Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus1

J. Mol. Biol. (1998) 276, 775±786

Thermodynamic Characterization of Non-sequencespecific DNA-binding by the Sso7d Protein from Sulfolobus solfataricus Thomas LundbaÈck1, Henrik Hansson2, Stefan Knapp1 Rudolf Ladenstein1 and Torleif HaÈrd2* 1

Department of Biosciences Karolinska Institute and 2

Department of Biochemistry and Biotechnology, Royal Institute of Technology Novum, S-141 57, Huddinge Sweden

We used isothermal titration calorimetry and ¯uorescence spectroscopy to investigate the thermodynamics of non-sequence-speci®c DNA-binding by the Sso7d protein from the archaeon Sulfolobus solfataricus. We report the Sso7d-poly(dGdC) binding thermodynamics as a function of buffer composition (Tris-HCl or phosphate), temperature (15 to 45 C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/2H2O), and compare it to poly(dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represented by the McGhee± von Hippel model for non-cooperative binding, with a binding site size of four to ®ve DNA base-pairs and binding free energies in the range
G  ÿ 7 to
G  ÿ 10 kcal molÿ1, depending on experimental conditions. The non-speci®c nature of the binding is re¯ected in similar thermodynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynamics. Sso7d binding to poly(dGdC) is endothermic at 25 C with a binding enthalpy
H  10 kcal molÿ1 in both phosphate and Tris-HCl buffers at pH 7.6, indicating that
H does not include large contributions from coupled buffer ionization equilibria at this pH. The binding enthalpy is temperature dependent with a measured heat capacity change
Cp ˆ ÿ 0.25(0.01) kcal molÿ1 Kÿ1 and extrapolations of thermodynamic data indicate that the complex is heat stable with exothermic binding close to the growth temperature (75 to 80 C) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on
G and the exchange of H2O for 2H2O has only a small effect on
H , consistent with the inference that complex formation is not accompanied by net changes in surface hydration. Thus, other mechanisms for the heat capacity change must be found. The observed thermodynamics is discussed in relation to the nature of non-sequence-speci®c DNA-binding by proteins. # 1998 Academic Press Limited

*Corresponding author

Keywords: protein± DNA interactions; thermodynamics; isothermal titration calorimetry; osmotic stress; dehydration

Introduction Abbreviations used: K, binding constant; n, binding site size in DNA bp; bp, DNA base-pair;
G , free energy of binding;
H , enthalpy of binding;
S , entropy of binding;
Cp , heat capacity change; Qmax, maximum ¯uorescence quenching; Qobs, observed ¯uorescence quenching; Qi, reaction heat content in calorimetric experiments; ITC, isothermal titration calorimetry;
Hd, heat of dilution; CD, circular dichroism; NMR, nuclear magnetic resonance; PEG, polyethylene glycol. 0022±2836/98/090775±12 $25.00/0/mb971558

The hyperthermophilic archaeon Sulfolobus solfataricus (Brock et al., 1972) expresses large amounts of several mutually similar DNA-binding proteins with a molecular mass of about 7 kDa (Thomm et al., 1982; Kimura et al., 1984; Grote et al., 1986; Choli et al., 1988a,b). These proteins can be classi®ed as ``histone-like'' based on their physical properties, i.e. size, basicity, relative abundance and DNA-binding properties. The exact biological roles of histone-like proteins in eukarya and archaea # 1998 Academic Press Limited

776 remain to be elucidated, but it is likely that they are involved in DNA packing maintenance and control (for reviews, see Dijk & Reinhardt, 1986; Drlica & Rouviere-Yaniv, 1987; Pettijohn, 1988; Grayling et al., 1994). We previously determined the structure of one protein, Sso7d (Choli et al., 1988a), within this group using nuclear magnetic resonance (NMR; Baumann et al., 1994). Sso7d consists of a triplestranded anti-parallel b-sheet onto which a doublestranded b-sheet is packed. This fold is actually highly similar to that of eukaryotic SH3 domains. NMR was subsequently used to identify the DNAbinding surface, which was found to consist of the triple-stranded b-sheet and a reverse turn connecting the two strands of the double-stranded b-sheet (Baumann et al., 1995). Biophysical characterizations of Sso7d have included studies of how Sso7d increases the thermal stability of DNA (Baumann et al., 1994) and also promotes DNA annealing (Guagliardi et al., 1997), a characterization of the Sso7d folding thermodynamics (Knapp et al., 1997), thermodynamic investigations of the salt concentration dependence of the DNA-binding (LundbaÈck & HaÈrd, 1996), and studies of arginine residue side-chain dynamics in the free protein and the DNA-complex, respectively (Berglund et al., 1995). Here we use isothermal titration calorimetry and ¯uorescence spectroscopy to characterize the thermodynamics of the binding of Sso7d to doublestranded poly(dGdC) and poly(dAdT). The studies address several issues related to the physical basis for Sso7d± DNA interactions as well as to the thermodynamics of non-sequence-speci®c DNA-binding in general, including: (1) a comparison of the binding to poly(dGdC) and poly(dAdT); (2) the temperature and pH dependence of the binding to poly(dGdC); (3) the role of accompanying dehydration effects upon non-sequence-speci®c DNAbinding; and (4) the effect of native lysine methylation on the DNA-binding. We ®nd that several aspects of the binding thermodynamics are consistent with the current view of non-sequence-speci®c protein ±DNA equilibria. These include similar thermodynamics for binding to different DNA sequences and the absence of large dehydration effects (reported here) and also the fact that the well-known salt effect of the DNA-binding af®nity is of entropic origin (LundbaÈck & HaÈrd, 1996). We do, on the other hand, ®nd an unexpected heat capacity change for the association process. This change contributes to make the DNA-binding by Sso7d optimum at high temperatures.

Results Thermodynamics of Sso7d ± DNA interactions We used isothermal titration calorimetry (ITC) to compare the binding to poly(dGdC) by the native heterogenously methylated Sso7d protein, puri®ed

Thermodynamics of Sso7d DNA Interactions

Figure 1. a, Calorimetric data (top) for the isothermal titration at 35 C of non-methylated Sso7d protein (0.77 mM) into a solution of poly(dGdC) (0.129 mM base-pairs) in a buffer containing 10 mM sodium phosphate and 20 mM NaCl at pH 7.6. Also shown is data (bottom) from a corresponding reference titration where Sso7d is injected into plain buffer solution. b, Integrated heats of binding ( & ) and integrated heats of dilution (*) for the data in a.

from S. solfataricus, with that of non-methylated Sso7d, obtained by overproduction in Escherichia coli cells. A calorimetric titration of poly(dGdC) with non-methylated Sso7d in 10 mM sodium phosphate and 20 mM NaCl at pH 7.6 and 25 C is illustrated in Figure 1a. The titration is initially characterized by an endothermic effect (heat uptake) which decreases as the DNA becomes saturated with bound protein and the last injections yield only very small instrumental responses re¯ecting heats of dilution. Included in Figure 1a is also a corresponding blank titration in which the protein is injected into plain buffer. The integrated heats for both titrations are shown in Figure 1b. The ITC binding isotherm is adequately described by the McGhee ± von Hippel model for non-cooperative DNA-binding, containing parameters for the microscopic binding af®nity (K) and the binding site size (n) in DNA bp (equation (6)). These parameters and the binding enthalpy,
H , were ®tted to experimentally observed heats of binding using equations (6) and (7) as described in Materials and Methods. The analysis shows that the DNA-binding is characterized by K ˆ (3.6  0.4)  106 Mÿ1,
H ˆ 9.8(0.5) kcal molÿ1 and n ˆ 4.3(0.4). Comparisons with identical experiments on the methylated Sso7d from S. solfataricus do not reveal any signi®cant differences in the binding thermodynamics, although small differences in
H (< 1 kcal molÿ1) and n (<10%) cannot be excluded (see Table 1, which contains

777

Thermodynamics of Sso7d DNA Interactions Table 1. Summary of calorimetric experiments on Sso7d binding to homopolymeric DNAa DNA

Sso7d sample

Bufferb (titrations)c

pH

Temperature

poly(dGdC) poly(dGdC) poly(dGdC)

E. coli native E. coli

B (2) B (2) B (2)

7.6 7.6 7.6

25 C 25 C 25 C

poly(dGdC) poly(dAdT)

native native

C (2) C (2)

7.5 7.5

25 C 25 C

poly(dGdC) poly(dGdC) poly(dGdC) poly(dGdC) poly(dGdC) poly(dGdC)

E. E. E. E. E. E.

A A A A A A

(3) (2) (2) (2) (2) (2)

7.6 7.6 7.6 7.6 7.6 7.6

25 C 25 C 25 C 25 C 25 C 25 C

poly(dGdC) poly(dGdC) poly(dGdC)

E. coli E. coli E. coli

B (2) B (2) B (2)

7.1 7.1 7.1

16 C 25 C 34 C

poly(dGdC) poly(dGdC) poly(dGdC) poly(dGdC)

E. E. E. E.

B B B B

(2) (3) (2) (2)

7.6 7.6 7.6 7.6

poly(dGdC) poly(dGdC) poly(dGdC)

E. coli E. coli E. coli

B (2) B (1) B (2)

8.0 8.0 8.0

coli coli coli coli coli coli

coli coli coli coli


G
H ÿT
S (kcal molÿ1) (kcal molÿ1) (kcal molÿ1)

Soluted

K (10ÿ6  Mÿ1)

n

100% 2H2O

3.6 4.0 2.9

4.3 4.0 4.5

ÿ8.9 ÿ9.0 ÿ8.8

9.8 8.9 10.2

ÿ18.7 ÿ17.9 ÿ19.0

0.92 0.41

4.5 5.1

ÿ8.1 ÿ7.7

10.0 10.9

ÿ18.1 ÿ18.6

0.19 0.19 0.20 0.20 0.18 0.22

4.7 4.6 4.8 5.0 4.5 4.7

ÿ7.2 ÿ7.2 ÿ7.2 ÿ7.2 ÿ7.2 ÿ7.3

10.4 10.8 10.2 10.1 11.0 10.0

ÿ17.6 ÿ18.0 ÿ17.4 ÿ17.3 ÿ18.2 ÿ17.3

5.5 8.1 10.6

4.6 4.5 4.4

ÿ8.9 ÿ9.4 ÿ9.9

12.6 10.7 8.5

ÿ21.5 ÿ20.1 ÿ18.4

15 C 25 C 35 C 45 C

2.9 3.7 4.4 4.9

4.4 4.3 4.3 4.3

ÿ8.5 ÿ9.0 ÿ9.4 ÿ9.7

11.7 9.8 7.1 4.3

ÿ20.2 ÿ18.8 ÿ16.5 ÿ14.0

16 C 25 C 35 C

4.9 7.7 7.0

4.1 4.1 4.0

ÿ8.9 ÿ9.4 ÿ9.6

10.9 9.0 6.5

ÿ19.8 ÿ18.4 ÿ16.1

100% 2H2O 0.5 osm sucrose 1.1 osm sucrose 1.2 osm betaine 3.0 osm betaine

a Possible systematic errors were evaluated as described in Materials and Methods. These were found to be approximately 10% (K and n), 0.5 kcal molÿ1 (
H ), 0.2 kcal molÿ1 (
G ) and 0.5 kcal molÿ1 (ÿT
S ). b Buffer compositions: A, 20 mM Tris-HCl, 20 mM NaCl and 4 mM MgCl2; B, 10 mM sodium phosphate and 20 mM NaCl; C, 50 mM Tris-HCl and 50 mM NaCl. c Number of independent calorimetric titrations. d Sucrose and betaine additions reported in osmolal units.

data from all calorimetric experiments). The accuracy of these parameters is related to the accuracy of determined DNA and protein concentrations and the small observed differences might, therefore, also be attributed to these latter uncertainties (about 5%). The possible existence of coupled protonation/ deprotonation equilibria can result in large contributions to
H (Murphy et al., 1993). Such effects can be examined by comparing results obtained in buffers with different ionization enthalpies. The results of experiments with non-methylated Sso7d in two buffers at very similar total salt concentrations and the same pH conditions are shown in Table 1. The buffering components are Tris-HCl for which
Hion  11 kcal molÿ1 (Janssen & Nelen, 1979) and phosphate for which
Hion  1 kcal molÿ1 (Murphy et al., 1993). The difference in
H (<1 kcal molÿ1) is small compared to the difference in ionization enthalpies between Tris-HCl and phosphate. Binding of Sso7d to poly(dGdC) is, therefore, not strongly coupled to a protonation equilibrium at pH 7.6. Calorimetric titrations at three different pH values (phosphate buffer with pH adjusted to 7.1, 7.6 and 8.0, respectively) and at three different temperatures (15, 25 and 35 C), which are described in more detail below, also show that the binding thermodynamics is only weakly pH-dependent in the interval 7.1 to 8.0. A comparison of the binding of native Sso7d to poly(dGdC) and poly(dAdT) con®rms a weak pre-

ference of Sso7d for poly(dGdC) (Baumann et al., 1994), for which the binding free energy
G ˆ ÿRT ln(K) is 0.4 kcal molÿ1 more favorable than for binding to poly(dAdT). The two binding processes show similar endothermic binding enthalpies: 10.0 and 10.9 kcal molÿ1 for poly(dGdC) and poly(dAdT), repectively. As a consequence, the thermodynamics for Sso7d binding to these two different DNA homopolymers is essentially the same, at least within the uncertainties of our measurements (Table 1). For the data in Table 1, we note a tendency for the best-®t value of the binding site size, n, to be somewhat smaller when the binding af®nity is higher. However, the precision in determined n values (10%) is not suf®cient to allow conclusions based on this observation. A similar tendency has also been observed for the binding of Sac7d to different DNA homopolymers (McAfee et al., 1996). Effect of osmotic stress We performed equilibrium titrations of Sso7d to poly(dGdC) at 20 C in 20 mM Tris-HCl, 20 mM NaCl, 4 mM MgCl2 and 0.1 mM octaethylene glycol monododecyl ether at pH 7.6 using ¯uorescence spectroscopy. The role of dehydration on this binding equilibrium was investigated by replacing part of the solvent water with different neutral solutes, thereby lowering the water activity of the

778

Thermodynamics of Sso7d DNA Interactions

Figure 2. Fluorescence titrations at 20 C where poly(dGdC) is added to a constant concentration of nonmethylated Sso7d (2 mM) in 20 mM Tris-HCl, 20 mM NaCl, 4 mM MgCl2 and 0.1 mM octaethylene glycol monododecyl ether at pH 7.6. The binding process is monitored by following the observed fractional quenching of the intrinsic tryptophan ¯uorescence as the protein binds to DNA. Qobs/Qmax is plotted as a function of the molar ratio of DNA base-pairs to Sso7d. The effect of having part of the solvent water replaced by glycerol with osmolal concentrations of 0 ( & ), 1.6 (*) and 4.1 (~), respectively, is illustrated. The solid lines represent the best ®t of binding isotherms as described in Material and Methods.

Figure 3. Logarithm of relative binding af®nities (ln[K/ K0]) as a function of solute osmolal concentration. K0 is the association constant of the Sso7d-poly(dGdC) reaction with no added solutes and K is the association constant in presence of solutes. Results obtained using both ¯uorescence spectroscopy and ITC are included and the reference K0 was determined independently for each titration series. Osmolytes used in the ¯uorescence experiments were glycerol ( & ), betaine (!) and sucrose (^) and in the ITC experiments dextran (~), betaine (*) and sucrose ("). The lines represent scenarios in which ®ve (broken) or 50 (continuous) water molecules are released in the binding process as calculated from equation (9).

binding buffer (see the review by Parsegian et al. 1995 and work cited therein). Experimental data at different concentrations of glycerol are shown in Figure 2 where the fractional ¯uorescence quenching is plotted as a function of the molar ratio of DNA base-pairs to Sso7d. The data demonstrate that the af®nity of Sso7d for poly(dGdC) is only very weakly dependent on the glycerol concentration. This is further illustrated in Figure 3 where the logarithm of the relative binding af®nities are plotted as a function of buffer osmolality. Included in this ®gure are the corresponding data observed for the addition of betaine and sucrose, and data obtained using ITC (at 25 C) in buffers containing different concentrations of sucrose and betaine. The latter experiments were carried out by ®rst lyophilizing the samples and then resuspending them in the desired solvent as described in Materials and Methods. The lyophilization had negligible effects on measured thermodynamics as judged from a comparison with samples that had not been lyophilized (not shown). Figure 3 shows that the relative binding af®nity depends only weakly on the osmolality of glycerol, betaine or sucrose. Two lines have been drawn in the Figure, which represent binding processes in which there are a net release of ®ve or 50 water molecules (equation (9)), respectively. Additional

information is expected when the binding free energy is decomposed into its underlying thermodynamic driving forces, i.e. the binding enthalpy and entropy. Measured
G ,
H and ÿT
S values for the calorimetric osmotic stress experiments are shown in Table 1. It is evident that osmolyte addition has only a small, if any, effect also on
H and ÿT
S . There is a small difference between binding af®nities estimated using ¯uorescence spectroscopy (K ˆ (5.6  2.6)  105 Mÿ1 at 20 C) and ITC (K ˆ (1.9  0.2)  105 Mÿ1 at 25 C in the same buffer, but without the octaethylene glycol monododecyl ether) and the measured binding site size is also slightly lower in the ¯uorescence experiments with an average of approximately 3.7 compared with 4.5 in the ITC experiments. We cannot fully explain this difference. However, the observation underscores the necessity to base conclusions on data obtained in experimental series using the same technique and the same protein batch. This procedure ensures that data are mutually comparable. We performed similar experiments in the presence of different concentrations of PEG300 and PEG600. In the ITC experiments, where the protein and DNA concentrations are somewhat higher, the addition of these polymers resulted in precipitation. Precipitation was not seen in the ¯uor-

Thermodynamics of Sso7d DNA Interactions

Figure 4. Fluorescence emission spectra of the free Sso7d (2.5 mM) protein (middle curve) and Sso7dpoly(dGdC) complex (bottom curve) recorded with excitation at 290 nm in 20 mM Tris-HCl and 0.1 mM octaethylene glycol monododecyl ether at pH 7.6 and 20 C. Illustrated is also the blue-shift and increased intensity observed in the Sso7d emission spectrum (upper curve) recorded in the presence of 1.6 osmolal (approximately 35 wt%) PEG600.

escence experiments, but we observe an apparent increase of the binding af®nity. There is a blueshift and an increased intensity in the ¯uorescence emission spectrum with increasing concentrations of PEG (Figure 4). A blue-shift in the emission spectrum is also observed upon binding to DNA. The shift indicates that the local environment of the single tryptophan in the Sso7d protein, which is positioned at the DNAbinding surface (Baumann et al., 1995) in the complex, has been altered (Lakowicz, 1983). The ¯uorescence properties of Sso7d were much less affected by the presence of the other solutes. These observations suggest that PEG300 and PEG600 might interact speci®cally with Sso7d. The PEG data were therefore not included in the evaluation of the osmotic stress effect. Cautious use of PEGs as osmotic agents is also recommended in the literature (Parsegian et al., 1995). Light versus heavy water The effect of replacing H2O with 2H2O on the thermodynamics of Sso7d binding to poly(dGdC) was measured using ITC in two different buffers (20 mM Tris-HCl, 20 mM NaCl, 4 mM MgCl2 at pH 7.6 and 10 mM sodium phosphate, 20 mM NaCl at pH 7.6). Again, this was accomplished by ®rst lyophilizing the samples and then resuspending them in the desired solvent. The results are summarized in Table 1. It is evident that the exchange of H2O for 2H2O has only a marginal effect on the binding thermodynamics in both buffers. In particular, there is only a very minor

779

Figure 5. Integrated heats of binding corrected for heats of dilution from titrations of non-methylated Sso7d protein (0.77 mM) into a solution of poly(dGdC) (0.129 mM base-pairs) at four different temperatures, 15 C (*), 25 C (~), 35 C (^) and 45 C ( & ). The experiments were carried out in a buffer solution containing 10 mM sodium phosphate, 20 mM NaCl at pH 7.6. Data presented here are the means of two independent titrations at each temperature, except at 25 C where data represent the mean of three titrations. The continuous lines represent the best-®t binding isotherms as described in Materials and Methods.

increase in binding enthalpy (

H 4 0.5 kcal molÿ1) in 2H2O. Temperature and pH dependence of binding The results of calorimetric titrations in 10 mM sodium phosphate, 20 mM NaCl at pH 7.6 are shown in Figure 5 for four different temperatures (15, 25, 35 and 45 C). The observed heats show a signi®cant temperature dependence, which is further illustrated in Figure 6 where
H measured at pH 7.1, 7.6 and 8.0 is plotted as a function of temperature. Also the af®nity of Sso7d for poly(dGdC) increases with temperature in the studied pH range (Table 1). These data clearly indicate that the Sso7d-DNA complex is ``heat stable'' with a maximum binding af®nity at a temperature higher than 45 C. It was not practically possible to carry out experiments at higher temperatures, because the heats of reaction decrease with increasing temperature. It should, in principle, be possible to measure an exothermic reaction at some temperature higher than TH (see below), but our calorimeter does not allow measurements at temperatures greater than 65 C. Quantitative evaluations of the heat capacity change (
Cp ) and the temperature dependence in
H and
G are described in Discussion.

780

Thermodynamics of Sso7d DNA Interactions

above. However, we still cannot exclude a minor pH dependence in
H .

Discussion Fluorescence spectroscopy and isothermal titration calorimetry have been used to characterize the DNA-binding by the Sso7d protein from S. solfataricus. The objective of this work is to obtain thermodynamic pro®les for equilibrium DNAbinding of this histone-like protein from a hyperthermophilic organism. The studies form a part of the on-going effort to understand the physical chemistry of biochemical processes in organisms that are adapted to life at high temperatures. In addition, Sso7d protein is a very suitable model system for the characterization of the physical principles of nonsequence speci®c DNA-binding. The present study covers the temperature and pH dependence of the binding parameters, the effect of the lysine methylation of Sso7d that occurs in vivo, the effects of osmotic stress and change of solvent from H2O to 2H2O, and a comparison of binding to poly(dGdC) and poly(dAdT). A calorimetric study of the salt dependence for binding to poly(dGdC) has recently been published elsewhere (LundbaÈck & HaÈrd, 1996). Lysine methylation Figure 6. A, Measured binding enthalpies as a function of temperature at pH 7.1 (open squares), pH 7.6 (®lled circles) and pH 8.0 (open diamonds) in 10 mM sodium phosphate, 20 mM NaCl. The results from all independent titrations are shown in this ®gure, in contrast to data in Table 1 and Figure 5, where averages are reported. The continuous line is a ®t of the temperature dependence in
H at pH 7.6 assuming a temperature independent
Cp . The broken line represents a corresponding ®t assuming a linear temperature dependence in
Cp . (See the text for further details.) B, Measured free energies of binding as function of temperature at pH 7.6. The continuous and broken lines represent best®t free energy pro®les assuming temperature independent or temperature dependent
Cp , repectively. (See the text for further details.)

Also included in Figure 6A are binding enthalpies observed in corresponding experiments performed at pH 7.1 and 8.0. These experiments were included to further examine the possible in¯uence of coupled protonation or deprotonation effects on the temperature dependence of
H , i.e. on the apparent
Cp (Murphy et al., 1993; Baker & Murphy, 1996). We ®nd that measured values of
H differ somewhat between the experiments at different pH, but that the variation with temperature is virtually identical in the three experiments. The latter ®nding is consistent with the absence of large protonation/ deprotonation effects, as are the comparison of binding enthalpies in different buffers described

Five of 14 lysine residues in Sso7d are subjected to speci®c, but heterogeneous, Nz-mono-methylation within the cell (Choli et al., 1988a,b). As shown in Table 1, we ®nd that the methylation has only minor effects on the DNA-binding thermodynamics. We previously showed that none of the ®ve possible lysine methylation sites are located at the DNA-interacting surface of Sso7d (Baumann et al., 1995), but instead they are exposed to the bulk solvent also in the complex. The independence of measured DNA-binding parameters, both in terms of free energy, enthalpy and entropy of binding, further indicates that the methylation is related to other functions than DNA-binding. Furthermore, thermodynamic studies of the stability of the Sso7d protein using both differential scanning calorimetry and circular dichroism (CD) spectroscopy (Knapp et al., 1997) show that the methylation has a negligible effect also on the stability of the Sso7d protein. Thus, any physical relevance of the methylation remains unclear. Thermodynamics of binding to poly(dGdC) compared to poly(dAdT) Using ¯uorescence spectroscopy we previously observed that the DNA-binding af®nity of Sso7d depends somewhat on the DNA sequence (Baumann et al., 1994). Here, we use ITC to make a more complete thermodynamic comparison of the binding to poly(dGdC) and poly(dAdT), respectively. The present study con®rms a weak preference (0.4 kcal molÿ1) for poly(dGdC). The two

781

Thermodynamics of Sso7d DNA Interactions

binding processes also display approximately equal binding enthalpies (Table 1). The overall similarity in the thermodynamics of binding to GC and AT-containing sequences suggests that the interactions stabilizing the complexes also are similar. Thus, it appears unlikely that the most important stabilizing interactions include contacts between the protein and the DNA-bases in the major groove, where the chemical nature of the surface is sequence dependent. The inference is consistent with our model of the Sso7d-DNA complex, which contains a cavity between the protein and the DNA at the center of the major groove that could ensure the nonsequence speci®c nature of the Sso7d-DNA complex (Baumann et al., 1995). Alternatively, Sso7d binds only in the minor groove. Osmotic stress and changes in macromolecular hydration The effect of osmotic stress on the Sso7dpoly(dGdC) binding equilibrium (Figure 3 and Table 1) was studied to estimate the net volume of released surface waters upon complexation. We observe only a very minor effect on the binding constant in the presence of increasing osmolalities of three different solutes (glycerol, betaine and sucrose). These three solutes have different molecular mass (85, 117 and 342 g molÿ1, respectively), and have different effects on the bulk water dielectric constant (Parsegian et al., 1995). The similarity of the observed small effects with the three solvents therefore suggests that the measurements re¯ect only the (very minor) effect of water activity on the equilibrium, and are not biased by effects of solute size or on the dielectric constant of the bulk solution. A quantitative estimate of the osmotic stress dependence yields a corresponding water release of about ®ve water molecules (equation (9); Figure 3), i.e. practically no net change in the volume of solute-excluded water. The small effect of added solutes is also emphasized by the relatively unaffected
H (Table 1). A similar observation has been made on other non-sequencespeci®c protein ±DNA equilibria, e.g. the binding of gal repressor to poly(dIdC) (Garner & Rau, 1995). As a comparison, a net release of approximately 140 water molecules was measured for sequence-speci®c DNA-binding of gal repressor (Garner & Rau, 1995). Similarly, non-sequencespeci®c DNA-binding by EcoRI sequesters about 110 waters more than the sequence speci®c binding (Sidorova & Rau, 1996). Thus, Sso7d appears to conform to the observation that non-sequencespeci®c binding is not associated with extensive surface dehydration. This inference is consistent with the generally held view that nonsequence speci®c protein ±DNA complexes do not involve tight packing of interacting surfaces (for reviews on this subject, see von Hippel & Berg, 1986; Record et al., 1991; HaÈrd & LundbaÈck, 1996).

A different experimental approach that measures the importance of solvent reorganization is provided by the solvent isotope effect, i.e. light water replaced by heavy water, on the measured enthalpy of binding (Connelly et al., 1993; Chervenak & Toone, 1994; Makhatadze et al., 1995). Generally, the substitution of H2O for 2H2O results in a less favorable binding enthalpy which is related to solvent reorganization (Chervenak & Toone, 1994). A correlation between the binding enthalpy difference and the heat capacity change, which is often interpreted in terms of dehydration, was also observed (Chervenak & Toone, 1994). The interpretation in terms of a solvent reorganization is further supported by a study of the water isotope effect on protein stability, where it is shown that changes in folding enthalpies can be correlated with the change in non-polar surface areas upon folding (Makhatadze et al., 1995). In the case of Sso7d binding to poly(dGdC), we observe a small solvent isotope effect on
H (Table 1), although we note that the difference is comparable with the experimental errors. Still, if the differential binding enthalpy (

H ˆ
H (2H2O) ÿ H (H2O) 4 0.5 kcal molÿ1) is interpreted in terms of change in non-polar solvent-accessible surface area,
Anpl, according to Makhatadze et al.(1995), we obtain Ê 2. This is a small number com
Anpl < 50 to 100 A pared to sizes of the interacting surfaces, and the observed effect of 2H2O on the binding enthalpy is, therefore, consistent with the interpretation of the osmotic stress dependence, i.e. that both protein and DNA essentially retain their hydration shells upon complexation. Temperature dependence of binding The binding af®nity of Sso7d for poly(dGdC) is clearly temperature dependent (Figure 6B), with stronger binding at higher temperatures and a maximum af®nity at some temperature higher than 45 C. Furthermore, the marked temperature dependence of
H indicates a signi®cant negative change in heat capacity (
Cp ) upon binding (Figure 6A). It would, of course, be desirable to obtain binding isotherms at the higher physiological relevant temperatures, but this was not practically possible for reasons already mentioned. However, the precision of the low-temperature data is suf®cient to attempt an extrapolation to higher temperatures. Assuming a temperatureindependent heat capacity change, we can ®t the temperature dependence of the binding enthalpy to obtain a value of
Cp ˆ ÿ0.26(0.01) kcal molÿ1 Kÿ1 for binding to poly(dGdC) in phosphate buffer at pH 7.6, and a temperature (TH) at which
H (TH) ˆ 0; TH ˆ 62(2) C. This ®t is indicated by a continuous line in Figure 6B. Given these values and well-known thermodynamic relationships one only needs a value for the temperature (TS) at which
S (TS) ˆ 0 to predict the temperature pro®le of
G . The continuous line in Figure 6B represents a ®t of
G as a function of

782

Thermodynamics of Sso7d DNA Interactions

temperature to the equation:     T  
G ˆ
Cp T 1 ÿ ln ÿ TH Ts

…1†

(with all temperatures in units of K) where
Cp and TH were ®xed to the values determined in the ®t of enthalpy versus temperature data. The best-®t value of the single adjustable parameter was obtained as TS ˆ 106(4) C, which corresponds to the temperature of the minimum in
G . Alternatively, it may be argued that the heat capacity change is not constant within a wide temperature range resulting in a non-linear temperature dependence in
H , in which case the optimal binding temperature estimated based on a constant
Cp may be misleading. Actually, a very weak non-linearity can be inferred also in the temperature range 15 to 45 C (Figure 6A, data at pH 7.6). A linear temperature dependence in
Cp , e.g.:
Cp …T† ˆ
Cp …303 K† ‡ a…T ÿ 303†

…2†

where the linear expansion is centered around 30 C, can be integrated to obtain the corresponding binding enthalpy: …T




Cp …T† dT ˆ …
Cp …303 K†


H …T† ˆ TH

a 2 ÿ 303a†…T ÿ TH † ‡ …T 2 ÿ TH † 2

…3†

and free energy: 

…T


G …T† ˆ TH


Cp …T†

…T dT ÿ T Ts


Cp …T† dT T

ˆ …
Cp …303 K† ÿ 303a†…T ÿ TH †  a 2 2 ‡ …T ÿ TH † ÿ T …
Cp …303 K† 2  T ÿ 303a† ln ‡ a…T ÿ Ts † Ts

…4†

where the reference temperatures again have been chosen so that the enthalpic and entropic contributions to
G are zero at TH and TS, respectively, i.e.
H (TH) ˆ 0 and
S (TS) ˆ 0. Fitting equations (3) and (4) to the temperature dependencies of
H and
G (at pH ˆ 7.6), respectively (broken lines in Figure 6), we obtain
Cp (303 K) ˆ ÿ 0.25(0.01) kcal molÿ1 Kÿ1, a ˆ ÿ 0.005(0.001) kcal molÿ1 Kÿ2, TH ˆ 57(2) C and TS ˆ 81(4) C. Again, TS represents the position of the
G (T) minimum, but this temperature is now predicted to be lower than if a temperatureindependent
Cp is assumed. (This latter exercise, therefore, illustrates the dif®culties associated with extrapolating binding free energies to higher temperatures.) In any case, it appears that maximum

binding af®nity, K ˆ exp(
G /ÿRT) at T ˆ TH, should be achieved at some temperature which is well above 45 C and which approaches the optimal growth temperature (75 to 80 C ) of S. solfataricus (Brock et al., 1972). It is also worth noting that
H , which takes a positive value at lower temperatures, is predicted to be negative, i.e. exothermic binding, at physiological temperatures. Significance of the observed binding thermodynamics and origin of the heat capacity change The observation of a heat capacity change and accompanying strong temperature dependence in the Sso7d ± DNA association equilibrium is unexpected. This is because heat capacity changes close to zero have been measured for several nonsequence-speci®c protein± DNA equilibria (e.g. see Takeda et al., 1989; Ladbury et al., 1994). Speci®c biomolecular interactions involving tight and solvent excluded interfaces are often associated with negative heat capacity changes, i.e.
Cp < 0, for the association reactions. The major contribution is believed to come from a dehydration of the interacting surfaces, a process which seems closely related to protein folding where a great part of the extended protein chain is buried from hydrating waters upon folding. Thus, previous measurements on non-sequence-speci®c protein ±DNA equilibria are consistent with the inference these complexes do not involve tight macromolecular interfaces and accompanying dehydration effects. Here, we con®rm the absence of large net dehydration effects for the Sso7d ±DNA equilibrium. We also ®nd very similar thermodynamics for binding to different DNA sequences. Previously, we have shown that the salt dependence of the binding af®nity has an entropic origin (LundbaÈck & HaÈrd, 1996). These observations are all consistent with the expected behavior of a protein which has evolved to function as a ``true'' non-sequencespeci®c DNA-binder. Still, we measure a signi®cant heat capacity change (
Cp ˆ ÿ 0.25(0.01) kcal molÿ1 Kÿ1 at 30 C) for the DNA-binding process. The effect may very well be speci®c to Sso7d or to the fact that the protein comes from a hyperthermophilic organism which has evolved some mechanism to ensure strong binding at high temperatures. On the other hand, Sso7d is, to the best of our knowledge, the ®rst non-sequence speci®c DNA-binding protein for which the binding thermodynamics have been fully characterized using calorimetric methods. The possibility that the thermodynamics observed are actually a general feature for non-sequence-speci®c DNA-binding, therefore, remains, at least until similar measurements on other proteins, e.g. histones, have been made. It may be argued that previous observations on nonsequence speci®c binding rules out this possibility. However, these observations have all been made with proteins that have evolved to bind precisely de®ned DNA sequences (i.e. not non-

783

Thermodynamics of Sso7d DNA Interactions

speci®cally). Proteins like Sso7d can in a similar way be thought of as proteins having evolved to bind strongly to all DNA sequences, i.e. to tolerate all sequences. Thus, just as the difference in function distinguishes, e.g. bacterial repressors and histone-like proteins, there might also be an accompanying difference in their respective nonsequence-speci®c DNA-binding thermodynamics. As for the actual mechanism for the observed
Cp (in the absence of dehydration effects) there are several possibilities which have been discussed in the literature and which are brie¯y reviewed here. The most plausible mechanisms are the coupling of the DNA-binding to some other equilibrium, such as conformational changes (Ha et al., 1989; Spolar & Record, 1994) or protonation/ deprotonation events (Baker & Murphy, 1996), or stiffening of vibrational modes (Sturtevant, 1977; Ladbury et al., 1994). A shift with temperature of an equilibrium between two or more states will appear experimentally as a contribution to the heat capacity. In the present case we can rule out a large contribution from a coupled protonation ±deprotonation equilibrium. We do not have any indication of a possible folding/unfolding of the Sso7d protein upon DNA-binding, although it cannot be completely ruled out. However, it was recently shown that the DNA-binding of the homologous Sac7d protein causes a signi®cant alteration of the CD spectrum of poly(dGdC), while identical CD spectra were obtained for the native Sac7d protein in both the complexed and uncomplexed state (McAfee et al., 1996). The changes in CD spectra were interpreted in terms of conformational changes in the DNA. If the suggested conformational change is temperature dependent, it could account for the
Cp observed here. A similar effect has been observed for the binding of the SSB protein to single-stranded DNA, where an apparent heat capacity change is due to a DNA base pair unstacking reaction which is coupled to the DNAbinding (Ferrari & Lohman, 1994). On the other hand, a potential contribution from a coupled process can be expected to result in a temperature dependence in
Cp that is stronger than what we observe between 15 and 45 C. Further studies, such as a structure determination of the Sso7dDNA complex, are clearly needed before any contribution of a conformational change in protein or DNA to the observed thermodynamics can be ®rmly established. An alternative molecular mechanism for heat capacity changes has been suggested in an analysis by Sturtevant (1977). This mechanism involves a restriction of soft vibrational modes and it has later been used to account for the
Cp observed for DNA-binding by the trp repressor (Ladbury et al., 1994). The dynamic restriction is in that case supported by structural data, because crystallographic temperature factors are uniquely and considerably lowered for the DNA-interacting fragments of the repressor. A dynamic restriction has actually been measured upon DNA-binding by Sso7d, both for

backbone atoms (H. Berglund et al., unpublished data) and, in particular, for an arginine residue side-chain at the DNA ± protein interface (Berglund et al., 1995). A similar restriction of dynamic ¯exibility has also been observed for DNA phosphate groups upon non-sequence-speci®c DNA-binding of the lac repressor (Botuyan et al., 1993). However, more comprehensive experimental and theoretical analyses are needed to elucidate the relation between observed changes in biomolecular dynamics and measured binding thermodynamics.

Materials and Methods DNA and protein preparation Poly(dGdC) and poly(dAdT) were purchased from Pharmacia and dialyzed against titration buffer A (20 mM Tris-HCl, 20 mM NaCl, and 4 mM MgCl2 at pH 7.6), B (10 mM sodium phosphate, and 20 mM NaCl at pH 7.6) or C (50 mM Tris-HCl, and 50 mM NaCl at pH 7.5). DNA concentrations were determined spectrophotometrically using the extinction coef®cients e260 nm ˆ 16.800 M bpÿ1 cmÿ1 for poly(dGdC) and e260 nm ˆ 13.200 M bpÿ1 cmÿ1 for poly(dAdT) speci®ed by the manufacturer. Native Sso7d, with heterogeneously Nz-mono-methylated lysine residues, was obtained by puri®cation of protein from S. solfataricus as described previously (Baumann et al., 1994). Homogeneous non-methylated protein samples were obtained by over-production of Sso7d in E. coli cells followed by puri®cation as described elsewhere (Knapp et al., 1997). Pure protein preparations were extensively dialyzed against titration buffers A, B or C in the same beaker as the DNA to avoid heat signals from mixing with nonequivalent buffers in the calorimetric titrations. Protein concentrations were determined spectrophotometrically using the extinction coef®cient e280 nm ˆ 8.300 Mÿ1 cmÿ1 calculated for tyrosine and tryptophan absorption (Cantor & Schimmel, 1980). Fluorescence spectroscopy The binding of Sso7d to DNA was examined by measuring the quenching of intrinsic tryptophan ¯uorescence upon DNA-binding. Titration experiments were performed as reverse titrations in which poly(dGdC) was added to a constant Sso7d concentration (2 to 2.5 mM) in the cuvette. The experimental set-up including spectro¯uorometer, cuvette temperature control, cuvette sample mixing, and corrections for background ¯uorescence, optical ®ltering and light scattering has been described previously (LundbaÈck et al., 1994). For the present experiments we used an excitation wavelength of 290 nm and emission intensities were sampled at 0.2 nm intervals within 330 to 360 nm of the emission spectrum. Three separate emission spectra were recorded at each titration point. The titrations were carried out at 20 C in buffer A (with or without additional solutes) with 0.1 mM octaethylene glycol monododecyl ether added to the cuvette to prevent protein adhesion to quartz surfaces. Sso7d was added to the cuvette after the background ¯uorescence/scattering had been recorded. The titrand DNA was then added in 1 ml aliquots using a microsyringe with a repeating adapter.

784

Thermodynamics of Sso7d DNA Interactions

A number of reaction buffers were prepared in which part of the solvent water was replaced by neutral solutes. These buffers were identical to buffer A in terms of salt and buffering agent concentrations. The solutes were sucrose, betaine, glycerol and polyethylene glycols (PEGs) with approximate molecular mass of 300 Da (PEG300) and 600 Da (PEG600), respectively. The osmolalities of the buffers were calculated based on data made available by the Laboratory of Structural Biology at the National Institutes of Health (NIH), Bethesda, MD (URL: http://www.mgsl.dcrt.nih.gov/docs/osmdata/osmdata. html). The fractional ¯uorescence quenching (Qobs) was calculated as (I0 ÿ I)/I0 where I0 and I represent the protein ¯uorescence observed in the absence and presence of DNA, respectively. The concentration of bound Sso7d was calculated as: ‰Sso7dŠbound ˆ ‰Sso7dŠtot

Qobs Qmax

…5†

where [Sso7d]tot is the total protein concentration and Qmax is the maximum quenching, i.e. the quenching observed when all the protein in the sample is bound to DNA. The analysis assumes that the fractional change in ¯uorescence quenching upon DNA-binding is equal to the fraction of bound protein. The validity of this assumption has been veri®ed for the homologous Sac7d protein (McAfee et al., 1996), using a model independent ligand binding density method (Bujalowski & Lohman, 1987). Isothermal titration calorimetry (ITC) Calorimetric titrations were carried out using the MCS-ITC instrument from MicroCal (Amherst, MA). All solutions were degassed before the titrations using equipment provided with the instrument. Protein and DNA samples for studies of temperature dependence were dialyzed against buffer B and aliquoted in appropriate volumes for each individual titration. The pH of buffer B was adjusted prior to dialysis to allow studies at pH 7.1 and 8.0. Dialyzed samples were stored in the freezer and were thawed and heated to the appropriate temperature immediately before use. Sso7d solutions (0.8 mM) were titrated into poly(dGdC) solutions (0.13 mM base-pairs) using a 130 ml syringe. Each titration consisted of a preliminary 2 ml injection followed by 14 subsequent 8 ml injections. Protein and DNA samples for the experiments comparing binding to poly(dGdC) and poly(dAdT) were dialyzed against buffer C. Sso7d solutions (0.14 mM) were in this case titrated into poly(dGdC) (65 mM base-pairs) or poly(dAdT) (73 mM base-pairs) solutions using a 300 ml syringe, and each titration consisted of 24 subsequent 12.5 ml injections. Heats of dilution (
Hd) were measured in corresponding blank titrations by adding protein to buffer and were found to be small and similar to the heats observed at the end of the DNA ± protein titrations. Still, small ¯uctuations (less than 0.5 kcal molÿ1) in the observed magnitude of
Hd could not be avoided and measured binding enthalpies were therefore corrected for
Hd as described below. Protein and DNA samples for the (calorimetric) osmotic stress experiments were dialyzed against buffer A and aliquoted in appropriate volumes for each individual titration, followed by freezing and lyophilization. Experiments were performed at different concentrations of neutral solutes (betaine and sucrose) by resuspending

the lyophilized samples in the appropriate solute ± water solution. Sso7d solutions (0.6 mM) were titrated into poly(dGdC) solutions (0.11 mM base-pairs) using a 130 ml syringe, where each titration consisted of a preliminary 1 ml injection followed by 19 subsequent 6 ml additions.
Hd values were measured in blank titrations by adding protein to buffer and were found to be similar to the heats observed at the end of the DNA± protein titrations. A small continuous change in
Hd (in no case larger than 0.7 kcal molÿ1 between the ®rst and the last injection) was observed for titrations with different reaction buffers. This variation was ascribed to the imprecision of matching the buffer conditions between the calorimeter cell and the injection syringe, as samples were prepared by resuspending lyophilized samples in different solvent volumes. Linear regression was in this case therefore applied to analyze the trends in
Hd and
H was subsequently corrected by subtracting the regression line. Small ¯uctuations (less than 0.5 kcal molÿ1) in the average magnitude of
Hd could also be seen and
H was therefore additionally corrected by subtracting a constant value as described in the following section. Analysis of binding isotherms The binding of Sso7d to polymeric DNA was evaluated using a model (McGhee & von Hippel, 1974) for binding of non-interacting ligands to a linear lattice of overlapping binding sites:  nÿ1 r 1 ÿ nr …6† ˆ K…1 ÿ nr† L 1 ÿ …n ÿ 1†r The equation is in the Scatchard form where r ˆ [Sso7d]bound/[DNA bp] and L ˆ [Sso7d]free. K is the association constant and n is the number of lattice residues, i.e. DNA base-pairs, covered by one ligand. All experimental data could be represented by this model and there was therefore no need to include a cooperative protein interaction parameter in the evaluation. (Noncooperative binding behavior has also been observed for interactions between the homologous Sac7d protein and different DNA polymers (McAfee et al., 1996).) In the case of equilibrium data based on ¯uorescence titrations there are three adjustable parameters: K, n and Qmax. These were determined by non-linear least-square ®ts of the binding model (equation (6)) to experimental data ([Sso7d]bound and [Sso7d]free was calculated from equation (5) using routines available within the Mathematica program package (Wolfram Research). Since this procedure includes ®nding a best-®t value of Qmax, the fraction of Sso7d protein bound to DNA was recalculated for each value of Qmax during the ®tting process. Reported equilibrium parameters and errors represent mean values and estimated standard deviations, respectively, for three independent experimental titrations. For equilibrium data obtained using ITC there are also three adjustable parameters: K, n and
H . The r parameter represents the fractional occupancy of DNA by ligand and the reaction heat content Qi of the calorimetrically sensed solution volume V0 at a particular value of ri is therefore: Qi ˆ ri ‰DNA bpŠ
H  V0 

…7†

where
H is the observed binding enthalpy. The experimental observable is the heat released and sensed by the instrument at the ith injection (
Qi). When corrections have been made for the solution displaced by each

785

Thermodynamics of Sso7d DNA Interactions injection, which contributes to the heat sensed by the titration cell before it passes out of the calorimetrically sensed volume, this observable is given by (MicroCal Users Manual):   Vi Qi ‡ Qiÿ1 ÿ Qiÿ1 …8†
Qi ˆ Qi ‡ V0 2 where Vi is the injection volume at the ith injection. K, n and
H were determined from nonlinear leastsquare ®ts of the binding isotherm (equation (8)) in which Qi is calculated from equation (7) and ri is calculated from equation (6). Unless otherwise noted, we report equilibrium parameters determined from two independent titrations at each experimental condition. Possible systematic errors in given equilibrium parameters were estimated from the dependence of ®tted parameters on uncertainties in protein and DNA concentrations (5%) and from the uncertainties associated with
Hd, as mentioned above. We chose to ®t an additional constant
Hd value, which is subtracted from the observed heats of binding, together with K, n and
H for all experiments. This is analogous to a procedure in which the heat of dilution is estimated based on the last points of each titration. As already mentioned, blank titrations were carried out to ensure that the heat of dilution remained constant throughout the titrations and close to the values observed at the end of the DNA ± protein titrations. The ®tted value of
Hd was in no case larger than 0.5 kcal molÿ1, which is comparable with other experimental errors. The binding free energy (
G ) and entropy (ÿT
S ) were calculated as
G ˆ ÿ RT ln(K) and T
S ˆ
H ÿ
G . The different thermodynamic quantities reported here are determined based on total macromolecular concentrations with a standard state of 1 M, neglecting non-ideality of the solution. Thus, they are valid only at the speci®ed conditions. Osmotic stress experiments A methodology to probe the amount of water that is released upon complexation is based on measurements of binding af®nities as a function of the solution osmotic pressure (Parsegian et al., 1995). By adding inert and neutral solutes to the binding buffer one can change the chemical potential of water in a direct and controlled manner. If the binding equilibrium is associated with a net release of waters and the added solutes are excluded from the hydration compartment surrounding the macromolecules, then the increased osmotic pressure will drive the equilibrium towards its more dehydrated state. The shift in the binding constant (K) with osmotic pressure (osm) is directly related to the (net) volume of water (
V) that is released in the reaction as: @‰ln KŠ
V ˆÿ …9† @osm osm ˆ0 kT Dividing
V by the volume of a water molecule Ê 3) gives the number of water molecules (nw  30 A released or taken up (
Nw ˆ
V/nw). The change in osmotic pressure (dosm) is directly related to the change in chemical potential of water through nwdosm ˆ dmw. In Figure 3, we report osmotic pressure in osmolal units, so that osm ˆ (kT/nw) (osmolality/55.6), where 55.6 is the molarity of pure water. A number of considerations have to be made before a shift in an equilibrium is interpreted in terms of hydration effects (Parsegian et al.,

1995). These include possible pH effects, changes in dielectric constant or activity of other co-solutes, protein denaturation or aggregation, and the fact that the solute may not be completely excluded from the immediate surroundings or even interacts directly with the macromolecules. To estimate if, and to what extent, these mechanisms in¯uence the analysis, one normally uses several solutes which differ in size and chemical character.

Acknowledgments This work was supported by the Swedish Natural Sciences Research Council and the Magnus Bergvall Foundation.

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Edited by A. R. Fersht (Received 20 August 1997; received in revised form 10 November 1997; accepted 26 November 1997)