J. Mol. Biol. (1995) 252, 133–143

Destabilization of a Protein Helix byElectrostatic Interactions

Stefan Walter 1, Bernd Hubner 2, Ulrich Hahn 2 and Franz X. Schmid 1*

Electrostatic interactions between charged residues and the helix dipole in1Laboratorium fur Biochemiea protein were investigated by protein engineering methods. In ribonucleaseUniversitat BayreuthT1, two surface-exposed acidic residues (Glu28 and Asp29) are located nearD-95440 Bayreuth, Germanythe carboxyl terminus of the a-helix between residues 13 and 29. They were2Institut fur Biochemie replaced, individually and in concert, by the uncharged amides Gln28 and

Medizinische Universitat zu Asn29, and the stabilities of the wild-type protein and its variants wereLubeck, D-23538 Lubeck determined as a function of pH. The effects of the two mutations are additive.Germany Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to

a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that thedeprotonations of Glu28 and Asp29 reduce the free energy of stabilizationof folded ribonuclease T1 by about 4 kJ/mol each. This destabilization isprobably caused by unfavorable electrostatic interactions of Glu28 andAsp29 with the negative end of the helix dipole. The activation energies forthe unfolding of the different variants of ribonuclease T1 change in parallelwith the differences in the thermodynamic stability when the pH is varied.This indicates that the unfavorable electrostatic interactions of Glu28 andAsp29 are lost very early in unfolding, and are not present in the activatedstate of unfolding.

7 1995 Academic Press Limited

Keywords: protein stability; protein folding; protein engineering;electrostatic interactions; a-helix*Corresponding author

Introduction

The conformational stability of a folded protein isusually very low and results from a multitude oflocal as well as non-local interactions. Two majorexperimental approaches have been used tocharacterize individual contributions to proteinstability: the analysis of protein variants with alteredamino acid sequences (Matthews, 1991, 1993, 1995;Fersht & Serrano, 1993), and the investigationof model peptides (Scholtz & Baldwin, 1992;Chakrabartty & Baldwin, 1995). In particular, thestabilities of helical peptides with varying sequenceshave been examined in great detail, and the factorsthat determine the stability of an isolated helix arenow well known from these studies (Gans et al.,1991; Scholtz & Baldwin, 1992; Park et al., 1993;Chakrabartty et al., 1994).

Helices can be divided into three regions; theamino-terminal region, the helix center, and the

carboxyl-terminal region. For the helix center,several scales of helix propensities have beenproposed to explain the effects of sequencevariations on stability (O’Neil & DeGrado, 1990; Lyuet al., 1990; Gans et al., 1991; Horovitz et al., 1992;Blaber et al., 1993; Park et al., 1993; Chakrabartty et al.,1994). For the helix termini, two aspects areimportant. Firstly, the three amino-terminal and thethree carboxyl-terminal residues are not engaged ini, i + 4 hydrogen bonding, and secondly, the helixmacro-dipole is orientated such that a partialpositive charge is located near the N terminus and apartial negative charge near the C terminus. As aconsequence, helices in model peptides are stabil-ized by hydrogen bond acceptors at the N-capposition, and by negatively charged amino acidsin the N-terminal region, which interact favorablywith the helix dipole (Chakrabartty et al., 1993; Lyuet al., 1993; Doig et al., 1994). The strength of theelectrical field at the amino terminus of a peptidehelix has been characterized by Lockhart & Kim(1992).

The statistical analysis of folded proteins supportsthese results, and shows that negatively chargedresidues are strongly preferred in the first turn ofhelices in proteins (Richardson & Richardson, 1988;

Present address: Ulrich Hahn, Institut fur Biochemie,Universitat Leipzig, D-04103 Leipzig, Germany.

Abbreviations used: RNase T1, ribonuclease T1; N,native protein; U, unfolded protein; GpC,guanylyl(3'-5')-cytidine.

0022–2836/95/360133–11 $12.00/0 7 1995 Academic Press Limited

Electrostatic Destabilization of a Protein Helix134

Figure 1. A drawing of the backbone conformation ofRNase T1 showing the residues of interest. The two acidicresidues, Glu28 and Asp29, are located near the C-terminalend of the single a-helix of RNase T1, which extends fromresidues 13 to 29. The subsequent loop contains anotherGlu at position 31. Lys25 and Glu82 are also shown. TheFigure was drawn with the program MOLSCRIPT(Kraulis, 1991) using the coordinates of Martinez-Oyanedel et al. (1991).

RNase T1 are engaged in unfavorable electrostaticinteractions with the macro-dipole of the 13-29a-helix. To detect and characterize such interactions,we compared the stability of four variants of theprotein: the wild-type protein with both acidicresidues, E28Q-RNase T1, in which Glu28 is replacedby Gln, D29N-RNase T1, in which Asp29 is replacedby Asn, and E28Q/D29N-RNase T1, which hasboth mutations. The individual contributions of thenegatively charged forms of Glu28 and Asp29 to thefree energy of stabilization of RNase T1, DGstab , can bedetermined when the DGstab values of the wild-typeand the mutant proteins are known as a function ofpH. Protein stability and ionization are linked, andtherefore the dependence on pH of the difference instability depends on the shifts in the pK values ofGlu28 and Asp29 upon unfolding. The doublemutant E28Q/D29N-RNase T1 is included to find outwhether the effects of these two neighboring acidicresidues on protein stability are additive.

Results

Characterization of the variant proteins

Despite its function as an RNA-cleaving enzyme,RNase T1 is an acidic protein with an isoelectric pointof 3.8 (Iida & Ooi, 1969). The wild-type proteindisplays about 11 negative charges at pH 9 (Pace et al.,1991), and differences in single charges can beidentified by native polyacrylamide gel electrophor-esis at pH 9.2 (Ornstein, 1964). The Glu28 : Glnand the Asp29 : Asn mutations led to identicaldecreases in the electrophoretic mobility of RNaseT1, and in the E28Q/D29N variant the retardationscaused by these two mutations were approximatelyadditive. An analysis by mass spectrometry gaveidentical Mr values for all variants, as expected.

The reduction of the net charge by theGlu28 : Gln and the Asp29 : Asn mutations leftthe enzymatic activity of RNase T1 virtuallyunchanged. The activities of the wild-type protein,the two single mutants and the double mutanttowards the dinucleotide GpC and towards thenatural substrate RNA were measured at pH 7.5and 20°C. Under these conditions, the measuredactivities were all identical within experimental error(Table 1).

Glu28 and Asp29 are located at the C-terminal endof the 13-29 helix. The CD of RNase T1 in the far-UVregion is dominated by the contribution of this helix.To detect potential differences in helicity betweenthe wild-type protein and the variants, their CDspectra were measured at pH 2.5 and pH 7.0. Thespectra of all forms at both pH values were foundto be virtually identical (data not shown). Thisdemonstrates that neither the deprotonations ofGlu28 and Asp29, nor their replacements with Gln28and Asn29, led to significant changes in helicalstructure.

The Glu28 : Gln and the Asp29 : Asn mutationsalso did not affect the absorbance or the fluorescence

Dasgupta & Bell, 1993; Harper & Rose, 1993).Experimentally, the roles of the residues at the N-capand in the N-terminal region of helices in severalproteins have been investigated by protein engin-eering methods, and a qualitative agreement with thestatistical analyses has been found (Nicholson et al.,1991; Sancho et al., 1992; Serrano et al., 1992; Bell et al.,1992).

In contrast to the strong preference for negativelycharged residues in the N-terminal regions of helicesin proteins, there is only a marginal trend towardspositively charged residues near the C termini, andit is unclear whether charge-helix dipole interactionsat the carboxyl ends of helices are of similarimportance for the stability of folded proteins. Inbarnase, a helix is stabilized by a C-terminal Hisresidue, which is involved in both electrostaticinteractions and hydrogen bonding (Sali et al., 1988).

In this work, we have investigated the roleof electrostatic interactions at the carboxyl terminusof a helix in protein stability by using ribonucleaseT1 (RNase T1) from Aspergillus oryzae as a modelprotein. RNase T1 is a small protein of 104 residueswith a single a-helix that extends from residues 13 to29 (Heinemann & Saenger, 1982; Martinez-Oyanedelet al., 1991). Two negatively charged residues, Glu28and Asp29, are located at solvent-exposed positionsnear its carboxyl terminus (Figure 1). These tworesidues are not conserved in evolution, and, in mostrelated RNases, small uncharged residues occur atthe positions equivalent to Glu28 and Asp29 ofRNase T1 (Heinemann & Hahn, 1989).

We investigate in this work whether the negativelycharged side-chains of Glu28 and Asp29 in native

Electrostatic Destabilization of a Protein Helix 135

Table 1. Relative enzymatic activities of the variantstowards the substrates GpC and yeast RNAVariant GpC Yeast RNA

Wild-typea 100 (28) 100 (28)E28Q 113 (24) 107 (22)D29N 100 (25) 100 (24)E28Q/D29N 106 (25) 88 (25)

The hydrolysis of GpC (0.025 mg/ml) was measured by thechange in differential absorbance A280 nm − A500 nm. The solventconditions were 50 mM Tris-HCl (pH 7.5), 2 mM EDTA at 20°C.The hydrolysis of yeast RNA (1.0 mg/ml) was measured by thechange in differential absorbance A300 nm − A350 nm. The solventconditions were 50 mM Tris-HCl (pH 7.5), 2 mM EDTA at 20°C.

a The enzymatic activity of the wild-type protein towards theindicated substrate was set to 100.

equal to the stability of the protonated form, DG0stab ,

modified by a pH-dependent energy term which isdetermined by the difference between pKN and pKU,the pK values of the acidic residue in the native andin the unfolded states, respectively.

K0stab·KU

a = K −stab·KN

a (1)

Kappstab = [N−] + [NH]

[U−] + [UH] = K0stab

[H+] + KNa

[H+] + KUa

(2)

DGstab = DG0stab − R·T·ln 10−pH + 10−pKN

10−pH + 10−pKU (3)

The expression in equation (3) indicates that thestabilizing or destabilizing effect of a charged groupcan be determined quantitatively by measuring DGstab

as a function of pH. Unfortunately, proteins containmany ionizable groups, and each of them caninfluence the conformational stability by an energyterm, as in equation (3).

Protein engineering can be used to determine thelinkage between the protonation of one particularcharged residue and protein stability (as in equa-tion (3)). In this approach, the residue of interest isreplaced by an uncharged amino acid, preferably ofsimilar size and polarity (e.g. the replacement of Aspand Glu residues with Asn and Gln, respectively).The stabilities of both the wild-type protein (DGwt

stab )and the variant (DGvar

stab ) are measured as a function ofpH, and the difference in stability (DDGstab ) shouldreflect the influence of the replaced residue on thestability of the wild-type protein. The analysis ofDDGstab as a function of pH should thus yield the pKvalues of this residue in the folded and unfoldedstates, and the effect of its ionization on proteinstability. The relation between DDGstab and pH isgiven in equation (4), which is formally equivalent toequation (3).

DDGstab = DGvarstab − DGwt

stab

= DDG0stab + R·T·ln 10−pH + 10−pKN

10−pH + 10−pKU (4)

Experimental DDGstab values will follow equation (4)only when pKN and pKU are independent of pH. ThepK values of ionizable groups in the native, as wellas in the unfolded, state depend on the local chargedistribution, which can change as a function of pH.Large changes can occur, in particular, between pH 3and pH 6, where most of the acidic groups of aprotein become deprotonated. In such a case, the twopK values cannot be determined exactly. Neverthe-less, the stabilizing or destabilizing effect of thedeprotonation of the residue of interest is given bythe difference in DDGstab between pH 2 and pH 7,provided that this residue is protonated at pH 2and deprotonated at pH 7, which is a reasonableassumption for Glu and Asp residues.

Dependence on pH of the thermalstability of RNase T 1

The conformational stability of RNase T1 isstrongly dependent on pH between pH 2 and pH 8

spectra of the protein (data not shown). Thesespectroscopic data, and the conserved enzymaticactivity, suggest that the mutations at the C-terminalend of the 13-29 a-helix did not change the foldedstructure of RNase T1.

Thermodynamic linkage betweendeprotonation and protein unfolding

The pK value of an ionizable residue in a proteinis influenced by its local environment. Because theenvironment changes upon unfolding, the pK valuewill be different in the native and in the unfoldedstate. As a consequence, ionization and unfoldingbecome thermodynamically coupled. Scheme Idescribes the linkage between the denaturation of aprotein and the ionization of a single acidic residue.The system is described by four equilibriumconstants: the stability of the protonated and thedeprotonated states, K0

stab and K −stab , respectively, and

the ionization constants of the folded and theunfolded states, KN

a and KUa , respectively. These four

equilibrium constants are linked by the relation inequation (1).

U− + H+

KUa|{

UH

gh

K0stabgh

N− + H+|{KN

a

NH

K−stab

Scheme I. Thermodynamic linkage between theprotonation of a single residue and protein un-folding. The protonated forms of the unfolded and of thenative protein are denoted by UH and NH and thedeprotonated forms by U− and N−, respectively.

Usually, the protonated and deprotonated states ofa protein do not differ in their spectral properties,and cannot be discriminated in unfolding exper-iments. Therefore, an apparent stability constant,Kapp

stab , is derived from the analysis of equilibriumunfolding transitions, which is equal to the ratio ofthe concentrations of all folded and all unfoldedforms ([N−] + [NH])/([U−] + [UH]). The dependenceon pH of Kapp

stab is given by equation (2). From thisequation, the dependence on pH of the stability of theprotein, DGstab , is readily derived (equation (3)). It is

Electrostatic Destabilization of a Protein Helix136

Figure 2. Thermal unfolding transitions of wild-typeRNase T1 at various pH values: (w) 10 mM glycine/HCl(pH 2.0); (q) 10 mM Na-acetate/acetic acid (pH 5.0); (r)10 mM Na-cacodylate/HCl (pH 6.5). The protein concen-tration was 10 to 15 mM in 1 cm cells. Unfolding wasmonitored by the differential absorption y(T ) = A286 nm

− A274 nm. The transition curves were analyzed by anon-linear least squares fit of y(T ) versus T according toequation (6). The decrease in the fraction of foldedmolecules is shown as a function of temperature.

Figure 3. The dependence on pH of the stabilities of thewild-type protein and of the E28Q variant, measured bythermal unfolding experiments, as in Figure 2. Thefollowing buffers (10 mM) were used: glycine/HCl(pH 2.0/3.0), Na-formate/formic acid (pH 3.5/4.0), Na-acetate/acetic acid (pH 4.5/5.0/5.5), Na-cacodylate/HCl(pH 6.0/6.5/7.0), and Hepes/HCl (pH 8.0). A, DGstab at55°C of wild-type protein (q) and E28Q variant (w) as afunction of pH. DGstab (55°C) was calculated according toequation (7) using the thermodynamic parameters derivedfrom the relevant transition curve. B, The difference instability (DDGstab ) at 55°C between the wild-type proteinand the E28Q variant. The data points were fitted toequation (4) by a non-linear least squares method (——).The pK values obtained for Glu28 from this fit are 5.86 inthe native protein and 5.25 in the unfolded protein.

(Kiefhaber et al., 1990; Pace et al., 1991; Hu et al., 1992;Yu et al., 1994), reflecting the influence of the 12 Aspand Glu residues and the three His residues of thisprotein. Representative thermal unfolding tran-sitions, as measured by the decrease in absorbance at286 nm, are shown in Figure 2. The stability of theprotein shows a maximum near pH 5, and decreaseswhen the pH is either increased to 6.5 or lowered to2.0. The reversibility of unfolding is generally good,except at pH values near the isoelectric point (pH 3.5to 4.5), where the thermally unfolded protein tendsto aggregate.

The buffer concentrations were low (10 mM) inthese experiments to minimize the screening ofelectrostatic interactions, and buffers with lowenthalpies of ionization (glycine, formate, acetate,and cacodylate) were used to keep the variation ofthe pH with temperature small. To calculate DGstab ,the thermal unfolding transitions of RNase T1 wereanalyzed as described in Materials and Methods.The DGstab values were compared, not at 25°C, but at55°C. This temperature lies within the transitionregion for all forms of RNase T1 at all pH valuesbetween pH 2 and pH 8 (cf. Figure 2), and thereforethe error prone extrapolation of the stability datacould be avoided.

Influence of the ionization of Glu28 on thethermal stability

To determine the influence of the negativelycharged side-chain of Glu28 on DGstab , we measuredthe thermal unfolding of both the wild-type proteinand the E28Q variant between pH 2 and pH 8. Thestabilities at 55°C of the wild-type protein and of theE28Q variant are compared in Figure 3A as a

function of pH. In both cases, bell-shaped curvesare observed, with maximal stabilities reached nearpH 5. Between pH 2.0 and pH 4.5, the DGstab valuesof the wild-type protein and the E28Q variant arevery similar, but above pH 5.0 the E28Q variantbecomes progressively more stable than thewild-type protein. This is clearly seen in Figure 3B,which shows the difference in stability between thetwo forms, DDGstab . Below pH 3.5, the E28Q variantis about 0.7 kJ/mol less stable than the wild-typeprotein, indicating that the replacement of theprotonated glutamic acid at position 28 by glutaminemarginally destabilizes RNase T1. With increasingpH, however, the E28Q variant becomes increasingly

Electrostatic Destabilization of a Protein Helix 137

more stable than the wild-type protein, and abovepH 7, the protein is stabilized by about 3.2 kJ/mol,due to the E28Q mutation. Taken together, thissuggests that the ionization of Glu28 leads to adestabilization of the folded wild-type protein byabout 4 kJ/mol.

If the folded conformation of a protein isdestabilized by an ionized carboxyl group, then thepK value of this group must decrease upon unfolding(cf. equation (4)). The data in Figure 3B followequation (4) very well. This indicates that the linkagebetween unfolding and the protonation of Glu28 isadequately represented by the thermodynamic cyclein Scheme I, and that its pK values in the folded andin the unfolded state are apparently independent ofpH. The fit of the experimental values for DDGstab toequation (4) is shown in Figure 3B. It yields pK valuesfor Glu28 of 5.25 in the unfolded protein, and of 5.86in the native state. This change in the pK of Glu28 by0.61 unit is equivalent to a decrease in proteinstability of 3.8 kJ/mol between pH 2 and pH 7 asobserved in Figure 3B.

Influence of the ionization of Glu28 on the rateof unfolding

The results in Figure 3B show that the ionized formof Glu28 reduces the difference in free energybetween the native and the unfolded state of RNaseT1. In the first approximation, we assume that thisdestabilization originates from an unfavorableelectrostatic interaction in the folded protein, and notfrom a favorable interaction in the unfolded state.A comparison of the rates of unfolding of thewild-type protein and the E28Q variant betweenpH 2 and pH 8 should reveal whether thisunfavorable interaction in the native protein is lostearly or late in the course of unfolding. If it is stillpresent in the activated state of the unfoldingreaction, then the difference in energy between thenative and the activated state should not change uponthe protonation of Glu28 or its mutation to Gln. Inthis case, the rate constants of unfolding of thewild-type protein and of the E28Q variant shouldfollow identical pH profiles. If the unfavorableinteraction is already disrupted in the activated state,then the energies of the native state and the activatedstate of unfolding should depend differently on pH.In this case, the difference in the rates of unfoldingof wild-type protein and the E28Q variant shoulddepend on pH in a fashion similar to the DDGstab

values in Figure 3B.The unfolding reactions of the wild-type protein

and the E28Q variant in 8 M urea were measuredas a function of pH by the decrease in proteinfluorescence. The time constants of unfolding of thetwo forms of RNase T1 (Figure 4A) follow closely theprofiles that were obtained for DGstab (cf. Figure 3A).With increasing pH, the time constants of unfold-ing of the two forms increase in parallel; unfoldingis slowest near pH 5, where protein stability ismaximal, and it then becomes faster again when the

Figure 4. The dependence on pH of the unfoldingkinetics of wild-type RNase T1 and the E28Q variant in 8 Murea, 25°C. Unfolding was initiated by a 80-fold dilution ofthe native protein into the cuvette to give a final proteinconcentration of 0.7 mM. Buffers as in Figure 3 were used.The kinetics were followed by the decrease in fluorescenceat 320 nm. Unfolding was, in all cases, a monoexponentialreaction. A, Time constants t of unfolding of the wild-typeprotein (q) and of the E28Q variant (w). B, The differencein log t between the wild-type protein and the E28Qvariant.

pH is further increased. It should be noted that thepH profiles for the time constants of unfolding areshifted by about 0.6 unit to higher pH values, relativeto the stability profiles in Figure 3A. This shift isobserved because the apparent pK values for protondissociation are increased by urea (Donovan et al.,1959). In a control experiment, it was found that thepK of acetic acid is increased from 4.7 in water to 5.4in 8 M urea (data not shown).

The difference between the logarithms of the timeconstants of unfolding of the wild-type protein andthe E28Q variant (D log t) is shown in Figure 4B asa function of pH. This curve is strikingly similar tothe pH profile for DDGstab in Figure 3B, when the shiftby 0.6 pH unit in the presence of 8 M urea is takeninto account. Under the assumption that theunfolding of RNase T1 can be described by trans-ition state theory, D log t is linearly related to the

Electrostatic Destabilization of a Protein Helix138

difference in activation energy of unfolding of thetwo forms, according to equation (5):

DDH$ = 2.303 RTD log t (5)

The change in D log t from −0.18 at pH 2 to +0.48at pH 8 is equivalent to a decrease by 3.8 kJ/mol inthe energy difference between the native state andthe activated state for unfolding caused by theionization of Glu28. This value agrees with theenergy difference of 3.8 kJ/mol between the nativestate and the unfolded state, as measured in theequilibrium unfolding experiments of Figure 3. Thiscorrespondence of the changes in equilibrium withactivation energies suggests that the destabilizinginteraction of the negatively charged Glu28 is alreadylost when the activated state of unfolding is reached.

Influence of the Asp29 : Asn mutation onstability and the unfolding kinetics

The thermal unfolding of D29N-RNase T1 was alsomeasured between pH 2 and pH 8, and the freeenergy of stabilization DGstab was determined at 55°Cas outlined for the E28Q variant. The DDGstab valuesare shown in Figure 5A. Again, the substitution ofthe protonated aspartic acid by an asparagineresidue is slightly destabilizing and the D29N variantis about 0.8 kJ/mol less stable than the wild-typeprotein below pH 3. The stability of the variantincreases more strongly with pH, however, andabove pH 7 it is about 3.3 kJ/mol more stable than thewild-type protein. Unlike the E28Q variant, the pHprofile for D29N-RNase T1 does not follow therelation in equation (4), presumably because the pKvalues of Asp29 in the folded and/or in the unfoldedwild-type protein change with pH. As a conse-quence, the shift in the pK of Asp29 upon unfoldingcannot be determined from the results in Figure 5A.Still, these results show that the ionized form ofAsp29 destabilizes folded RNase T1 by about4 kJ/mol, a value that is very similar to the de-stabilization caused by the ionization of Glu28(cf. Figure 3A).

The kinetics of unfolding by 8 M urea ofD29N-RNase T1 were also measured, and thedifference in the time constants of the unfoldingreactions of the wild-type and the D29N proteins,D log t, is shown in Figure 5B as a function of pH. TheD29N variant unfolds faster than the wild-typeprotein at pH 2.0, but slower than the wild-typeprotein at pH 7. As in the case of the E28Q variant,the dependences on pH of D log t in Figure 5B andof DDGstab in Figure 5A are very similar, and thedecrease in the energy of activation between pH 2and pH 8 that is caused by the ionization of Asp29and calculated in equation (5) amounts to about3.7 kJ/mol. This value is again very similar to thedecrease by 4 kJ/mol in the thermodynamic stability,as shown in Figure 5A.

Figure 5. A, The dependence on pH of the stabilities ofthe wild-type protein and the D29N variant. The stabilitiesof the proteins were measured by thermal unfolding andanalyzed as described in Figure 3. The difference instability (DDGstab ) at 55°C between the wild-type proteinand the D29N variant is shown as a function of pH. B, Thedependence on pH of the unfolding kinetics of wild-typeRNase T1 and of the D29N variant in 8 M urea at 25°C. Theunfolding experiments were carried out as described inFigure 4. The difference in log t of unfolding between thewild-type protein and the D29N variant is shown as afunction of pH.

Additivity of the effects of the Glu28 : Gln andthe Asp29 : Asn mutations

Glu28 and Asp29 are adjacent in sequence, and theobserved changes in stability after mutating theseresidues individually could be caused by a directunfavorable interaction between them, or by aninteraction of either one with the helix dipole in amutually exclusive fashion. In these cases, the effectsof both mutations combined in the same moleculeshould not be additive. Accordingly, a variant withboth mutations was constructed, and its stability andthe kinetics of its unfolding were investigated. Theresulting DDGstab values between the E28Q/D29Nvariant and the wild-type protein are shown inFigure 6, and compared with the sum of the DDGstab

values obtained for the single mutants (taken fromFigures 3B and 5A). To a first approximation, the

Electrostatic Destabilization of a Protein Helix 139

Figure 6. Comparison between the differential stabilityof the E28Q/D29N double mutant (W) and of the sum ofthe differential stabilities of the E28Q and the D29N singlemutants (w). The stability of the E28Q/D29N doublemutant was determined as a function of pH from thermalunfolding transitions as described in Figure 3. The data forthe stabilities of the single mutants were taken fromFigures 3B and 5A.

the rate of unfolding of the wild-type protein, asexpected for independent mutations.

Discussion

The replacement of the two negatively chargedresidues, Glu28 and Asp29, near the carboxylterminus of the single helix of RNase T1 by theuncharged residues Gln28 and Asn29 influenced thestability of the protein in a pH-dependent manner.At pH 2 both mutations were almost neutral, but atpH 7 they stabilized the protein significantly. Thissuggests that in the ionized form, the side-chainsof Glu28 and Asp29 participate in destabilizinginteractions in the native wild-type protein. Theseunfavorable interactions are already lost when theactivated state of unfolding is reached, and thechanges in activation energy parallel closely thechanges in thermodynamic stability when pH isvaried.

Ionization and unfolding are thermodynamicallycoupled, and thermodynamic cycles such as thatshown in Scheme I can be used to evaluate thecontributions of individual ionizable groups toprotein stability. Many amino acids of the same type(Asp and Glu in our case) exist in a protein, andtherefore a comparative analysis of protein variantswith single substitutions is necessary to derive theseindividual contributions experimentally. Such vari-ants are, however, chemically different species, andin principle they cannot be related by thermodyn-amic cycles similar to the one for protonation inScheme I. In this work, we have minimized thisproblem by making the smallest changes possible(the substitutions of the carboxylic acids by thecorresponding amides) at solvent-exposed sites.These changes did not alter the molecular weight orthe number of non-hydrogen atoms. As a conse-quence, at pH 2, where Glu28 and Asp29 areprotonated, the protein stability remained almostunchanged after the mutations to Gln28 and Asn29.When the original amino acid and its substitutediffer in the number of atoms or covalent bonds, thenthe molecular interpretation of the changes in proteinstability after a substitution is generally much moredifficult (Tidor & Karplus, 1991).

At pH 7, Glu28 and Asp29 are negatively charged,and both destabilize the folded conformation ofRNase T1 by about 4 kJ/mol each in an additivefashion. In the case of Glu28, this decrease in stabilitycould be correlated with an increase in pK from 5.25in the unfolded state to 5.86 in the folded state, anda good fit of the experimental data to the simplemodel in Scheme I was obtained, indicating that, toa first approximation, these pK values are indepen-dent of pH between pH 5 and pH 7, where the majorchanges in stability occur (cf. Figure 3B). In this pHrange, the net charge of the protein changes onlyslightly, because most of the other acidic residues arealready negatively charged.

In contrast, the changes in stability caused by theionization of Asp29 begin at pH 3, and continue untilpH 7 is reached (cf. Figure 5A). This suggests that the

stabilizing effects of the two mutations in theE28Q/D29N variant are equal to the sum of theirindividual effects in the single mutants, whichindicates that the two mutations are independent ofeach other.

A similar result was obtained when the kinetics ofunfolding of the wild-type protein, the two singlemutants and the double mutant were compared atpH 3.0 and at pH 7.0 (Table 2). At pH 3.0, theunfolding of the E28Q and the D29N variants is 1.7-and 1.8-fold faster, respectively, and the unfolding ofthe E28Q/D29N double mutant is 2.7-fold faster thanthe unfolding of the wild-type protein. A threefoldincrease in the rate of unfolding would have beenexpected if the effects of the two mutations werestrictly additive. At pH 7.0, all variants unfold moreslowly than the wild-type protein. Unfolding of theE28Q and the D29N variants are 0.4- and 0.5-foldas fast, respectively, and the unfolding of theE28Q/D29N double mutant decreased to 0.2 times

Table 2. Kinetics of urea-induced unfolding at 25°CpH 3 pH 7

Variant t (s)a Rate factorb t (s) Rate factor

Wild-type 39.8 1.00 218 1.00E28Q 23.3 1.71 565 0.39D29N 22.5 1.77 479 0.46E28Q/D29N 14.6 2.73 1040 0.21

Unfolding experiments were carried out at 25°C in 10 mMglycine/HCl (pH 3), 7.0 M urea, and in 10 mM Na-cacodylate/HCl (pH 7), 8.0 M urea, as described in Figure 4.

a Time constant of unfolding; t was determined by fitting thetime course of the unfolding reaction to a monoexponentialfunction.

b The rate factor of a variant protein is equal to the ratiot(wild-type)/t(variant).

Electrostatic Destabilization of a Protein Helix140

pK values of this residue in the unfolded and/or inthe folded state are fairly low in an unchargedenvironment at low pH. The pK values increase,however, when the pH increases, and the environ-ment becomes more negatively charged. Thisvariation of the pK values leads to the observedbroad change in DDGstab with pH in Figure 5A.

Charged side-chains can destabilize a foldedprotein: (1) when they are less exposed to solvent inthe folded state than in the unfolded state, (2) whenlocal electrostatic repulsions with other charges ofthe same sign occur, (3) when they interactunfavorably with partial charges, such as peptidedipoles or the helix macro-dipole, and (4) whenexisting hydrogen bonds are disrupted by deproto-nation. Both Glu28 and Asp29 are accessible tosolvent in the folded state; therefore, desolvationshould not be a major source of destabilization.Also, there is no evidence for hydrogen bondinginteractions that could stabilize the protonated formsof Glu28 and Asp29 from the X-ray structuredetermination (Martinez-Oyanedel et al., 1991).

Increased repulsive interactions with neighboringnegative charges probably contribute to the observeddestabilization. Even in the unfolded protein, thenegative charge density is high, because Glu28,Asp29 and Glu31 are close in sequence. This couldexplain why the pK value of Glu28 is higher then fivein the unfolded protein. In the folded state (cf.Martinez-Oyanedel et al., 1991), Glu82, the negativeend of the helix dipole, and the amino group of Lys25are located near the side-chains of Glu28 and Asp29.The distances between the amino group of Lys25and the carboxylate groups of Glu28 and Asp29 areboth about 4 A. The carboxylate groups of Glu28 andAsp29 are separated by about 7 A, and the distancesfrom them to the carboxylate of Glu31 are 13 and 7 A,respectively. Glu82 is 10 and 13 A distant from Glu28and Asp29, respectively.

In folded RNase T1, the carboxyl-terminal end ofthe 13-29 helix is thus immersed into a region of highnegative charge density (cf. Figure 1). The stability ofthe protein increases when the number of negativecharges in this region is reduced by the protonationor the replacement of one or two acidic residues.These stabilizing effects are additive, which rules outthe possibility that Glu28 and Asp29 specificallyinteract with each other, or with a third residue orstructural element, in a mutually exclusive fashion.Interactions between charged groups at the proteinsurface are expected to be very weak, because of theentropic cost of immobilizing the side-chains(Matthews, 1995). This explains the lack ofinteraction between the carboxylate groups of Glu28and Asp29. For the same reason, their interactionswith the amino group of Lys25 should be very weak.

The simplest explanation for the observeddestabilization of wild-type RNase T1 by deproto-nated Glu28 and Asp29 involves their unfavorableinteractions with the dipole of the 13-29 helix. Theintroduction of negative charges near the aminotermini of helices has been found to increase thestability of several proteins (Nicholson et al., 1991;

Sancho et al., 1992; Serrano et al., 1992; Bell et al.,1992). These effects can be additive, and the Tm valueof a polyalanine helix increased by 44 K when a blockof 20 Glu residues was moved from the carboxyl tothe amino terminus (Ihara et al., 1982). Regarding thiscomplexity, it remains a challenging task to developexperimental strategies to test the theoretical modelsfor the electrostatic interactions in proteins (Stigter &Dill, 1990; Yang & Honig, 1993).

Interestingly, the changes in the unfolding kineticsof both variants follow the changes in thethermodynamic stability. This suggests that theunfavorable interactions of Glu28 and Asp29 thatdestabilize the native protein do not destabilize theactivated state of unfolding, i.e. they are no longerpresent in this state. The repulsive interactions ofthese two residues with other negative charges andwith the helix dipole are apparently lost in a rapidlocal unfolding reaction. This reaction could involvethe fraying of the 13-29 helix at its C-terminal endbefore global unfolding takes place.

The loss in stability of 8 kJ/mol after thedeprotonation of Glu28 and Asp29 is significant,because at pH 7 and 25°C, the total free energy ofstabilization of RNase T1 is only about 20 kJ/mol(Pace et al., 1991). We speculate that these two acidicresidues have been conserved in RNase T1 becausethey are involved in some aspect of the function ofthis protein. Negative charges in this region, whichis remote from the catalytic region (cf. Figure 1),could help to direct the negatively charged substratesto the active site on the other side of the protein. Itis worth noting that Glu28 and Asp29 of RNase T1 arenot conserved in other microbial RNases, but in thesespecies Glu and Asp residues often occur at positionsthat are equivalent to Asn84 and Gln85 of RNase T1

(Heinemann & Hahn, 1989). These residues are nearthe 28-29 region in the folded proteins (cf. Figure 1).

Materials and Methods

Materials

RNase T1, and the variant proteins, were expressed inEscherichia coli strain DH5a transformed with the plasmidpA2T1, which harbored a chemically synthesized gene forRNase T1 (Quaas et al., 1988a,b). The respective mutationswere introduced by using the polymerase chain reaction asdescribed previously (Landt et al., 1990), except that bothreactions were carried out with one unit Vent DNApolymerase (New England Biolabs, Beverly, MA) under theconditions proposed by the manufacturer, with 100 ngpA2T1 as template, 1 mM of each primer and 400 mM ofeach dNTP. Primers used were 5'-TACGGATTCACTG-GAACT-3' (5' universal primer), 5'-CATCTTAGCAGCCT-GAAC-3' (3' universal primer), 5'-CAGTTTCACCG-TCTTGGTGAAGTT-3' (E28Q mutagenesis primer), 5'-CAGTTTCACCGTTTTCGTGAAGTT-3' (D29N mutagen-esis primer), and 5'-CAGTTTCACCGTTTTGGTGAA-GTT-3' (E28Q/D29N mutagenesis primer; base substi-tutions are underlined). The wild-type protein and thevariants were purified as described (Mayr & Schmid,1993).

Urea (ultrapure) was obtained from ICN (Aurora, OH).Yeast RNA was obtained from Boehringer Mannheim

Electrostatic Destabilization of a Protein Helix 141

(Mannheim, Germany), and guanylyl (3'-5')cytidine (GpC)from Pharma Waldhof (Dusseldorf, Germany). All otherchemicals were obtained from Merck (Darmstadt,Germany). The concentrations of wild-type RNase T1 andits variants were determined spectrophotometrically byusing A1 cm

278 = 1.9 for a 1 mg/ml solution of the wild-typeprotein and its variants (Takahashi et al., 1970; Yu et al.,1994). To remove low molecular mass impurities, allsolutions of RNase T1 were filtered over NAP-10 columns(Pharmacia, Uppsala, Sweden) and equilibrated with therespective buffer. For optical measurements, a HitachiF-4010 fluorescence spectrometer, a Jasco J600A spectro-polarimeter, a Hewlett-Packard 8452A diode arrayspectrophotometer, and a Kontron Uvikon 860 spectropho-tometer were used.

Gel electrophoresis under native conditions

The differences in the net charge that result fromthe E28Q and D29N substitutions in RNase T1 wereinvestigated by discontinuous electrophoresis with SDS-15% (w/v) polyacrylamide gels under non-denaturingconditions by using the buffer system of Ornstein (1964).The electrophoresis was performed at 10°C in a Midgetgel apparatus (Hoefer, San Francisco, CA). The pHvalue of the separation gel was 9.2. Gels were fixed in20% (w/v) trichloroacetic acid and stained with 0.1%(w/v) Coomassie brilliant blue G in 10% (v/v) glacialacetic acid and 50% (v/v) methanol. The destainingsolution contained 10% (v/v) glacial acetic acid and 5%(v/v) methanol.

Activity assays

The enzymatic activities of wild-type RNase T1 and itsvariants were determined using two different assays, withthe dinucleotide GpC and yeast RNA as substrates(Oshima et al., 1976; Grunert et al., 1991). A 990 ml sampleof the assay solution (0.025 mg/ml GpC or 1 mg/ml yeastRNA in 50 mM Tris-HCl (pH 7.5), 2 mM EDTA) werepreincubated in a 1 cm cell with a stirring bar at 20°C forat least five minutes. The reaction was started by adding10 ml RNase T1 solution to give final protein concentrationsof 2.7 nM (GpC assay) and 10 nM (yeast RNA assay). Thetime course of hydrolysis was measured in a HP 8452Adiode array spectrophotometer for five minutes by usingthe differential absorptions A280 nm − A500 nm (GpC assay)and A300 nm − A350 nm (RNA assay) to follow the reactions.The initial slopes of Al1 − Al2(t) were calculated.Enzymatic activities were measured five times, andaveraged.

Circular dichroism

The CD spectra of the different variants of RNase T1

were measured in a JASCO J600A spectropolarimeter inthermostatically controlled cells at 25°C. The buffers were10 mM Na-phosphate (pH 2.5) and 10 mM Na-phosphate(pH 7.0). The protein concentration was 15 mM in a 1 mmcell. The bandwidth was 1 nm, and the scan rate was20 nm/min. The spectra were measured ten times, andaveraged.

Thermal denaturation

The conformational stabilities of wild-type RNase T1 andthe three variants at various pH values were determinedfrom thermal unfolding. The transitions were recorded

with a Hewlett-Packard 8452A diode array spectropho-tometer equipped with a Peltier element and a temperaturesensor which was inserted into the cell. The followingbuffers were used: glycine/HCl (pH 2.0/3.0). Na-for-mate/formic acid (pH 3.5/4.0), Na-acetate/acetic acid(pH 4.5/5.0/5.5), Na-cacodylate/HCl (pH 6.0/6.5/7.0),and Hepes/HCl (pH 8.0). The protein concentration was10 to 15 mM in 1 cm cells, and the buffer concentrationswere 10 mM.

The samples were heated at a rate of 0.5 deg.C perminute in the baseline regions of the native and theunfolded state, and at a rate of 0.25 deg.C per minute in thetransition region. Spectra from 250 nm to 350 nm wererecorded at intervals of 1 deg.C in the baseline region, andof 0.5 deg.C in the transition region. The reversibility ofdenaturation was examined by cooling the samples to thestarting temperature.

The transition curves were analyzed by a non-linear least squares fit of the differential absorptiony(T ) = A286 nm − A274 nm as a function of temperature withthe program GraFit 3.0 (Erithacus Software, Staines, UK)according to equation (6) (Mayr et al., 1993). Equations (6)and (7) relate the change in differential absorption y withtemperature to the thermodynamics of a two-state foldingreaction, under the assumption that the enthalpy of foldingis a linear function of temperature (Privalov, 1979). Inaddition, it is assumed that the absorptions of the nativeprotein (yN ) and of the unfolded protein (yU ) dependlinearly on temperature. The values of y0

U and y0N are those

at T = 0 K, and mU and mN define the dependences ontemperature of yU and yN , respectively.

y(T ) = y0N + mN·T −

y0N + mN·T − y0

U − mU·T

1 + exp6 − DGstab (T )R·T 7

(6)

DGstab (T ) = DHm·01 − TTm1 − DCp·0Tm − T + T·ln0 T

Tm11(7)

In equation (7), Tm is the midpoint of thermal unfolding,DHm is the van’t Hoff enthalpy at Tm, and DCp is the changein heat capacity upon unfolding. A temperature-indepen-dent value of DCp = 5.2 kJ/(K·mol) was assumed for thewild-type protein and for its variants (Kiefhaber et al.,1990; Hu et al., 1992; Yu et al., 1994). The results obtainedfor DHm and Tm were used to calculate the conformationalstability at 55°C, DGstab (55°C), with equation (7). The set ofDGstab (55°C) values at a given pH was then used tocalculate the differential stabilities of the variant proteins(cf. equations (1) to (4)). An extrapolation of the values forDGstab could thus be avoided, and the accuracy of thedifferences in DGstab be improved.

pH dependence of unfolding

The unfolding rates of wild-type RNase T1 and itsvariants E28Q and D29N in 8 M urea were measured in thesame pH range and with the same buffers as the thermalstabilities. The kinetics were recorded with a HitachiF-4010 spectrofluorimeter. The wavelength of excitationwas 268 nm (1.5 nm band width), and the emission wasmeasured at 320 nm (10 nm band width). The responsetime of the instrument was 0.5 second. A 1580 ml sample of8.1 M urea in 10 mM buffer was thermostaticallymaintained at 25°C in a 1 cm × 1 cm fluorescence cell.

Electrostatic Destabilization of a Protein Helix142

Unfolding was started by the addition of 20 ml RNase T1 togive final concentrations of 8 M urea, 10 mM buffer and0.7 mM RNase T1. The solutions were mixed by a magneticstirrer in the cell, and the dead time of mixing was abouttwo seconds. The additivity of the mutational effects on theunfolding kinetics were measured in 10 mM glycine/HCl(pH 3), 7.0 M urea, and in 10 mM Na-cacodylate/HCl(pH 7), 8.0 M urea. All unfolding kinetics weremonoexponential reactions, and fitted to single exponentialfunctions by using the program GraFit 3.0.

AcknowledgementsThis work was supported by grants from the Deutsche

Forschungsgemeinschaft (to F.X.S. and U.H.) and theFonds der Chemischen Industrie (to S.W., F.X.S. andU.H.). We thank R. L. Baldwin, C. Frech, M. Mucke, andT. Schindler for discussions.

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Edited by P. E. Wright

(Received 17 March 1995; accepted 15 June 1995)