Localized, stereochemically sensitive hydrophobic packing in an early folding intermediate of dihydrofolate reductase from Escherichia coli1

Article No. jmbi.1999.3403 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 295, 737±744

COMMUNICATION

Localized, Stereochemically Sensitive Hydrophobic Packing in an Early Folding Intermediate of Dihydrofolate Reductase from Escherichia coli John C. O'Neill Jr and C. Robert Matthews* Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University University Park, PA 16802, USA

Mutational analysis was performed to probe the development of hydrophobic clusters during the early events in the folding of dihydrofolate reductase. Replacements were made in several hydrophobic subdomains to examine the roles of hydrophobicity and stereochemistry in the formation of kinetic intermediates. Amide protons in two of these clusters, including residues I91, I94, and I155, have been shown to be protected against solvent exchange within 13 ms of folding. Additional hydrophobic clusters were probed by substitutions at residues I2, I61, and L112; these residues are not protected from exchange until later in the folding reaction. Valine and leucine replacements at positions I91, I94, and I155 signi®cantly diminish the formation of the burst phase kinetic intermediate, relative to the wild-type protein. In contrast, I2 and I61 are insensitive to these substitutions in the ®rst 5 ms of the folding reaction, as is the replacement of L112 with either isoleucine or valine. These results demonstrate that the tightly packed core of dihydrofolate reductase is acquired in a non-uniform fashion, beginning in the submillisecond time frame. The progressive development of speci®c side-chain packing in localized hydrophobic clusters may be a common theme for complex protein folding reactions. # 2000 Academic Press

*Corresponding author

Keywords: protein folding; stopped-¯ow circular dichroism; burst phase intermediate; side-chain packing interactions; hydrophobic clusters

Although it has long been known that the amino acid sequence of a protein contains all the information required to determine the native threedimensional fold (An®nsen et al., 1961), it is less clear how the side-chains guide this process in the early stages of folding reactions. Stable, molten globule-like models of early folding intermediates in many proteins suggest that these transient species are compact, contain signi®cant secondary structure, have hydrophobic surfaces exposed to solvent and have marginal stability (Roder et al., 1988; Kuwajima, 1989; Jennings & Wright, 1993; Raschke & Marqusee, 1997). Because the sideAbbreviations used: BPA, burst phase amplitude; CD, circular dichroism; DHFR, dihydrofolate reductase from Escherichia coli; K2EDTA, ethylenediaminetetraacetic acid, dipotassium salt; FL, ¯uorescence; MTX, methotrexate; SF-CD, stopped-¯ow circular dichroism; SVD, singular value decomposition; UV, ultraviolet. E-mail address of the corresponding author: [email protected] 0022-2836/00/040737±8 $35.00/0

chains in these partially folded forms are often not constrained, these models suggest that native-like side-chain packing is only achieved at a later stage in the folding reaction. For example, the product of a microsecond contraction reaction for cytochrome c is a compact form that contains elements of native secondary structure and has marginal stability (Sauder & Roder, 1998; Shastry & Roder, 1998). A previous mutational analysis had shown that the replacement of a buried hydrophobic residue, Leu94, with either Ile or Val has marginal effects on the stability of this species (Colon et al., 1996). By contrast, the stability of a later intermediate, formed on the 10 ms time scale, is measurably affected by these conservative replacements. These studies demonstrated that non-random side-chain interactions develop within the ®rst few tens of milliseconds of folding (Shastry & Roder, 1998). The progressive development of side-chain packing parallels the enhanced protection patterns of backbone amide # 2000 Academic Press

738

Hydrophobic Packing in an Early Folding Intermediate of DHFR

protons against solvent exchange for the subsequent intermediate (Sauder & Roder, 1998). Previous quench-¯ow hydrogen exchange NMR results for dihydrofolate reductase suggest that the side-chains are involved in the formation of the burst phase intermediate on the millisecond time scale (Jones & Matthews, 1995). Eleven amide protons in the eight-stranded b-sheet and two amide protons in two different a-helices that dock on the sheet have protection factors that range from 2 to >100 after 13 ms of folding. The side-chains corresponding to these protons comprise two different hydrophobic clusters, one on each side of the sheet, that together span seven of the eight strands and overlap along two of the central strands (Figure 1) (Bystroff & Kraut, 1991). Although these clusters are dominated by branched b-carbon side-chains, Ile and Val, and aromatic side-chains, Phe and Tyr, the high bstrand propensity and hydrophobicity of these residues cannot account for the development of the complex strand topology that is inferred in this early folding intermediate. Rather, native-like, speci®c side-chain packing interactions in the early intermediate of DHFR must facilitate the rapid organization of the complex strand topology (‡2, ‡1, ‡1, ÿ4, ÿ2, ÿ2, ‡1) and account for the observed protection pattern. Mutational strategy Mutagenesis combined with stopped-¯ow CD spectroscopy provides a simple test of the hypothesis that stereochemically sensitive side-chain packing is achieved in two hydrophobic clusters in DHFR within 5 ms. Six aliphatic residues (principally isoleucine) located in several hydrophobic clusters in DHFR were selected for replacement with leucine or valine. Leu maintains the hydrophobicity but moves the branch point of the

side-chain to the g-carbon while Val conserves the b-carbon stereochemistry but reduces the hydrophobicity (Creighton, 1993). Residues I91, I94, and I155 were chosen to probe the packing of two hydrophobic clusters postulated to form in an early kinetic intermediate (Jones & Matthews, 1995). I91 is located in b-strand E and is a member of a ®ve residue hydrophobic cluster that presumably stabilizes the adenine-binding domain (Tsai & Nussinov, 1997; Tsai et al., 1998), residues 38-108. I94 and I155 are located in bE and bH and are at either end of a seven-member cluster on the opposing face of the sheet. Presumably, this cluster serves to organize the discontinuous domain, residues 1-37 and 107-159 (Sawaya & Kraut, 1997), and integrate it with the adenine-binding domain. Three additional buried hydrophobic residues, I2, I61, and L112, which are located in bA, bC, and bF, were chosen as control sites. Their amide protons are not protected from exchange in the ®rst few milliseconds of folding; however, L112 does show protection from exchange in native DHFR (Jones & Matthews, 1995). Influence of Leu and Val replacements on the equilibrium properties of DHFR Equilibrium studies were performed to examine the effects of steric and hydrophobic replacements at positions I2, I61, I91, I94, L112, and I155 on the structural and thermodynamic properties of native DHFR. The far-UV CD spectra of the native conformations of this set of proteins, with the exception of L94 and V94, are similar to wild-type DHFR with a shoulder at 222 nm and a minimum at 218 nm. The secondary structures are not signi®cantly distorted by these amino acid replacements (data not shown). The spectra for L94 and V94 display minima around 225 nm and 208 nm, suggesting that these mutations either perturb the

Figure 1. Stereo view of wild-type DHFR, based upon crystallographic data (Bystroff & Kraut, 1991), highlighting residues that comprise several hydrophobic clusters. The six residues that served as mutagenic probes are labeled, as well as two of the ®ve tryptophan residues, W47 and W74. Evidence from this work and previous hydrogenexchange NMR data (Jones & Matthews, 1995) support the development of native-like contacts within the burst phase intermediate for two of these hydrophobic clusters, colored in blue and green. A third cluster, colored in yellow, involves W47 and W74 and becomes structured several orders of magnitude later in the folding reaction (Garvey et al., 1989; Kuwajima et al., 1991).

739

Hydrophobic Packing in an Early Folding Intermediate of DHFR

b-sheet to some extent or alter the packing of aromatic side-chains (Kuwajima et al., 1991). However, the retention of greater than 85 % of the normalized CD signal intensity and 60 % of the speci®c activity of the wild-type protein by these mutants (Table 1) implies that the native conformation is largely conserved. Far-UV CD and steady-state ¯uorescence spectroscopy were used to monitor the urea-induced equilibrium unfolding transitions of wild-type and mutant DHFR. The coincidence of the normalized unfolding curves monitored by CD and FL (data not shown) is consistent with a two-state unfolding process in which only the native and unfolded forms are signi®cantly populated. The two-state model used to ®t the data yielded the apparent free energy of folding,
Gapp(H2O), the denaturation midpoint, Cm, and the sensitivity of the free energy to the denaturant concentration, m value, for each mutant (Table 1). Either one or both of the replacements at each position reduces the stability by approximately 1 to 2 kcal molÿ1 relative to wild-type DHFR (Touchette et al., 1986). The mutants at positions 61 and 91 are also particularly sensitive to the substitutions based upon their signi®cantly reduced m values, about 1.45 kcal molÿ1 Mÿ1, relative to the 2.1 kcal molÿ1 Mÿ1 determined for wild-type DHFR. These lower m values may

re¯ect minor populations of folding intermediates (Luo et al., 1995). These destabilizations are similar to those observed for conservative replacements in other proteins (Kellis et al., 1989; Lorch et al., 1999) and are consistent with a decrease in favorable side-chain packing interactions and/or the differences in hydrophobicities of the side-chains. The tight packing evident around these side-chains in the native conformation, therefore, offers an opportunity to study the temporal development of structures during folding. Effects of Leu and Val mutations on the burst phase intermediate The perturbation of the burst phase intermediate of DHFR by mutational replacement was determined by measuring the amplitude of the far-UV CD signal at 222 nm at 5 ms into the folding reaction as a function of the ®nal urea concentration. For the wild-type protein (Figure 2(a)), a signi®cant signal intensity develops within the dead time of the mixing reaction under strongly refolding conditions, 0.5 M ®nal urea. This burst phase amplitude is substantial, accounting for approximately 25 % of the total change in ellipticity expected during the refolding process (Table 1). The signal continues to develop for the wild-type protein as

Table 1. Physical properties of wild-type and mutant DHFR DHFR

Specific activity (units mgÿ1)


G (H2O)a (kcal molÿ1)

ma (kcal molÿ1 Mÿ1)

Cm (M)

BPAb []222*10ÿ3 (deg-cm2 dmolÿ1)

Percentc burst phase (%)

wt I2Le I2V I61L I61V I91L I91V I94L I94V L112I L112V I155L I155V

83 (11)d ndf 71 (12) 49 (7) 50 (3) 72 (9) 85 (2) 64 (4) 63 (11) 88 (19) 81 (2) 80 (9) 78 (11)

6.37 (0.23) nd 5.65 (0.40) 4.91 (0.18) 4.53 (0.12) 3.94 (0.12) 3.88 (0.14) 5.67 (0.26) 5.59 (0.27) 6.23 (0.26) 4.71 (0.04) 4.43 (0.16) 6.51 (0.10)

2.13 (0.08) nd 2.02 (0.15) 1.46 (0.03) 1.43 (0.06) 1.45 (0.06) 1.35 (0.03) 2.12 (0.07) 2.06 (0.03) 2.22 (0.10) 1.98 (0.03) 1.95 (0.08) 2.27 (0.04)

3.0 nd 2.8 3.4 3.1 2.7 2.9 2.7 2.7 2.8 2.4 2.3 2.9

3.56 (0.13) nd 3.43 (0.03) 3.41 (nd) 3.41 (0.14) 2.93 (0.03) 2.80 (0.12) 3.05 (0.06) 2.85 (0.18) 3.27 (0.04) 3.66 (0.23) 2.73 (0.33) 2.92 (0.26)

25 (3) nd 28 (1) 23 (nd) 23 (3) 14 (1) 8 (3) 14 (1) 11 (4) 23 (1) 30 (4) 13 (5) 13 (4)

Protein puri®cation: Protein was over-expressed and puri®ed as described (Kuwajima et al., 1991; Jennings et al., 1993). The concentrations of proteins were determined spectroscopically using e280 ˆ 3.11  104 Mÿ1 cmÿ1 (Touchette et al., 1986). Speci®c activity: The speci®c activities of wild-type DHFR and the mutants were measured at 30  C by the method of Hillcoat (Hillcoat et al., 1967). The reported activity of wild-type (wt) DHFR is 85 units mgÿ1 in 0.1 M imidazole chloride (pH 7.0) at 30  C (Baccanari et al., 1977). Spectroscopy: Equilibrium CD experiments were performed at 15  C on an Aviv 62DS CD spectrometer (Lakewood, NJ). Spectra were recorded from 300 to 216 nm with a 1 nm bandwidth. The path length of the cuvette varied from 0.1 to 0.2 cm, depending on the protein concentration. Steady-state ¯uorescence emission spectra were recorded at 15  C on an Aviv model 105 spectrometer. The samples were excited at 295 nm and the emission scans were recorded from 305 to 500 nm with a 1 nm bandwidth. The slit width for both excitation and emission were 5 nm. The path length was 1 cm. Data were collected at 54.7  from the vertical position to detect the total intensity of the ¯uorescence signal using excitation and emission polarizers (Lakowicz, 1983). Protein concentrations ranged from 3 to 18 mM. a Calculated from CD and FL data assuming a two-state unfolding transition and a linear dependence of the free energy on the denaturant concentration:
G ˆ
G (H2O) ÿ m[urea]. b The burst phase amplitude (BPA) was determined under strongly refolding conditions (0.5 M urea). c Calculated from the fractional recovery of the burst phase [y]222 at 0.5 M urea, using a normalized unfolding transition curve for each protein. d Error estimates shown in parentheses represent one standard deviation based upon multiple independent measurements. e The I2L mutant was not studied because of its poor expression properties; this implies that I2L is substantially destabilized by the amino acid replacement. f nd, not determined.

740

Hydrophobic Packing in an Early Folding Intermediate of DHFR

Figure 2. Refolding kinetics of wild-type DHFR in (a) (*), I155L in (b) (&), and I61V in (c) (~), monitored by stopped-¯ow CD at 222 nm. The fast kinetics are shown to highlight the burst phase amplitudes for the representative proteins. Arrows indicate both the predicted ellipticities of the unfolded forms at 0.5 M urea and the ®nal amplitudes of the kinetic traces. The refolding experiments were performed at 15  C in 10 mM potassium phosphate buffer (pH 7.8) containing 0.2 mM K2EDTA and 1 mM b-mercaptoethanol. The refolding reactions were initiated by tenfold dilution from 5 M urea into standard buffer. Urea concentrations were calculated from refractive indices (Pace et al., 1989). Kinetic experiments were performed using a Bio-Logic (Grenoble, France) SFM-3 stopped-¯ow drive train installed on the Aviv 62DS CD spectrometer. The dead time for the kinetic experiments was 5 ms. Protein concentrations ranged from 3 to 18 mM. Kinetic refolding data were ®t by a non-linear least-squares procedure to the equation:

the reaction progresses and passes through transient intermediates (Jennings et al., 1993). Because these subsequent folding reactions are several orders of magnitude slower, the initial signal provides a measure of the concentration of the burst phase kinetic intermediate and an estimate of its stability (Kuwajima et al., 1991). Thus, the sensitivity of the burst phase CD amplitude to mutational replacement can serve as a probe of the effects of side-chain interactions on the formation of early kinetic species in DHFR. To test the importance of stereochemistry and hydrophobicity on the development of the burst phase intermediate in DHFR, SF-CD kinetic data were collected for Leu and/or Val mutants at positions I2, I61, I91, I94, and I155 and for Ile and Val replacements at L112. In contrast to the wild-type protein (Figure 2(a)), the mutants at positions 91, 94, and 155 have diminished burst phase amplitudes under strongly refolding conditions for both the Leu and Val substitutions. A representative example of such data is shown in Figure 2(b) for I155L. To insure that the reduced amplitudes observed for positions 91, 94, and 155 re¯ected properties of the burst phase intermediate and not simply the effects of mutating any buried hydrophobic residue, similar replacements were made at three other buried Ile or Leu positions. I2 and L112 reside in b-strands on the opposing face of the b-sheet as that of the large cluster previously identi®ed as participating in the early burst phase intermediate (Jones & Matthews, 1995). I61 also resides in a b-strand but on the opposing face of the sheet from the smaller cluster; it participates in the formation of the adenine-binding site that also contains W47 and W74. The burst phase amplitudes of the mutants at positions I2, I61, and L112, are similar to that for wild-type DHFR; a representative example is provided in Figure 2(c) by I61V. The absence of an effect on the burst phase amplitude implies that the side-chains at positions 2, 61, and 112 do not participate in the corresponding early intermediate. By monitoring the burst phase amplitude as a function of urea concentration, the stability of the burst phase kinetic species can be estimated (Kuwajima et al., 1991). In the case of wild-type DHFR, the burst phase intensity decreases sigmoidally as a function of urea until ®nally merging with the unfolded baseline at approximately 3 M

A…t† ˆ A1 ‡

X i

  ÿt Ai exp ti

…1†

where A(t) is the total amplitude at time t, A1 is the amplitude at in®nite time, Ai is the amplitude of the individual phase, i, and ti is the corresponding relaxation time. Kinetic traces were ®tted to a sum of three exponentials. Extrapolation of the data to 5 ms provided the ellipticity of the burst phase intermediate.

Hydrophobic Packing in an Early Folding Intermediate of DHFR

urea (Figure 3). A two-state ®t of the burst phase amplitude of wild-type DHFR as a function of urea yielded an apparent free energy of folding of 1.5(0.5) kcal molÿ1. The burst phase intensities at ®nal urea concentrations below 1.5 M for the mutants at positions 91, 94, and 155 are consistently less than the wild-type amplitudes. For example, under strongly folding conditions, 0.5 M urea, the burst phase amplitudes for I155L or I155V are about half the amplitude of wild-type DHFR (Figure 3). Unfortunately, the absence of folded baselines under strongly refolding conditions precluded the determination of the stabilities of the burst phase intermediates for these mutants. Additionally, qualitative comparisons of their stabilities may also be complicated by nontwo-state folding reactions observed in marginally stable intermediates (Schulman et al., 1997). Thus, the altered urea-dependence of the burst phase amplitudes may re¯ect a change in the cooperativity of the unfolding transitions. Nonetheless, the

Figure 3. Unfolding transitions of DHFR monitored by CD (222 nm) as a function of urea concentration for both equilibrium (®lled symbols) and burst phase kinetic species (open symbols) for wild-type (*), I155L ( & ), and I155V (~). Continuous lines indicate the goodness of the ®t to a two-state equilibrium model. In the model, the stability was assumed to depend linearly on the denaturant concentration (Schellman, 1978). Short broken lines represent the unfolded baseline of wildtype DHFR extrapolated to 0 M urea concentration. Long broken lines indicate the goodness of ®t of the folding transition for the burst phase intermediate as a function of urea concentration to a two-state model. Errors in the burst phase amplitudes, shown for the wild-type protein, represent one standard deviation based upon three or more individual experimental measurements. Errors for the mutant proteins are comparable to the wild-type protein. The folding reactions were performed with the standard buffer conditions as for Figure 2.

741

collective loss in burst phase amplitudes of the mutants at positions 91, 94, and 155, does indicate, qualitatively, that these early kinetic species are less stable than their wild-type counterpart. The reduction in the burst phase amplitude for both the Leu and Val amino acid substitutions at positions 91, 94, and 155 demonstrates that both the stereochemical con®guration of the side-chain and the hydrophobicity are critical to the formation of the earliest detectable folding intermediate. Evidence to support speci®c hydrophobic packing interactions in marginally stable intermediates has recently been shown for apomyoglobin (Kay et al., 1999) and a-lactalbumin (Song et al., 1998; Wu & Kim, 1998). Moreover, the results for a-lactalbumin demonstrate that folding intermediates characterized as highly ¯exible and dynamic, can possess cores with distinct native-like packing contacts (Wu & Kim, 1998). The absence of any effects on the burst phase CD intensity at positions 2, 61, and 112 (Table 1) indicates that the formation of tight packing in the ®rst few milliseconds of folding of DHFR is localized to portions of the protein molecule and is not a global phenomenon. The correlation between early protection against hydrogen exchange (Jones & Matthews, 1995) and the perturbation of the burst phase amplitudes at positions 91, 94, and 155 (Figure 4) provide strong support for the critical role of two hydrophobic clusters in organizing the complex strand topology in DHFR. These results are also consistent with previous ¯uorescence (Garvey et al., 1989) and CD (Kuwajima et al., 1991) studies on DHFR that demonstrated the formation of native-like sidechain packing interactions between W47 and W74 during the next few hundred milliseconds of folding. Because these aromatic side-chains are part of a hydrophobic cluster that includes I61 (Figure 1), these mutational results support the progressive development of hydrophobic clusters in DHFR. Apparently, the I94/I155 and I91 clusters form ®rst and provide a template for the development of the W47/I61/W74 cluster. The sensitivity of DHFR's burst phase to mutational replacement support a partially structured species The observation of ``burst phase'' behavior, as proteins are refolded by dilution from concentrated denaturant, has generally been interpreted as the formation of productive folding intermediates (Roder & Colon, 1996). Recent reports have suggested that the variation in signal intensity on the microsecond time scale during truncated cytochrome c (Sosnick et al., 1997) and modi®ed RNase A (Qi et al., 1998) refolding reactions re¯ect a solvent-dependent change of the unfolded polypeptide rather than the formation of distinct partially folded forms. Although protein reorganization due to altered solvent conditions could result in a concomitant change in

742

Hydrophobic Packing in an Early Folding Intermediate of DHFR

Figure 4. Three-dimensional diagram highlighting the percent burst phase amplitude for DHFR under strongly refolding conditions (Table 1). The burst phase intensities fall into two categories: mutants at positions I91, I94, and I155 (grey bars), which have diminished amplitudes relative to wild-type DHFR, and mutants at positions I2, I61, and L112, which have similar burst phase intensities to wild-type protein (black bars). These results correlate with the pattern of protection seen for the pulse-quench hydrogen exchange NMR experiments on DHFR (Jones & Matthews, 1995); the positions that are protected in the early kinetic intermediate (13 ms) were observed to be sensitive to mutational replacement in their burst phase amplitudes. These results suggest that hydrophobic and steric interactions of speci®c residues in the core of DHFR are contributing to the formation and stability of this early kinetic intermediate.

signal intensity, the principal cause of the burst phase behavior observed in the case of DHFR appears to be the formation of a partially folded intermediate. The CD spectrum of the burst phase species (Kuwajima et al., 1991), the protection pattern displayed in pulse-quench NMR experiments (Jones & Matthews, 1995), and the sensitivity of the signal intensity to mutational replacement observed in this study, all demonstrate that the burst phase intermediate in DHFR is structurally more developed than simply a compact ``unfolded'' form. Moreover, the correspondence of the amide protection pattern (Jones & Matthews, 1995) with the changes observed here in the burst phase amplitudes is consistent with, but does not prove, that this early kinetic species is a productive, on-pathway intermediate in the folding mechanism. The development of progressive hydrophobic clusters may lead to complex kinetics The speci®c intramolecular recognition observed for DHFR within the submillisecond time range is consistent with the fast folding of small proteins via a two-state mechanism (Huang & Oas, 1995;

Schindler et al., 1995). In these systems, the sidechains achieve a native-like packing environment within several milliseconds. Larger proteins like DHFR may fold by a similar but more complex process, in which packing interactions participate in the development of microdomains at early stages of the reaction. The evidence presented here, along with several other protein models, including cytochrome c (Shastry & Roder, 1998), and Cd2.d1 (Lorch et al., 1999), support this hypothesis. An early folding core has also been suggested for several other proteins, including barnase (Fersht, 1993), apomyoglobin (Gilmanshin et al., 1998), and CheY (Lopez-Hernandez & Serrano, 1996). In particular, the heterogeneous development of partially folded domains has been postulated in the folding mechanisms of apomyoglobin (Fink et al., 1998), hen lysozyme (Matagne & Dobson, 1998), and phosphoglycerate kinase (Parker et al., 1996). In these systems, considerable structure is developed within distinct subdomains that fold more rapidly than other elements of the polypeptide. The ratelimiting steps arise from docking of marginally stable microdomains as the proteins attain the native fold. Thus, the non-uniform acquisition of structure in partially folded forms observed for DHFR appears to be a common feature observed in complex protein folding reactions. Ultrafast folding experiments are required to ascertain precisely when stereochemically sensitive packing is achieved in DHFR. If this packing occurs in the same time range as the microsecond collapse reaction observed for cytochrome c (Shastry & Roder, 1998), it is possible that the collapse is not random. Rather, it may be guided by the formation and association of simple elements of secondary structure through stereo-speci®c sidechain interactions (Ballew et al., 1996; Chan et al., 1997; Gilmanshin et al., 1998; Sauder & Roder, 1998).

Acknowledgments The authors express their appreciation to Dr Osman Bilsel for his assistance in data ®tting and analyses. The authors also thank Drs Jill A. Zitzewitz, Virginia Smith, and Roxana Ionescu for their critical reviews and comments. This work was supported by NSF grant MCB 9604678 (to C.R.M.).

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Edited by P. E. Wright (Received 16 September 1999; received in revised form 16 November 1999; accepted 16 November 1999)