Histidine interaction in α-helices 1

J. Mol. Biol. (1997) 267, 184±197

The Tryptophan/Histidine Interaction in a -Helices Juan FernaÂndez-Recio1, Ana VaÂzquez2, ConcepcioÂn Civera2, Paz Sevilla2 and Javier Sancho1* 1

Departamento de BioquõÂmica y BiologõÂa Molecular y Celular Facultad de Ciencias Universidad de Zaragoza Zaragoza 50009, Spain 2

Departamento de QuõÂmica FõÂsica II, Facultad de Farmacia Universidad Complutense de Madrid, Madrid 28040, Spain

Contacts between aromatic and charged residues are often found in proteins. Mutational studies have shown that a tryptophan/charged histidine pair can stabilise a protein by more than 1 kcal molÿ1. We have used circular dichroism and nuclear magnetic resonance to study the helical content of several peptides in which a tryptophan/histidine pair was placed at i, i ‡ 3 or i, i ‡ 4 in either the N to C or the C to N orientation. Our data indicate that the WH i, i ‡ 4 geometry is the most stabilising one (when the histidine is protonated) and gives rise to the highest helical content. Little preference is observed among the three other orientations. The energy of the WH‡ i, i ‡ 4 interaction (calculated with AGADIR and SCINT) is about 1 kcal molÿ1. A statistical analysis of the occurrence of tryptophan/histidine pairs in a-helices of natural proteins reveals that, although the WH i, i ‡ 4 pairs are not more abundant than the others, in most of the WH i, i ‡ 4 pairs the two side-chains are in contact, but not in the other three orientations. These results suggest that the conformational stability of proteins could be increased by means of solvent-exposed intrahelical i, i ‡ 4 tryptophan-histidine bridges and that these bridges could be useful to tailor the stability of helical peptides at physiological pH. # 1997 Academic Press Limited

*Corresponding author

Keywords: protein stability; a-helix stability; protein folding; protein design; aromatic/charged residue interactions

Introduction Both native and denatured proteins form in solution numerous interactions involving protein and solvent atoms. From the balance of these interactions emerges a native conformation that is only marginally more stable than the corresponding unfolded conformation. The accurate prediction of protein stability from protein structure is an important goal yet to be achieved. Towards this end, much effort has to be devoted to a quantitative understanding of the interactions that stabilise the native conformations of proteins. The protein folding problem, i.e. prediction of the structure from the sequence, is even more challenging. There is, however, a stage of the folding reaction, the beginning, which is more amenable to prediction. This is so because the earliest folding Abbreviations used: SD, standard deviation; TFE, tri¯uoroethanol; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root-mean-square deviation; COSY, correlated spectroscopy; HOHAHA, homonuclear-Hartmann-Hahn; ROESY, rotating frame Overhauser enhancement spectroscopy. 0022±2836/97/110184±14 $25.00/0/mb960831

stages are likely to consist of the formation of regions of secondary structure, the stability of which, especially in the case of a-helices, will be dominated by local interactions. Algorithms predicting early-folding regions are available (Moult & Unger, 1991; Rooman et al., 1992) and methods have been developed that allow, for a given sequence, prediction of the a-helices that would be populated in the absence of tertiary interactions (Scholtz et al., 1993; MunÄoz & Serrano, 1994). Since one of the factors contributing to the stability of a-helices is the formation of favourable interactions between sidechains located in consecutive turns of the helix, it is desirable to identify and quantify these interactions and to determine for each type of interaction its preferred geometry. Knowledge gathered through these types of studies will be useful in the de novo design of proteins and peptides and in engineering the stability of naturally occurring proteins. Contacts between aromatic and positively charged residues are often found in proteins (Singh & Thornton, 1992; Dougherty, 1996). The electronic distribution in an aromatic ring (Hunter & Sanders, 1990) favours a neighbouring positively charged side-chain to be frequently located on top # 1997 Academic Press Limited

The Tryptophan/Histidine Interaction in a-Helices

185

of the ring, as has been observed in proteins (Singh & Thornton, 1992). The contribution of aromatic/ charged residue interactions to protein stability was studied in barnase for the case of a histidine and the three aromatic residues (Loewenthal et al., 1992). Protonation of the histidine residue strengthens its interaction with a neighbouring tryptophan and increases the stability of the protein by 1 kcal molÿ1. Weaker but signi®cant interactions were observed when the tryptophan residue was replaced by either a tyrosine or a phenylalanine. A stabilising phenylalanine/histidine i, i ‡ 4 interaction has been described in model peptides (Armstrong et al., 1993). Here we study the in¯uence of the tryptophan/histidine interaction on the stability of isolated a-helices. After exploring the four possible relative orientations of the two residues at an i, i ‡ 3 or i, i ‡ 4 spacing, we have found that WH i, i ‡ 4 is the only geometry where histidine residue protonation stabilises the helical conformation. Analysis of a database (Hobohm & Sander, 1994) of 285 proteins with low sequence homology indicates that of all the tryptophan/histidine pairs found in a-helices only those with the WH i, i ‡ 4 orientation have close contact between their side-chains.

Results Design of peptides The peptide series WH3, WH4, HW3 and HW4 (Table 1) are alanine-based peptides. In a polyalanine framework we have introduced a few groups that make the peptides water soluble (R3, E6 and R12). The peptide amino and carboxy ends were acetylated and amidated, respectively, in order to avoid destabilising interactions with the helix dipoles. The three N-terminal and C-terminal amino acid residues of each peptide are identical so that capping effects are kept constant through the series. In each peptide, either a histidine or a tryptophan residue is placed at position 5 and the other residue of the pair at either position 8 or 9. All four peptides have the same amino acid composition and they will be referred to as the WH series. Two control peptides have also been designed and studied: WAH4 aims to exclude a possible helixstabilising interaction between E6 and H9 as the source of the helix stabilisation effect observed in WH4 at acidic pH (see below); WAH3, together Table 1. Peptide sequences and notation Sequence acetyl-AARAWEAHAARA-amide acetyl-AARAWEAAHARA-amide acetyl-AARAHEAWAARA-amide acetyl-AARAHEAAWARA-amide acetyl-AARAAWAAHARA-amide acetyl-AARAWAAAHARA-amide acetyl-AARWAEAAHARA-amide

Figure 1. Mean residue ellipticity (220 nm) of the WH peptides as a function of pH. (*) WH4, (*) WH3, (&) HW4, (}) HW3. (a) 2 C; (b) 25 C.

with WAH4, permit comparison of WH (i, i ‡ 3) and (i, i ‡ 4) spacing while keeping the histidine residue equally close to the carboxy end of the helix (see Discussion). We shall refer to these two control peptides as the WAH series. In addition, a peptide with a WH (i, i ‡ 5) spacing (termed WH5), where the interaction between the tryptophan and the histidine is not possible in a helical conformation, has been studied.

Notation WH3 WH4 HW3 HW4 WAH3 WAH4 WH5

pH and temperature dependence of the helical content in the WH and in the WAH series The ellipticity of peptides HW3, HW4, WH3 and WH4 as a function of pH is shown in Figure 1. At pH levels higher than 8.0, the peptides exhibit similar ellipticity values. Around pH 7.0 there is an in-

186

Figure 2. Circular dichroism spectra of WH4 at pH 5.0 (*) and 9.5 (*) and WH3 at pH 5.0 ( & ) and 9.5 (&). Temperature was 2 C.

¯ection in the helical content that separates the peptides into two groups: the helical contents of HW3, HW4 and WH3 decrease while WH4 becomes more helical. The pH dependencies of peptide ellipticities at 2 C (Figure 1(a)) are very similar to those at 25 C (Figure 1(b)) but, as expected, the helical contents are higher at 2 C. For example, the calculated helical content (Chen et al., 1974) of WH4 at pH 5.0 is 25% at 2 C, but only 15% at 25 C. The CD spectra of WH4 and WH3 at pH 5.0 and 9.5 are compared in Figure 2. The ellipticity of the control peptides WAH3 and WAH4 as a function of pH is shown in Figure 3. The behaviour of WAH4 is similar to that of WH4: its helical content sharply increases from pH 8.0 to 6.0. The helical content of the second control peptide, WAH3, is similar to that of WAH4 at pH levels higher than 8.0 but it is much lower below pH 6.0. A small increase in the ellipticity of WAH3 is observed from pH 8.0 to 6.0. Since the in¯ections in the helical content observed around pH 7.0 (Figures 1 and 3) very probably re¯ect the protonation of the histidine residues in the peptides, the apparent pKa of the histidine residues can be calculated by ®tting the ellipticity data (in the pH 5.0 to 9.0 internal) to the simple equation for an ionisation equilibrium. We obtain the number of protons involved to be 1.0(0.1) for the six peptides analysed. The calculated apparent pKa values are shown in Table 4. The histidine residues of the WH series have higher pKa values than those of the control peptides. This was expected because the control peptides lack the glutamic acid residue present in the others. Importantly, within each series, the histidine in peptides with the WH4 spacing (peptides WH4 and WAH4) display higher apparent pKa values than the histidine residues in the other peptides. This indicates that, in WH4 and

The Tryptophan/Histidine Interaction in a-Helices

Figure 3. Mean residue ellipticity (220 nm) of the WAH peptides as a function of pH. (*) WAH4, (*) WAH3. Temperature was 2 C.

WAH4, the protonated form of the histidine is stabilised relative to the neutral form (Loewenthal et al., 1992; Sancho et al., 1992) to a greater extent than in the other peptides. These apparent pKa values represent, for each peptide, an average of the pKa of the histidine residues in helical and disordered conformations (Huyghues-Despointes et al., 1993). Since, in our case, a signi®cant percentage of peptide molecules are in disordered conformations the calculated apparent pKa values may signi®cantly differ from the microscopic pKa values of the helical histidine residues. Calculation of the interaction energies of Trp/His pairs using AGADIR Helical peptides in solution consist of an ensemble of helices of different lengths together with more disordered conformations. Because of this, the simple two-state model does not describe well the conformational equilibrium. More realistic descriptions are provided by the classical Zimm-Bragg (Zimm & Bragg, 1959) and Lifson-Roig (Lifson & Roig, 1961) theories. The calculation of intrahelical side-chain/side-chain interaction energies from comparison of the helical contents of peptides where the interaction is present, and peptides without the interaction, requires the use of a helix/coil theory which includes side-chain interactions. Fortunately, two such theories have been recently developed and implemented in the computer programs AGADIR (MunÄoz & Serrano, 1994) and SCINT (Stapley et al., 1995). We have used an improved version of AGADIR, AGADIR1s (MunÄoz & Serrano, 1996) to calculate the interaction of the Trp/His pairs as follows. First the helical content of each peptide was calculated with AGADIR1s. Then the default value of the Trp/His interaction used in the program was

187

The Tryptophan/Histidine Interaction in a-Helices Table 2. Energies of Trp/His interactions calculated by AGADIR

Peptide WH4 WH3 HW4 HW3 WAH4 WAH3

% Helixa pH 5.0 pH 9.5 25 7 6 8 32 15

14 13 10 13 17 13


Gb (kcal molÿ1) pH 5.0 pH 9.5 ÿ0.8(0.1) ÿ0.2(0.5) 0.2(1.5) ÿ0.1(0.5) ÿ0.8(0.1) 0.0(0.3)

ÿ0.2(0.2) ÿ0.2(0.2) ÿ0.1(0.2)c ÿ0.3(0.2) ÿ0.1(0.2)c ±

a

Determined by the method of Chen et al. (1974). When AGADIR cannot predict the observed helical content no energy value is reported. c AGADIR cannot predict a helical content three percentage units lower than the measured one. In these cases the error is simply the difference between the energy value that predicts the observed helical content and the value that predicts three percentage units higher. b

changed until the observed helical content was predicted. Since accurate determination of helical content by circular dichroism is particularly dif®cult for peptides where aromatic residues are present (Chakrabartty et al., 1993), we have assumed for our calculations with AGADIR1s that the percentage of helix that we measure by circular dichroism may be three percentage units higher or lower. This means that for a peptide with a measured helical content of 20% we modify the value of the interaction energy implemented in AGADIR1s until we ®nd interaction energies that lead to 17%, 20% and 23% helical content. In this example, the interaction energy value that predicts a 20% helical content would be taken as the output of the calculation; the error would be calculated as the biggest of the differences between the output and the value of interaction energy that predicts either 17% or 23% helical content for the peptide. This method yields, for many of the interactions analysed, big errors, which indicates that in those cases the uncertainty in peptide concentration is too big to allow a precise determination of the interaction energy. A similar method has been used by Viguera & Serrano (1995) to calculate errors, and similar problems have been encountered. The WH (i, i ‡ 4) interaction is fortunately calculated with acceptable accuracy (Table 2). At low pH, where the histidine is protonated, this interaction is worth ÿ0.8(0.1) kcal molÿ1 regardless of peptide (WH4 or WAH4) while at high pH the interaction energy is close to zero. The three other orientations show small interaction energies at low and high pH although in these cases the errors are big. We have compared the measured helical contents of peptide WH5, where no interaction between the tryptophan and histidine residues can take place in an a-helix, with the contents predicted by AGADIR1s. The helical content of WH5 changes very little with pH (not shown). The observed helical content is 10% (at both pH 5.0 and 9.5) and AGADIR1s calculates 12% (at either pH). The differences are thus within the errors estimated for the

Figure 4. Calculation of the pKa of helical histidine in WH4 using AGADIR. Measured ellipticities (*); calculated ellipticities using ÿ0.74 kcal molÿ1 as the value of the WH‡ interaction and 7.0 as the pKa of helical histidine (*); the continuous lines are ®ts to the equation of a single ionisation equilibrium. The broken line is an equivalent ®t of the calculated ellipticities of WH4 using ÿ0.8 kcal molÿ1 as the value of the WH‡ interaction and the default value of 6.5 for the histidine pKa.

determination of the helical content from CD measurements. The calculations of histidine interactions in AGADIR are performed assuming a default pKa of 6.5 for the helical histidine (plus a correction to account for helix dipole effects; MunÄoz & Serrano, 1995). This causes the predicted helical content as a function of pH to be shifted with respect to the observed one (Figure 4). The values of the interaction energies calculated by AGADIR and the histidine pKa used in the calculations can be re®ned as follows. First, the calculated value of the interaction energy is introduced in AGADIR and the default value of the histidine pKa is modi®ed to optimise the agreement between the predicted helical content as a function of pH and the observed one. Then the new pKa value is used to recalculate the interaction energy. We have obtained a good prediction of the helical content of WH4 as a function of pH using a pKa of 7.0 and an interaction energy of ÿ0.74 kcal molÿ1 (Figure 4). The ®tting is however poorer at high pH. We suggest that this could be related to the fact that in AGADIR the values of the interaction energies of histidine at high pH are set at 40% of the value at low pH but the real value of this particular Trp/His interaction at high pH could be lower (Table 2). Calculation of the interaction energies of Trp/His pairs using SCINT The interaction energies of the Trp/His pairs have also been calculated using SCINT. The value of the

188

The Tryptophan/Histidine Interaction in a-Helices

Table 3. Energies of Trp/His interactions calculated by SCINT

Peptide WH4 WH3 HW4 HW3 WAH4 WAH3


Gb (kcal molÿ1) pH 5.0 pH 9.5

% Helixa pH 5.0 pH 9.5 25 7 6 8 32 15

14 13 10 13 17 13

0.1(0.5)c 0.3(0.8) ± 0.2(0.7) ± ±

ÿ1.5(0.2) ± ± ± ÿ1.0(0.2) ±

a Determined by the method of Chen et al. (1974). These helical contents have been multiplied by 1.1 before performing calculations with SCINT because this program predicts helical content at 0 C and the helical content of the peptides was determined at 2 C. The 1.1 factor derives from the ratio of helical content predicted by AGADIR at 0 C and at 2 C for these peptides. b When SCINT cannot predict the observed helical content (after correction, seea) no energy value is reported. c SCINT cannot predict a helical content three percentage units lower than the measured one. In this case the error is simply the difference between the energy value that predicts the observed helical content and the value that predicts three percentage units higher.

interaction energies and the errors have been calculated following the method described above for the calculations with AGADIR1s. Most interaction energies have big errors but the Trp/His (i, i ‡ 4) interaction is also calculated with acceptable accuracy (Table 3). At low pH this interaction is calculated to be ÿ1.5(0.2) kcal molÿ1 in WH4 and 1.0(0.2) in WAH4. We take 1.2(0.4) kcal molÿ1 (mean of the two values  standard deviation (SD)) as the value of this interaction calculated by SCINT. At high pH the calculated interaction energy in WH4 is small as with AGADIR1s but the error is bigger. For some of the peptides no value of the interaction energy is given because SCINT cannot calculate the rather low observed helical contents. For WH5 SCINT predicts 11.5% helix at pH 5.0 (10% measured, 11% after temperature correction: see Table 3) and 14% at pH 9.5 (10% measured, 11% after correction). The differences between the observed helical contents of this pep-

tide and those calculated by SCINT are similar to the differences found with AGADIR. However for some of the other peptides the helical contents predicted by SCINT, before introduction of energy values for side-chain interactions, diverge from the measured helical contents more than the values predicted by AGADIR (not shown). These initial bigger divergences may explain why SCINT does not reproduce the observed helical contents of some of the peptides. Calculation of the interaction energy of the Trp/His i, i ‡ 4 pair assuming a two-state model The two-state model is usually appropriate to describe the folding equilibrium of proteins but has been reported to underestimate the value of intrahelical side-chain interactions in peptides (Shalongo & Stellwagen, 1995). However, there may be peptides where this approximation may yield fair values of the interaction energy. The calculations using a two-state model are as follows. First, the helical content of each peptide is calculated by the method of Chen et al. (1974) at several pH values. From these helical contents, apparent stability constants (K ˆ % helix/(100 ÿ % helix) for the helix/coil equilibria are derived. The fact that the protonation of the histidine residue alters the helical content of the peptides demonstrates that the equilibrium of protonation and the equilibrium of helix formation are linked. Equation 1 (Fersht, 1985; Kippen et al., 1994) shows the relationship between K, the ionisation constant for the helical histidine residues (Kahelix) and the equilibrium constants for the helix/coil transitions of protonated (KHis) and unprotonated (KHis‡) peptides: K ˆ ……‰H‡ Š ‡ Ka helix †KHis KHis ‡ †= …Ka helix KHis ‡ ‡ ‰H‡ ŠKHis †

…1†

Peptide helical contents as a function of pH have been ®tted to equation (1) and the stability constants of the protonated (KHis‡) and unprotonated

Table 4. Energies of Trp/His (i, i ‡ 4) interaction calculated by the two-state method

Peptide

(His‡)


Gh/ca (kcal molÿ1)

l

(His)



Gh/cb (kcal molÿ1)



Gh/c rel (kcal molÿ1)

pKa appc

WH4 WH3 HW4 HW3

0.65(0.10) 1.55(0.46) 1.72(0.79) 1.50(0.39)

1.06 (0.17) 1.17(0.20) 1.34(0.28) 1.15(0.19)

ÿ0.41(0.07) 0.38(0.26) 0.38(0.51) 0.35(0.20)

ÿ0.8(0.3)d 0.0d 0.0(0.6)d 0.0(0.3)d

7.01(0.03) 6.71(0.07) 6.88(0.07) 6.82(0.05)

WAH4 WAH3

0.44(0.08) 1.03(0.15)

0.92(0.13) 1.14(0.19)

ÿ0.48(0.05) ÿ0.11(0.04)

ÿ0.4(0.1)e 0.0e

6.81(0.04) 6.62(0.07)

The Table shows the apparent pKa values of the histidine residues in WH and WAH peptides. a Derived from the ®tted values of the equilibrium constants in equation (1). b Difference between the stability of the helix/coil equilibria with protonated histidine and those with neutral histidine using data in a. c Apparent pKa of the histidine residues calculated by direct ®tting of ellipticity versus pH data to the equation of a simple ionisation equilibrium. d Difference stabilities in b minus the difference stability of WH3. e Difference stabilities in b minus the difference stability of WAH3.

189

The Tryptophan/Histidine Interaction in a-Helices

histidine on protonation. Subtraction of this value from the difference energy found for WH4 (where the protonated histidine is in contact with the tryptophan, see below) yields the value of the interaction energy between the protonated histidine and the tryptophan in a WH i, i ‡ 4 orientation. This value turns out to be ÿ0.8(0.3) kcal molÿ1. A similar analysis of the WAH4 peptide using WAH3 as a reference peptide yields a value of ÿ0.4(0.1) kcal molÿ1 for the Trp/His‡ interaction in WAH4. We take 0.6(0.3) kcal molÿ1 (mean of the two values  SD) as the value of this interaction calculated by the two-state method. Trifluoroethanol titrations Peptide helical contents of the WH series have been determined, at pH 5.0 and pH 9.5, as a function of tri¯uoroethanol (TFE) concentration (Figure 5). The helical content of WH4 in water at acidic pH is higher than those of any of the other peptides at either acidic or basic pH, but the maximal helical contents obtained at high TFE concentration are similar in all of them and they do not change much with pH. Fluorescence spectra

Figure 5. Mean residue ellipticity (220 nm) of the WH peptides as a function of TFE concentration. (*) WH4, (*) WH3, (&) HW4, (}) HW3. Temperature was 2 C. (a) pH 5.0; (b) pH 9.5.

(KHis) helices have been calculated. From these equilibrium constants free energies of the helix/ coil equilibria are derived and are shown in Table 4. Errors are calculated as in previous sections allowing the measured ellipticity values to be three percentage units lower or higher than the observed ones. The data in Table 4 indicate that the differences in helix stability on protonation in peptides WH3, HW4 and HW3 are similar (0.35 to 0.38 kcal molÿ1). Our NMR data suggest (see below) that in these peptides there is no contact between the histidine and the tryptophan. The common difference in helix stability should thus represent the combined effect of the interaction of the charged histidine with the other residues in the peptide, and the change in helical propensity of

Comparison of the ¯uorescence emission spectra of WH4 and WH3 (Figure 6) at pH 5.0 and 9.5 shows quenching of the tryptophan residues at acidic pH relative to pH 9.5. The quenching is more pronounced in the case of WH4 than in WH3. In contrast, the intensity of the ¯uorescence emission spectra of N-acetyl tryptophan amide (an analogue of tryptophan residues) is slightly higher at pH 5.0 than at pH 9.5 under identical conditions (not shown). This indicates that the observed quenching of peptide ¯uorescence at acidic pH is not a solvent effect. Protonated histidine residues are known to exert a strong quenching effect on neighbouring tryptophan residues (Shinitzky & Goldman, 1967; Loewenthal et al., 1991). The fact that the quenching of the tryptophan residue in WH4 is stronger than in WH3 suggests that the side-chains of the histidine and tryptophan residues are in closer contact in WH4 than in the other peptide. The ¯uorescence spectra of HW4 and HW3 are similar to those of WH3 (not shown). Nuclear magnetic resonance of the WH series The structure of WH4 in solution has been studied by NMR at several temperatures, pH values and water/TFE ratios. Data in 100% H2O, at pH 4.3 and 5 C (Figure 7(a)) include three weak ai ! bi ‡ 3 NOEs (2 ! 5, 3 ! 6 and 5 ! 8) that are indicative of a helical conformation. This is further con®rmed by the markedly negative
da values observed from R3 to H9 (Figure 8(a)). No side-chain/sidechain cross-peaks are observed in the spectra. From the chemical shifts of the Ha (see Materials

190

The Tryptophan/Histidine Interaction in a-Helices

Figure 6. Fluorescence emission spectra of WH4 (*) and WH3 (*) at 2 C. Continuous lines, pH 9.0; broken lines, pH 5.0.

and Methods) we calculate that the helical content of WH4 at pH 4.3 and 5 C is about 28%. Data in 15% (v/v) TFE, at the same pH and temperature as above (Figure 7(b)), contain seven ai ! Ni ‡ 3 or ai ! bi ‡ 3 NOEs. These helical NOEs, spanning from R3 to R11, are stronger than those found in water. Markedly negative
da values are observed from R3 to H9 (Figure 8(b)). Importantly, a crosspeak connects the tryptophan and the histidine rings (not shown); excluding the alanine residues, this is the only side-chain/side-chain cross-peak observed under these conditions. The calculated helical content in 15% TFE is 37 %. Data in 30%

Figure 8. Conformational shifts of the CaH in WH4 with respect to the random coil values. (a) 0% TFE, (b) 15% TFE, (c) 30% TFE. Temperature was 5 C and the pH was 4.3.

Figure 7. Sequential and medium range NOEs in WH4. (a) H2O, (b) 15% (v/v) TFE, (c) 30% TFE. Temperature was 5 C and the pH was 4.3.

TFE at the same pH and temperature as above (Figure 7(c)), contain ten strong ai ! Ni ‡ 3 or ai ! bi ‡ 3 NOEs from A2 to R11 and markedly negative
da values are observed throughout the peptide (Figure 8(c)). The calculated helical content in 30% TFE is 53%, which agrees well with the ellipticity observed at the same TFE concentration (Figure 5). Chemical shift data for the histidine b protons are shown in Table 5. The value of
db for one of the b protons in WH4 is quite negative (and it becomes more negative as the concentration of TFE is raised) suggesting the proximity of the tryptophan aromatic ring. The other b proton has chemical shifts similar to those assigned to random coil. Negative
db values were described for the b

191

The Tryptophan/Histidine Interaction in a-Helices Table 5. Chemical shift differences Peptide WH4 WH4 WH4 WH3 WH3 WH3

% TFE

b1

30 15 0 30 15 0

ÿ0.07 ÿ0.08 ÿ0.07 ÿ0.02 ÿ0.04 ÿ0.02


db

b2 ÿ0.61 ÿ0.55 ÿ0.46 ÿ0.02 ÿ0.00 ÿ0.02

The Table shows differences between the chemical shifts at pH 4.3 (5 C) of the assigned proton resonances for the Cb protons of the histidine residues in WH4 and WH3 and those proposed as random coil parameters in polypeptides.

protons of the histidine residue in the S-peptide from ribonuclease (Gallego et al., 1983). This histidine is located at i ‡ 4 from a phenylalanine residue and the negative
db values were attributed to the proximity of the phenylalanine ring to the histidine. The WH4 proton chemical shift data (not shown) were used to assign nuclear overhauser enhancement spectroscopy (NOESY) cross-peaks. According to their volume intensity, the peaks were classi®ed as weak, medium, or strong, and the corÊ, responding upper distance constraints were 3.0 A Ê and 5.0 A Ê . Identi®cation of unambiguous 4.0 A NOESY peak correlations provided a set of 79 distance restraints; only seven of them were non-relevant for structure calculation. As many as 35 restraints involved Trp or His protons. Of these restraints, there were six involving protons of the tryptophan ring and ®ve involving protons of the histidine ring. We identi®ed a strong cross-peak between the tryptophan and histidine rings that ®x well the relative position of the rings. Pseudoatom corrections were used to de®ne distances when protons in methyl and methylene groups were involved. By use of a variable target function algorithm, as implemented in the DIANA program (GuÈntert et al., 1991), 200 random starting structures yielded a total of 35 convergent ones. The maximum violation for the upper distance limit Ê and for van der Waals radii 0.14 A Ê . The was 0.44 A mean root-mean-square deviation (RMSD) of the ten structures with the lowest target function was Ê for the backbone, or 1.41(0.25)A Ê if 0.62(0.18) A all heavy atoms are included. A plot of the global backbone RMSD per residue is shown in Figure 9. Arginine and glutamate side-chains are poorly de®ned while the tryptophan and histidine sidechains are very well de®ned, as expected from the high number of restraints derived from the NOESY spectrum. Figure 10 shows a plot of the best ®t for the ten ®nal structures. The structure in solution of WH4 has also been studied at pH 8.0. Only in the presence of 30% TFE have the spin systems been identi®ed (except for some of the alanine residues). We detect ®ve ai ! bi ‡ 3 NOEs spanning from A2 to A10 and ®nd low
da values from R3 to H9. We do not see however any side-chain/side-chain cross-peak, which suggest that all side-chains, in-

Figure 9. RMSD per residue of the side-chains in the calculated structures of WH4 at 5 C, pH 4.3 and 30% TFE.

cluding the tryptophan and the histidine, are quite mobile at pH 8.0. The WH3 peptide has also been extensively studied by NMR. This peptide has been chosen as a representative of the peptides of the WH series that do not increase their helical content at acidic pH (as observed by CD) because the sequence of WH3 is closer to that of WH4 than are those of HW4 or HW3. Spectra of WH3 at 5 C have been obtained in water, 15% TFE and 30% TFE at either pH 4.3 or pH 8.0. In no case has a sequential assignment been possible, because of extensive overlapping of the alanine resonances. Only the W5 to H8 region has been assigned because an ai ! Ni ‡ 1 NOE between A7 and H8 was detected in the spec-

Figure 10. A family of the best ten structures of WH4 at 5 C, pH 4.3 and 30% TFE as calculated with the program DIANA. The only side-chains shown are those of W5 and H9.

192

The Tryptophan/Histidine Interaction in a-Helices

Table 6. Statistical analysis of W/H contacts in a-helices of proteins

Sequence W***H W**H H***W H**W

Helical sequences founda

Helical sequences expectedb

Relative occurrencec

sc-sc WH contactsd

Contact probabilitye

Atomic WH contactsf

6 4 8 7

3.8 4.4 3.8 4.4

1.6 0.9 2.1 1.6

5 1 1 0

0.83 0.25 0.13 0.00

36 4 1 ±

a

Number of sequences found in a-helices of the database. Number of sequences expected according to the number of tryptophan and histidine residues located in a-helices of the database. c Helical sequences found divided by helical sequences expected. d Ê from an atom of Number of sequences with WH side-chain/side-chain contacts (at least one atom of a partner at less than 2.5 A the other partner). e Helical sequences with a WH side-chain/side-chain contact divided by all helical sequences found. f Number of contacts between W and H side-chain atoms observed in the helical database. b

tra. The
da values are low for the Trp, the Glu and the Ala. No side-chain/side-chain cross-peaks are detected under any of the conditions studied. Unlike in WH4, the
db values for the b protons of the histidine in WH3 are similar to those assigned to random coil suggesting that the tryptophan is not close to this histidine. The HW3 peptide has also been studied in water at acidic pH. Similarly to WH3, no peak characteristic of a helix appears in the spectra and only unique spin systems can be identi®ed because of the overlapping of several alanine residues.

Occurrence of i, i ‡ 3 and i, i ‡ 4 tryptophan/ histidine pairs in helical regions of proteins We have searched a database of 285 proteins with low sequence homology to one another (see Materials and Methods). The database contains examples of the four possible WH pairs (Table 6). There are four cases of the less common pair (WH; i, i ‡ 3) and eight cases of the most common one (HW; i, i ‡ 3). The relative occurrence of the pairs (number of pairs found divided by number expected in a random distribution) varies from roughly one to two and we do not observe any correlation between the relative occurrence and the helix-stabilising character of the pairs as calculated from circular dichroism data. Ê ) between side-chain Analysis of contacts (<2.5 A atoms of the two residues of the pair shows a clear cut difference between the number of contacts found for WH (i, i ‡ 4) pairs and for the other pairs. In ®ve out of six WH (i, i ‡ 4) pairs, the sidechains of the two residues are in contact (Table 6 and Figure 11(a)). For the three other pair types (19 pairs in total) there are only two pairs in contact (Table 6 and Figure 11(b)). Besides, the WH (i, i ‡ 4) contacts seem to be closer than the other contacts as judged from the number of atoms involved (Table 6). From our data we calculate that the probability that a helical tryptophan residue forms a contact with a helical histidine residue located at i ‡ 4 is higher than 0.8 while for the three other orientations it varies from 0.0 to 0.2.

The rotamers involved in WH (i, i ‡ 4) contacts are well de®ned. The ®ve tryptophan side-chains adopt the same trans conformation (w1 ˆ ÿ174(5)) with w2ˆ 90(6). Three of the contacting histidine side-chains are also trans (w1 ˆ ÿ179(3); w2 ˆ 78(7)) and the two other gauche‡ (w1 ˆ ÿ60(4); w2 ˆ81 or ÿ80). In the non-helical WH interaction in barnase (Loewenthal et al., 1992) the tryptophan and histidine rotamers are different: the tryptophan is gauche‡ (w1 ˆ ÿ50; w2 ˆ 100) and so is the histidine (w1 ˆ ÿ74; w2ˆ ÿ172). In the calculated structure of WH4 in 30% TFE the w1 and w2 angles do not de®ne common rotamers. We suggest this could be due to the lower resolution in the peptide calculated structure, compared to protein structures, or to conformational averaging in the peptide.

Discussion Preferred geometry of a WH bridge in helical peptides Inspection of Figures 1 and 3 shows that peptides WH4 and WAH4 exhibit a higher helical content at all pH values from pH 9.0 to 5.0. A distinction should, however, be made between the basic and the acidic pH intervals. In the basic region the ellipticities of all peptides are quite low even at 2 C, and the differences in helical content between peptides are small: for example, WH4 is only 20% more helical than HW3 or WH3, and WAH4 is only 15% more helical than WAH3. Around pH 7.0 a transition is observed in all cases (Figures 1 and 3) which is associated with the binding/release of one proton. This is most likely a consequence of the protonation of the histidine, since it occurs in the proximity of the pKa of histidine residues, which is around 6.5 (Sancho et al., 1992). In three peptides of the series, HW3, HW4 and WH3, protonation of the histidine decreases the helical content. The reason for this behaviour is not clear. A possible reason is the reported lower helical propensity of protonated histidine versus neutral histidine (Chakrabartty et al., 1994). If this were the case, peptide WAH3 should in principle behave

The Tryptophan/Histidine Interaction in a-Helices

193

Figure 11. Superposition of (a) WH4 and (b) WH3 pairs in nonhomologous proteins.

similarly, and it does not. Peptide WAH3, however, bears the histidine closer to the C terminus than HW3, HW4 and WH3, and on protonation the proximity of the histidine to the helix dipole could compensate its lower helical propensity. The lower helical content of HW3, HW4 and WH3 at low pH could also be due to a combination of other reasons such as a destabilising HR (i, i ‡ 3) interaction in WH3 (not present in WAH3) and the location of the histidine residue in the amino half of peptides HW3 and HW4. It is also possible that the protonated histidine is preferentially stabilised by the glutamic acid residue in the unfolded conformation as suggested by the fairly high apparent pKa values in these peptides of low helical content compared to the pKa of histidine residues in unfolded proteins and in protein fragments (6.5: Sancho et al., 1992; Kippen et al., 1994). The other peptide of the series (WH4) behaves quite differently on protonation since its helical content doubles from pH 8.0 to pH 6.0 becoming, at low pH, three to four times more helical than the other peptides. Comparison of the four peptides suggests that, on protonation, a helical histidine residue located at i ‡ 4 from a tryptophan residue is able to interact with the indole ring so stabilising the helical conformation. But before we can attribute the observed increase in helical content at acidic pH to a favourable interaction between the tryptophan and the protonated histidine at i ‡ 4 other possible reasons should be ruled out. For example, WH4 could be more stable at acidic pH than the other peptides because a salt bridge is formed between H9 and E6 on protonation. However, the drop in helical content below pH 4.0 due to protonation of the glutamate is small (Figure 12). Moreover, in the peptide WAH4 where the glutamate was replaced by an alanine residue, the increase in helical content from pH 9.5 to pH 5.0 is similar to that in WH4 (Figure 12). This rules out signi®cant helix stabilisation on histidine protonation due to an

interaction between the histidine and the glutamate. A second reason that could, in principle, explain why peptide WH4 is more stable than the other peptides of the WH series is that in WH4 the histidine residue is closer to the C terminus of the helix than any of the histidine residues in the other peptides. Because of this, protonation of H9 in WH4 could stabilise the helix by interacting with the helix dipole (Sancho et al., 1992). To assess this possibility, the pH dependence of the helical content of a second control peptide (WAH3) was compared to that of WAH4. These two peptides have the same amino acid composition and an identical location of the histidine residue; they differ only in the position of the tryptophan (Table 1). At basic pH the helical content of the two peptides is simi-

Figure 12. Comparison of the helical contents of WH4 (*) and WAH4 (*) from pH 9.5 to pH 2.0 at 2 C. The lines are ®ts to the equations of a double ionisation equilibrium (WH4) or a single ionisation equilibrium (WAH4).

194 lar (WAH4 is about 15% more helical) but as soon as the histidine residue becomes protonated WAH4 becomes more helical, and at acidic pH its helical content is more than twice that of WAH3. This indicates that the stabilising effect associated with the protonation of the histidine is not connected to its proximity to the end of the helix but is due to a favourable interaction with a tryptophan residue at i ÿ 4. Our NMR data fully support the CD evidence indicating that the WH4 geometry is the more favourable one for a tryptophan/histidine pair in an ahelix. We have compared the structure in solution of WH4 at low and high pH with the structure of WH3 under the same conditions. WH3 has been chosen as a reference because its sequence is the closest to that of WH4 among the peptides of the WH series. In water, at pH 4.3 there is a small helical population in WH4 that is not observed in WH3. As the TFE concentration is raised, the observed helix content of WH4 increases. At 15% TFE WH4 displays a helical region spanning amino acid residues 3 to 11 and a tryptophan/histidine cross-peak appears. At 30% TFE, WH4 seems to form a helix from A2 to R11 and the tryptophan/ histidine cross-peak remains as the only interaction between amino acid residues with a long sidechain. As for WH3, the dispersion of the peaks is so low even at 30% TFE that the sequence cannot be assigned. The resonances of the protons of the histidine and tryptophan side-chains are, however, well resolved and no cross-peak is observed between them. The CD spectra of WH4 and WH3 in 30% TFE show, however, a similar helical content. The poor dispersion of the WH3 spectra in 30% TFE and the lack of cross-peaks between the sidechains suggest that its helical structure may be an average of many ¯uctuating conformations while the structure of WH4 is better de®ned and contains a stable side-chain/side-chain interaction. From the NOEs observed in the spectra of WH4 in 30% TFE we have calculated the structure in solution of its helical conformation (Figure 10). Con®rming our predictions the tryptophan and the histidine sidechains are ®xed while all other long side-chains (the two arginine residues and the glutamate) are highly mobile (Figure 9). The effect of histidine protonation on the structure of WH4 is also clear from the NMR data. At pH 8.0 in water, and even in 15% TFE, there is little dispersion in the WH4 spectra. Only in 30% TFE are some NOEs found that are indicative of a helix, but no side-chain/ side-chain cross-peaks appear in the spectra. The effect of this tryptophan/histidine interaction has been observed in both the ¯uorescence of the tryptophan and the pKa of the histidine in WH4. A quenching effect of protonated histidine residues on the ¯uorescence of neighbouring tryptophan residues has been described (Loewenthal et al., 1992; Shinitzky & Goldman, 1967). We ®nd that the ¯uorescence emission of the tryptophan in WH4 is quenched at acidic pH to a higher extent than that of the other tryptophan residues (those in WH3,

The Tryptophan/Histidine Interaction in a-Helices

HW4 and HW3) suggesting that the proximity of the two rings is higher in the WH4 orientation. On the other hand, the interaction between the charged histidine and the tryptophan raises by 0.2 pH units the apparent pKa of the histidine residues in WH4 and WAH4 with respect to the pKa of the histidine residues in the corresponding peptides with the same amino acid composition. Energy of the WH (i, i ‡ 4) interaction The interaction energy of the Trp/His helical pair has been calculated using helix/coil theories that include side-chain interactions. Only in the case of the WH (i, i ‡ 4) orientation enough accuracy has been achieved as a consequence of the higher helical content of the corresponding peptides. For the three other orientations the calculated interaction energies are small in agreement with ¯uorescence, circular dichroism and nuclear magnetic resonance evidence that suggests that in those orientations the Trp/His bridge is not formed or is weak. The WH (i, i ‡ 4) interaction is stronger when the histidine is protonated as described for the FH (i, i ‡ 4) interaction in the C-peptide and in model peptides (Armstrong et al., 1993). Using AGADIR1s the WH‡ (i, i ‡ 4) interaction is calculated to be ÿ0.8(0.2) kcal molÿ1 and using SCINT the value is ÿ1.2(0.4) kcal molÿ1. The poorer two-state approximation yields a somewhat lower value of ÿ0.6(0.3) kcal molÿ1 (mean of WH4 and WAH4  SD). The values calculated by helix/coil theories are not far from the value found in barnase (Loewenthal et al., 1992) for a tertiary interaction between a charged histidine and a tryptophan (ÿ1.2 to ÿ1.4 kcal molÿ1). The interaction of a tryptophan with a protonated histidine in barnase was shown to be a particular case of a more general aromatic/charged histidine interaction since tyrosine and phenylalanine residues were also found to interact with a protonated histidine. We have found in the literature several reports on peptides whose helical content is increased on histidine protonation, the effect being attributed to interaction of the histidine with an aromatic residue, either a phenylalanine (Rico et al., 1986; Armstrong et al., 1993) or a tyrosine (Bradley et al., 1990). In all these cases the orientation is aromatic residue/charged histidine (i, i ‡ 4). WH bridges in protein helices We have analysed the occurrence of Trp/His sequences (at i, i ‡ 3 or i, i ‡ 4) in protein helices and we have not found any correlation between the stability of the interaction (as measured in our peptide models) and the frequency of the sequence in protein helices. Similar results have been reported for other interactions (Viguera & Serrano, 1995; Padmanabhan & Baldwin, 1994). As described for phenylalanine/cysteine and phenylalanine/methionine interactions (Viguera & Serrano, 1995), we observe a clear difference between the stabilizing

195

The Tryptophan/Histidine Interaction in a-Helices

orientation and the three other types. Most WH4 sequences (®ve out of six) form a bridge that brings the two side-chains into close contact. Hardly any of the WH3, HW4 and HW3 sequences (two out of 19) establishes a similar contact. This suggests that in protein helices only the WH4 orientation allows the formation of a stable interaction between the two side-chains. To test this point we have modelled the four types of WH bridges in polyalanine helices. For each orientation, all combinations of gauche‡ and trans rotamers of the two residues have been modelled. Only in the WH (i, i ‡ 4) orientation are the two side-chains in contact with favourable w2 angles (not shown). Our ®ndings with model peptides indicate that, in this orientation, protonated helical histidine residues can bind to tryptophan residues and form a stable interaction worth around 1 kcal molÿ1. These results suggest that engineering of WH4 bridges into solvent-exposed helices could be a simple strategy to increase the conformational stability of proteins. These bridges could also be useful in tailoring the stability of helical peptides in the physiological pH range.

Materials and Methods Peptide synthesis and quantification Peptides were synthesised either by the Protein & Peptide Service at the EMBL (WH3, WH4, HW3 and HW4) or by NEOSYSTEM S.A. (WAH3, WAH4 and WH5). Each peptide was >95% pure by reverse-phase HPLC. Peptide concentration was determined from the absorbance at 280 nm using for each peptide an extinction coef®cient of 5690 Mÿ1 cmÿ1 (Gill & von Hippel, 1989). Aliquots of peptide stock solutions were always dispensed with Hamilton syringes that had been calibrated spectrophotometrically using a concentrated solution of a dye. Test of peptide aggregation The mean residue ellipticity (220 nm) of the peptides at pH 5.0 and 2 C did not change in the 2 mM to 50 mM peptide concentration range (not shown). Circular dichroism Circular dichroism (CD) measurements were done in a thermostat ®tted Jasco 710 spectropolarimeter. The instrument calibration was routinely checked with (‡)-10camphorsulphonic acid (Chen & Yang, 1977). The ellipticity of each peptide at 220 nm was determined at several pH values. Water-jacketed circular cells with a 10 mm path length were used. The temperature was measured with a thermocouple immersed in the solution. Experiments were performed either at 25.0(0.1) C or at 2.0(0.1) C at a 9 mM peptide concentration. The buffer was in all cases 1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate, and 25 mM NaCl. The pH was adjusted as required with concentrated HCl or NaOH. Peptide samples were prepared for CD analysis by diluting the corresponding stock solutions with buffer of the appropriate pH. The pH of each sample was measured, with a Crison electrode calibrated according

to manufacturer's speci®cations, immediately after recording its ellipticity at 220 nm. All ellipticity data are reported as mean residue ellipticity [y] in degree cm2 dmolÿ1. Fluorescence Peptide ¯uorescence emission (excitation at 295 nm) was measured in a Kontron SFM 25 ¯uorimeter at 2 C. The concentration of peptide was 9 mM and the buffers used were sodium phosphate (pH 5.0; 25 mM ionic strength) and sodium borate (pH 9.0; 25 mM ionic strength). 1

H Nuclear magnetic resonance

Aqueous samples were prepared by dissolving lyophilised peptide in 90%H2O/10%2H2O (v/v). When required, deuterated tri¯uoroethanol (TFE-d3) was added to the aqueous peptide solution up to 15 or 30% (v/v). Typical sample volumes were 500 ml, and ®nal peptide concentrations were between 1.0 and 1.5 mM. All pH measurements were done at room temperature with a Crison micropH 2000 electrode. The pH of each sample was ®rst adjusted to 4.3 (with 2HCl) and a series of experiments were performed. Then the pH was raised to 8.0 (with NaO2H) and a new series of spectra were recorded. Sodium 3-(trimethylsilyl) propionate-2,2,3,3 was used as an internal reference. All spectra were recorded at 5 C on a Bruker AMX spectrometer, operating at 500 MHz 1H frequency. For WH4 in water at pH 4.3 the following spectra were acquired: correlated spectroscopy (COSY; Aue et al., 1976); homonuclear-Hartmann-Hahn (HOHAHA; Bax & Davis, 1985) with tm ˆ 70 ms; NOESY (Macura & Ernst, 1980) with mixing times of 200 ms, 250 ms, 300 ms and 350 ms; and rotating frame Overhauser enhancement spectroscopy (ROESY; Rance, 1987) with mixing times of 100 ms and 150 ms. For WH4 in other conditions and for WH3 in all conditions the following spectra were acquired: COSY, HOHAHA and NOESY, with mixing times of 200 ms and 250 ms, and ROESY with 150 ms. Time-proportional phase-incrementation mode was used in all cases. Preirradiation (during 1 s) was used to suppress the water resonance. Recorded data size was 256 (t1) and 2048 (t2) data points and 256 scans. Before Fourier transformation, zero ®lling in the t1 dimension and a phase-shifted square sine-bell were applied. Calculation of helical content Peptide helical contents were calculated from CD data by the method of Chen et al. (1974) assuming (at 220 nm and 2 C) an ellipticity value of ÿ30613 degree cm2 dmolÿ1 for a fully helical conformation and 310 degree cm2 dmolÿ1 for a random conformation. Aromatic residues in helical peptides have been reported to contribute to the observed ellipticity at 220 nm (Chakrabartty et al., 1993) and a method has been described to subtract this contribution from the observed ellipticity at 220 nm (Chakrabartty et al., 1994). The calculated helical contents of the WH and WAH peptides (after subtracting the contribution of the tryptophan residue) were close to those calculated by the simpler method of Chen et al. (1974; not shown). The helical content of WH4 in water, 15% TFE and 30% TFE were also calculated from chemical shift data according to Wishart et al. (1991). Since the tryptophan ring can in¯uence the chemical shift of neighbouring Ca

196 protons these helical contents should be regarded as approximate. Trifluoroethanol titrations The effect of TFE on the ellipticity of the peptides was studied at pH 5.0, in sodium phosphate (10 mM ionic strength) containing 18 mM NaCl, and at pH 9.5, in sodium borate (10 mM ionic strength) containing 18 mM NaCl. Aliquots of concentrated peptide stocks were added to buffered solutions containing increasing TFE concentrations. A 9 mM peptide concentration was used, and the ellipticity at 2 C was measured as above. Protein database analysis A protein database of 285 proteins with less than 25% homology (Hobohm & Sander, 1994) was used. The database contains, for each amino acid, a secondary structure determination made with the program DSSP (Kabsch & Sander, 1983). This database is currently implemented in the program WHAT IF (Vriend, 1990). Sequence and conformational searches were made with the SCAN3D option (Vriend et al., 1994) of this program. The random probability of ®nding a certain amino acid residue at any given position of an a-helix is de®ned as the number of amino acid residues of that kind found in helices of the database divided by the total number of amino acid residues found in helices of the database. In our case the probabilities of tryptophan and histidine are 0.0162 and 0.0193 respectively. The probability of ®nding any two speci®c residues at any two positions within an a-helix is the product of the individual probabilities, which for tryptophan/histidine pairs is 0.000312. The expected number of W***H (or H***W) sequences in the database is the product of the probability of this pair by the number of ®ve-residue helical segments (12,338) in the database. Similarly, the expected number of W**H (or H**W) sequences in the database is the product of the probability of the pair by the number of four-residue helical segments found (14,109). We de®ne the relative occurrence of a speci®c sequence as the number of cases found divided by the number of cases expected.

Acknowledgements This work was supported by grants PB94-0599, PB910368 and PB93-0073 from the DGICYT (Spain) and by grant 212-39 from the Universidad de Zaragoza, Spain. We thank Drs S. Padmanabhan, H. J. E. Loewenthal and R. Loewenthal for their comments on the manuscript. We thank Drs MunÄoz and Serrano for a preprint of MunÄoz & Serrano (1996), for making the source code of AGADIR1s available to us and for advice on its use. We also thank Dr Rohl for making SCINT available through the Internet.

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Edited by F. E. Cohen (Received 22 July 1996; received in revised form 26 November 1996; accepted 26 November 1996)