alekseev et al., 1998
). It was established that the EF2, EF3 and EF2,3 mutants, with the modified ability to bind Ca2+, were practically unable to interact with photoreceptor membranes and did not inhibit the rhodopsin phosphorylation in the micromolar range of Ca2+ concentrations. The +EF4 mutantt showed a high affinity to membranes and inhibited rodopsin kinase even more effectively than the wild-type (wt) protein (Alekseev et al., 1998
).
Here we present results of detailed studies of the structural properties and Ca2+-binding capabilities of these mutants. The recoverin species, the wt protein and its four mutants, were studied by intrinsic fluorescence spectroscopy, circular dichroism spectroscopy and differential scanning microcalorimetry in the absence and presence of Ca2+. Several interesting features of the recoverin species were revealed. In particular, the data presented are consistent with the suggestion that the binding of calcium ions to recoverin is a sequential process, with EF3-hand being filled first. Also, recoverin exhibits spectral effects very uncommon for most calcium-binding proteins.
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Materials and methods |
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Absorption spectra were measured on a Specord UVVIS spectrophotometer (Karl Zeiss, Jena, Germany) or a spectrophotometer designed and manufactured in the Institute for Biological Instrumentation (Pushchino, Russia).
Circular dichroism (CD) measurements were carried with a JASCO-600 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan), using cuvettes with pathlengths of 0.19 and 5.0 mm for far- and near-UV CD measurements, respectively. The protein concentration was kept at 0.64 mg/ml throughout all the experiments.
Fluorescence measurements were carried out on a laboratory-built spectrofluorimeter described earlier (Permyakov et al., 1977). All spectra were corrected for the spectral sensitivity of the instrument and fitted to log-normal curves (Burstein and Emelyanenko, 1996
) using non-linear regression analysis (Marquardt, 1963
). The maximum positions of the spectra were obtained from the fits. The temperature inside the cell was monitored with a copperconstantan thermopile.
The apparent binding constants for Ca2+ were evaluated from a fit of the fluorescence titration data to the specific binding scheme using non-linear regression analysis (Marquardt, 1963). The binding scheme was chosen on the `simplest best fit' basis, also taking into consideration fluorescence phase plots (Burstein, 1977
). The quality of the fit was judged by the randomness of the distribution of residuals. The accuracy of the determination of the calcium-binding constants was about half order of their values. The temperature dependence of intrinsic fluorescence was analyzed according to Permyakov and Burstein (1984).
Scanning microcalorimetric measurements were carried out on a DASM-4M differential scanning microcalorimeter (Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Russia) in 0.48 ml cells at a 1K/min heating rate. An extra pressure of 1.5 atm was maintained in order to prevent possible degassing of the solutions on heating. Protein concentrations were in the range 0.50.7mg/ml. The heat sorption curves were baseline corrected by heating the measurement cells filled with the solvent only. Specific heat capacities of the proteins were calculated as described (Privalov, 1979; Privalov and Potekhin, 1986
).
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Results |
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pH dependence of tryptophan fluorescence
Our results on the pH dependence of the tryptophan fluorescence of recoverin and its mutants (Permyakov et al., 1999) revealed the pH limits for the native recoverin structure, and also the regions of acidic and alkaline denaturation of the proteins. We found no spectral changes for any of the recoverin species studied in the pH range 7.58.5, so all measurements were carried out at pH
8.
Thermal denaturation
It is also important to determine the regions of thermal denaturation for the recoverin species in both the presence and absence of calcium ions. This procedure excludes the possibility of Ca2+-binding measurements at the middle of thermal transition and also enables us to compare the stability of the mutant proteins relative to the wild-type.
Figures 1 and 2 show the temperature dependences of the fluorescence maximum position and calorimetric scans for wt recoverin and the four mutants in the absence (1 mM EGTA) and presence (1 mM CaCl2) of calcium. The heat sorption peaks and the temperature-induced red shifts of the fluorescence spectra demonstrate cooperative thermal unfolding of the proteins causing the tryptophans to be more accessible to water.
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Figure 1 shows that all apo-forms of the proteins, except the apo-+EF4, have similar melting curves. The differences in the mid-temperatures of their transitions are within ~5°C. The apo-mutants are slightly more stable than the wt recoverin. The least thermostable mutant is +EF4, which is also characterized by the most red-shifted fluorescence spectrum at low temperatures. All this demonstrates that the mutations, except those in site 4, have only insignificant effects on the energetics of the apo-recoverin structure. This means that the mutations in the second and third Ca2+-binding sites have not caused deleterious structural changes, which frequently take place if chelating residues are substituted for Ala.
All calcium-loaded forms of recoverin can be divided into three groups according to their fluorescence maximum positions at low temperatures (Figure 2). +EF4 is characterized by the most red-shifted spectrum. The wt protein together with the EF2 form the second group and the EF3 and EF2,3 have the most blue-shifted spectra. The proteins of the last group are the least thermostable. This thermostability is correct if additional high-temperature peaks for EF2,3 are not taken into consideration, as these peaks are probably due to the melting of specific associates.
The binding of calcium to +EF4 causes the most pronounced stabilization of the protein (by ~35°C). For wt recoverin the calcium-induced shift of the thermal transition to higher temperatures is not very pronounced (~15°C) in comparison with the shifts for other calcium-binding proteins. The calcium-induced shift of the thermal transition for the mutant EF2 is ~7°C, which seems to be due to a reduced calcium affinity of this species (see below). However, in this case the calcium binding leads to a decrease in the area under the main heat sorption peak and in the appearance of an additional transition at about 65°C. The addition of calcium to the EF3 mutant shifts the main thermal transition to lower temperatures and induces an appearance of two small additional heat sorption peaks at 45 and 55°C. The addition of calcium to the mutant EF2,3 results in a small shift of the main heat sorption peak to lower temperatures and the formation of two additional peaks at 87 and 96°C, which is most unusual. The appearance of the additional heat sorption peaks in the case of the EF3 and EF2,3 mutants seems to be due to a low-affinity calcium binding (see below) or aggregation effects (Kataoka et al., 1993).
The results of these studies demonstrate that at pH 8 all the proteins are stable up to at least 30°C regardless of calcium content, thus allowing us to use room temperature for all titration experiments.
Calcium binding to recoverin species
Figure 3 presents the results of the spectrofluorimetric titration of calcium-free wt recoverin and its mutants by calcium and then by the calcium chelator EGTA. The dependence of the fluorescence maximum position of the proteins on the relative calcium and EGTA concentrations is shown in Figure 3A
. Figure 3B
shows the corresponding dependences of the fluorescence intensities at fixed wavelengths. The measurements were carried out at pH 8.0 and 14°C.
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The mutation in the third EF-hand abolishes the high calcium affinity of recoverin and the mutation in the second EF-hand does not cause any essential drop in calcium affinity. This is consistent with assumption that the binding of calcium ions to recoverin is sequential, with the third EF-hand being filled first:
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The data on the calcium and EGTA titrations were fitted simultaneously and the fit was achieved by variation of the binding constants K1 and K2 (Permyakov et al., 1999). In the case of the EGTA titration an equilibrium equation for the binding of calcium to EGTA with the well-known binding constant KEGTA (Permyakov et al., 1977
) was added:
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The values of K1 and K2 which give the best fit are presented in Table I, which contains similar data for the mutant proteins. It should be noted that the first binding constant for the EF2 is determined with poor accuracy because the fluorescence response to the filling of the third EF-hand is very small.
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It should be noted that EF3 and EF2,3 have no high-affinity calcium-binding sites, yet they can bind calcium with low affinity. Calcium titration of EF3 and EF2,3 in the region of millimolar concentrations (not shown) causes small spectral effects allowing the evaluation of the binding constants for the low-affinity binding sites (~103 M1). The existence of such sites is reflected in the different patterns of the thermal transitions for these mutants in the presence and absence of calcium (see above).
Calcium-induced changes of CD spectra of wt recoverin and its mutants
Figure 4 shows the near- (A) and far-UV (B) CD spectra for recoverin and its mutated forms in the absence of calcium (1 mM EGTA). It is known that a native protein with a rigid tertiary structure is characterized by a pronounced near-UV CD spectrum owing to the asymmetric environment of its aromatic amino acid residues. The far-UV CD spectra allow the characterization of the secondary structure of the protein. Figure 4
shows that the CD spectra of wt recoverin, EF2, EF3 and EF2,3 in the absence of calcium are practically coincidental with each other, in both the near- and far-UV regions. This suggests that neither the tertiary nor secondary structure of the recoverin molecule is affected by the amino acid substitutions.
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An unusual feature of recoverin is that the calcium binding induces a pronounced red shift in the tryptophan fluorescence spectrum. Such behavior can be explained by an increase in the exposure of tryptophan residues to the solvent. It is logical to assume that such intramolecular transformations should be accompanied by changes in the protein near-UV CD spectrum. Figure 5A shows the CD spectra for all the species of recoverin in the presence of 1 mM CaCl2. Comparison of these data with the results presented in Figure 4A
shows that the interaction with calcium induces significant changes in both shape and intensity of the recoverin near-UV CD spectrum. Interestingly, the largest calcium-induced effect is observed above 270 nm, which is the region where the largest tryptophan contribution is expected. At the same time, Figure 5A
indicates that the interaction with calcium does not significantly affect the environment of other aromatic amino acid residues (phenylalanines and tyrosines).
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The same situation is observed for the far-UV CD spectra of the proteins (Figure 5B). However, in this case the largest spectral changes are observed for +EF4. Analysis of the data presented in Figures 4B and 5B
allows one to conclude that for all the proteins the interaction with calcium is accompanied by minor changes in the far-UV CD spectrum intensity. This observation is consistent with a very small increase in
-helical structure in the protein molecule.
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Discussion |
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It should be noted that Kataoka et al. (1993) have shown that in the absence of calcium, myristoylated recoverin is monomeric and globular, whereas addition of calcium ions brings about aggregation. The aggregation effect should perturb experimental estimations of calcium binding constants.
Our data are in line with the results of Matsuda et al. (1998) on E85M and E121M mutants of a recoverin subfamily member, S-modulin. According to their data, E121M, which has a mutation in the EF3-hand, neither binds calcium nor inhibits phosphorylation, whereas E85M binds one calcium and has the same membrane affinity as wt S-modulin. However, this mutant has lost the ability to inhibit rhodopsin phosphorylation.
Comparison of NMR structures obtained both for Ca2+-loaded and apo-forms of recoverin (Tanaka et al., 1995; Ames et al., 1997
) reveals that loop regions of both functional EF-hands of the protein are equally accessible to solvent, thus making them equally attractive for calcium ions. If this is the case, then why is the third EF-hand filled first? Binding of two Ca2+ by recoverin results in a substantial change in the relative position of helices for all EF-hands of the protein, except the last (Yap et al., 1999
). Hence such structural rearrangements are necessary for both working EF-hands to form geometries of residues capable of Ca2+ binding. At the same time, all the helices of the EF-hands 1 and 2 in Ca2+-free recoverin are involved in forming a hydrophobic cavity with the myristoyl group (Tanaka et al., 1995
). This can potentially prevent them from making any displacements facilitating Ca2+ binding. On the other hand, the third EF-hand is located in the interdomain region of the polypeptide chain: the N-terminal helix is involved in the formation of the same hydrophobic pocket (Tanaka et al., 1995
), while the C-terminal helix is located in the C-terminal domain. The position of the EF3 hand is likely to promote relative displacement of its helices, assisting Ca2+ binding. The binding of the first calcium ion induces some structural changes, which alter the position of the EF2 helices so that the binding of the second Ca2+ becomes geometrically favorable. The myristoyl group might be exposed to water upon the binding of the first ion, but our previous study of the ability of EF2 recoverin mutant to bind to photoreceptor membranes (Alekseev et al., 1998
) showed a significant decrease in comparison with wt protein. Nevertheless, this effect may be the case for some members of the recoverin subfamily. For example, an S-modulin mutant with modified EF2-hand binds one Ca2+ and has the same membrane affinity as wt S-modulin (Matsuda et al., 1998
), indicating the extrusion of the myristoyl group upon loading of the EF3 hand.
The idea that the hydrophobic pocket, comprising the myristoyl group of recoverin, could prevent the EF2-hand helices from relative displacement, facilitating Ca2+ loading of the EF2-hand, is supported by data of Baldwin and Ames (1998). Hydrophobic core mutations W31K and I52A/Y53A aimed at destabilization of the N-terminal domain resulted in an increase in the apparent calcium-binding constant and a decreased cooperativity of binding (Hill coefficient in the range 1.01.2).
Analysis of the near-UV CD spectra of recoverins shows that the interaction of these proteins with Ca2+ is accompanied by a decrease in the asymmetry of the tryptophan residue environment. It is important to note that such a structural transformation is very unusual. Since the rigidity of the environment for other aromatic residues is minimally affected by the calcium binding (Figures 4A and 5A), we can assume that such spectral changes reflect only the increased solvent accessibility of Trp residues. This suggestion is confirmed by the fact that the binding of calcium induces a red shift in the tryptophan fluorescence spectrum. This is also unusual for calcium-binding proteins.
Examination of the tertiary structure of recoverin (Tanaka et al., 1995; Ames et al., 1997
) shows that the spectral changes observed can be due to a calcium-induced increase in the degree of solvation of all the tryptophan residues (Trp31, Trp104 and Trp156). This is also in accordance with the finding that the quenching of the tryptophan fluorescence of myristoylated recoverin by acrylamide is more efficient for the calcium-loaded state than the apo-state (Hughes et al., 1995
). The more complete exposure to solvent of Trp31 and Trp104 is likely to be a consequence of Ca2+-induced disruption of the hydrophobic cavity, formed by the myristoyl group along with helices of the first three EF-hands in calcium-free recoverin (Tanaka et al., 1995
).
Figure 6 compares the effects of calcium binding on the near-UV CD spectra of different recoverin species with those on the far-UV CD spectra ([
]277 versus [
]222 dependences). It is seen that the binding of calcium to any recoverin species is accompanied by an increase in the far-UV CD spectrum intensity and by a decrease in the aromatic CD signal. The observed increase of the far-UV CD signal reflects an increase in the
-helical structure of recoverin upon Ca2+ binding. This indicates that the Ca2+-loaded protein is more ordered than the apo-form. A Ca2+-induced decrease in the total size of the protein (Kataoka et al., 1993
) also supports this idea. Thus, in spite of the manifestation of extraordinary spectral effects of Ca2+, the overall structural behavior of recoverin is very similar to that of other Ca2+-binding proteins. The only peculiarity of the behavior of recoverin is the exposure of hydrophobic residues on calcium binding. This is manifested in the appearance of hydrophobic ANS binding surfaces in both myristoylated and unmyristoylated recoverin (Hughes et al., 1995
). The myristoyl group becomes solvent exposed upon Ca2+ binding, thus making the N-terminal domain residues, which interact with myristoyl in the apo-form, available for binding to membrane targets.
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Another important point is the existence of a very good correlation between calcium-induced changes in fluorescence and the near-UV CD spectra of recoverin. Figure 7 shows the [
]277 versus
Trpmax dependence obtained from the analysis of near-UV CD and fluorescence spectra of different species of recoverin in the absence and presence of calcium. One can see that the more red shifted the tryptophan fluorescence spectrum of the protein, the less intense is the CD spectrum in the near-UV region.
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Notes |
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5 To whom correspondence should be addressed. E-mail: uversky{at}hydrogen.ucsc.edu
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Acknowledgments |
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References |
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Ames,J.B., Porumb,T., Tanaka,T., Ikura,M. and Stryer,L. (1995) J. Biol. Chem., 270, 45264533.
Ames,J.B., Ishima,R., Tanaka,T., Gordon,J.I., Stryer,L. and Ikura,M. (1997) Nature, 389, 198202.[ISI][Medline]
Baldwin,A.N. and Ames,J.B. (1998) Biochemistry, 37, 1740817419.[ISI][Medline]
Blum,H.E., Lehky,P., Kohler,L., Stein,E.A. and Fisher,E.H. (1977) J. Biol. Chem., 252, 28342838.[Abstract]
Burstein,E.A. (1977) Intrinsic Protein Fluorescence: Origin and Applications. Biophysics, Vol. 7. VINITI, Moscow.
Burstein,E.A. and Emelyanenko,V.I. (1996) Photochem. Photobiol., 64, 316320.[ISI]
Chen,C.K., Inglese,J., Lefkowitz,R.J. and Hurley,J.B. (1995) J. Biol. Chem., 270, 1806018066.
Dizhoor,A.M., Ericsson,L.H., Johnson,R.S., Kumar,S., Olshevskaya,E., Zozulya,S., Neubert,T.A., Stryer,L., Hurley,J.B. and Walsh,K.A. (1992) J. Biol. Chem., 267, 1603316036.
Flaherty,K.M., Zozulya,S., Stryer,L. and McKay,D.B. (1993) Cell, 75, 709716.[ISI][Medline]
Gray-Keller,M.P., Polans,A.S., Palczewski,K. and Detwiler,P.B. (1993) Neuron, 10, 523531.[ISI][Medline]
Hughes,R.E., Brzovic,P.S., Klevit,R.E. and Hurley,J.B. (1995) Biochemistry, 34, 1141011416.[ISI][Medline]
Ikura,M. (1996) Trends Biochem. Sci., 21, 1417.[ISI][Medline]
Kataoka,M., Mihara,K. and Tokunaga,F. (1993) J. Biochem., 114, 535540.[Abstract]
Kawamura,S. (1993) Nature, 362, 855857.[ISI][Medline]
Kawamura,S., Hisatomi,O., Kayada,S., Tokunaga,F. and Kuo,C.H. (1993) J. Biol. Chem., 268, 1457914582.
Klenchin,V.A., Calvert,P.D. and Bownds,M.D. (1995) J. Biol. Chem., 270, 1614716152.
Marquardt,D.W. (1963) J. Soc. Ind. Appl. Math., 11, 431441.[ISI]
Matsuda,S., Hisatomi,O., Ishino,T., Kobayashi,Y. and Tokunaga,F. (1998) J. Biol. Chem., 273, 2022320227.
Permyakov,E.A. (1993) Calcium Binding Proteins. Nauka, Moscow.
Permyakov,E.A. and Burstein,E.A. (1984) Biophys. Chem., 19, 265271.[ISI][Medline]
Permyakov,E.A., Burstein,E.A., Sawada,Y. and Yamazaki,I. (1977) Biochim. Biophys. Acta, 491, 149154.[ISI][Medline]
Permyakov,E.A., Yarmolenko,V.V., Emelyanenko,V.I., Burstein,E.A., Gerday,C. and Closset,J. (1980) Eur. J. Biochem., 109, 307315.[Abstract]
Permyakov,E.A., Yarmolenko,V.V., Kalinichenko,L.P., Morozova,L.A. and Burstein,E.A. (1981) Biochem. Biophys. Res. Commun., 100, 191197.[ISI][Medline]
Permyakov,S.E. et al. (1999) Bioorg. Chem. (Moscow), 25, 742746.
Privalov,P.L. (1979) Adv. Protein Chem., 33, 167241.[Medline]
Privalov,P.L. and Potekhin,S.A (1986) Methods Enzymol., 131, 451.[Medline]
Senin,I.I., Zargarov,A.A., Aiekseev,A.M., Gorodovikova,E.N., Lipkin,V.M. and Philippov,P.P. (1995) FEBS Lett., 376, 8790.[ISI][Medline]
Tanaka,T., Ames,J.B., Harvey,T.S., Stryer,L. and Ikura,M. (1995) Nature, 376, 444447.[ISI][Medline]
Yap,K.L., Ames,J.B., Swindells,M.B., Ikura,M. (1999) Proteins: Struct. Funct. Genet., 37, 499507.[ISI][Medline]
Zozulya,S. and Stryer,L. (1992) Proc. Natl Acad. Sci. USA, 89, 1156911573.[Abstract]
Received December 12, 1999; revised August 2, 2000; accepted September 23, 2000.