Native tertiary structure in an A-state1

J. Mol. Biol. (1998) 275, 379±388

Native Tertiary Structure in an A-state Jennifer L. Marmorino, Melissa Lehti and Gary J. Pielak* Department of Chemistry University of North Carolina at Chapel Hill, Chapel Hill NC 27599-3290, USA

The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, ¯uctuating tertiary structure. We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state. Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities,
GA>D and
GN>D. We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants. The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface. We also examined the pH and sulfate concentration dependencies and found that while these factors affect the farUV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants. A
GA>D versus-
GN>D plot for the interface variants has a slope near unity and the control variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, con®rming our preliminary report. We determined that the heat capacity change for A-state denaturation is 60% of the value for native-state denaturation, indicating that the A-state interior is native-like. We discuss our results in relation to ferricytochrome c folding kinetics. # 1998 Academic Press Limited

*Corresponding author

Keywords: circular dichroism; cytochrome c; molten globule; protein folding; protein stability

Introduction Protein folding cannot be a random process because it would take 1027 years for a 100 residue protein to sample even three conformations per residue (Zwanig et al., 1992). A popular idea is that early intermediates constrain the number of subsequent conformations a protein must sample as it proceeds to the native state. Early intermediates can be detected using rapid-mixing and other techniques, but are dif®cult to characterize because of their ¯eeting existence. The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway intermediate with native secondary structure and non-native, ¯uctuating tertiary structure (Ptitsyn, 1992). Certain kinetic intermediates are similar to the molten globule (Jennings & Wright, 1993; ColoÂn & Roder, 1996; Raschke & Marqusee, 1997). Thus, determining the Abbreviations used: CD, circular dichroism; wt, wildtype; 2 , secondary; 3 , tertiary. 0022±2836/98/020379±10 $25.00/0/mb971450

interactions that stabilize A-states may help explain protein folding. The A-state forms because low pH (2) denatures a protein by increasing its positive charge, while added salt screens charge repulsion so that the protein collapses to a compact form. At pH 2 in the absence of salt, most proteins are aciddenatured. For some proteins, decreasing the pH to <1.5 creates such a high acid concentration that the dissociated ions act as a salt to form the A-state (Fink et al., 1994). In general, A-states contain native or nativelike secondary (2 ) structures (Ptitsyn, 1992) and many A-states are nearly as compact as native proteins. Until recently, the extent to which native tertiary (3 ) interactions stabilize A-states (and molten globules) was unclear. We set out to explore 3 interactions in the A-state of yeast iso1-ferricytochrome c. Cytochromes c are small (108 amino acid residues, 12,700 Da) globular proteins. They contain a covalently linked heme that allows the protein to # 1998 Academic Press Limited

380

Helix-Helix Packing in Cytochrome c Table 1. Thermodynamic parameters for the N>D transitions Protein wt A7Y A7F A7L A7G

Tm (K)


Hm (kcal molÿ1)


Cp (kcal molÿ1 Kÿ1)


GN>D, 327.9 K (kcal molÿ1)

327.9a 329.1 327.6b 326.7 323.9b

84.8a 88.2 89.5b 84.2 82.8b

1.38  0.23 1.38  0.16 1.49  0.12 1.51  0.25 1.46  0.07

0 0.32  0.29 ÿ0.08  0.30 ÿ0.31  0.29 ÿ1.06  0.31

Data were acquired in 50 mM sodium acetate (pH 4.6). The uncertainties in Tm and
Hm(from repetition of the experiment) are 1.1 K and 3.9 kcal molÿ1 (Cohen & Pielak, 1994). Uncertainties in
Cp are from linear leastsquares ®ts of
Hm versus Tm plots. Uncertainties in
GN>D were estimated by using equation (16) from Cohen & Pielak (1994). a Average of seven determinations. b Average of two determinations.

shuttle between the reduced, diamagnetic [Fe(II),d6] ferro form and the oxidized, paramagnetic [Fe(III), low spin d5] ferri form. Cytochrome c, the only soluble protein of the eukaryotic respiratory chain, transfers electrons to cytochrome c oxidase. Yeast iso-1-cytochrome c is ideal for model studies because high-resolution X-ray crystal structures are available (Brayer & Murphy, 1996), the native state gives good 1H-NMR data (Pielak et al., 1996), and exhibits reversible, two-state denaturation (Cohen & Pielak, 1994, 1995). In addition, variants can be readily produced and variant functionality is easily tested in vivo. The most conserved feature of the cytochrome c fold is the interaction between the N and C-terminal helices (Matthews, 1985). In 94 of the 106 known wild-type (wt) eukaryotic cytochrome c sequences the interface includes the three conserved residues Phe10, Leu94, and Tyr97, and one invariant residue, Gly6. The helix-helix interaction is implicated in early kinetic folding intermediates (Roder et al., 1988; ColoÂn et al., 1996). Variants with amino acid substitutions at the interface have been made (Auld & Pielak, 1991; Fredericks & Pielak, 1993; Beasley & Pielak, 1996; ColoÂn et al., 1996) and native-state stabilities have been determined (Pielak et al., 1995; ColoÂn et al., 1996). The fold of these variants is the same as that of the wt protein because these variants are biologically active and NMR studies show little difference in 3 structure relative to the wt protein (Fredericks, 1993; Gochin & Roder, 1995). In a preliminary report we showed that the contact between the N and C-terminal helices in native iso-1-cytochrome c also stabilizes the A-state (Marmorino & Pielak, 1995). We accomplished this by comparing the A-state stabilities of interface variants to their native-state stabilities. To check if stability changes are due to interface disruption, two control variants with substitutions at Ala7, a position near (but not at) the interface were also tested. ColoÂn et al. (1996) have shown that the interaction of these helices stabilizes a kinetic intermediate. Tertiary A-state interactions have been reported for a-lactalbumin (Wu et al., 1995; Schulman et al., 1997) ubiquitin (Khorasanizadeh et al., 1996), myoglobin (Kay & Baldwin, 1996), and

RNase H (Raschke & Marqusee, 1997). See Baldwin (1996), Ptitsyn (1996) and Roder & ColoÂn (1997) for reviews. Our preliminary result warrants further investigation for three reasons. First, most experiments were performed only once. Second, far-UV CD spectra of the less stable variants depend on pH and ionic strength. Third, only two control variants were examined.

Results N>D transition N>D transitions were monitored by CD (ellipticity at 222 nm (222 nm)) as a function of temperature. Data were ®t to a two-state model using van't Hoff analysis to obtain Tm and
Hm. Values of
Cp are the slopes of
Hm versus Tm plots, where Tm is perturbed by changing pH and assuming that changes in heats of ionization are small and pH-independent.
GN>D at temperature T was calculated using equation (1) (Elwell & Schellman, 1977):
GN!D ˆ
Hm …1 ÿ T=Tm † ÿ
Cp ‰…Tm ÿ T† …1† ‡ T ln…T=Tm †Š Tm,
Hm,
Cp and
GN>D values for the control variants are given in Table 1. Values for the interface variants are from Pielak et al. (1995). Reversibility was assessed in two ways: return of the native-state signal (structural reversibility); and by comparing thermodynamic parameters from two consecutive experiments (thermodynamic reversibility). From pH 3.2 to 5.0, denaturation is >75% reversible by the ®rst criterion. The A7L variant was not tested using the second criterion, but is 85% reversible by the ®rst criterion. For all other variants, Tm values were reproducible to 0.5 K, and 23 of the 31
Hm values are reproducible to within 5.0 kcal molÿ1 (1 cal ˆ 4.184 J). These values are close to the standard deviations in Tm and
Hm from repetition of experiments, indicating that the N>D transitions are thermodynamically reversible. The A7G variant is the least stable control variant. This is expected because glycine has a ten-

381

Helix-Helix Packing in Cytochrome c

Figure 1. [222 nm] versus pH for the wild-type protein in Na2SO4/H2SO4 buffer at 1 C (*). Upper and lower limits of [222 nm] at 30 C and 85 C at pH 1.9 ( & ). A solution was used to pH 1.85. Below 10 mM SO24 pH 1.85, additional acid was required. The curve is of no theoretical signi®cance.

dency to break helices (Creighton, 1993) and give more compact denatured states (NeÂmethy et al., 1966). On the other hand, the A7Y variant is as stable as the wt protein even though alanine has a higher helix propensity (MunÄoz & Serrano, 1994; Myers et al., 1997). Similarly, the A7Y and A7F variants are more stable than the A7L variant even though leucine has a higher helix propensity than tyrosine or phenylalanine. In summary, the A7Y, A7F and A7L variants are similar thermodynamically to the wt protein but the A7G variant is 1 kcal molÿ1 less stable. In addition, comparisons of CD spectra and growth of yeast harboring mutant strains suggest that the Ala7 variants are structurally and functionally similar to the wild-type protein (Marmorino, 1996).

Dependence of A-state and acid-denatured state CD spectra on pH and sulfate concentration Previously, we characterized the A-state of yeast iso-1-ferricytochrome c in 0.33 M Na2SO4/H2SO4 buffer (pH 2.1). This A-state exhibits molten globule-like characteristics with a native-like helix content and a non-native heme environment. Unlike the equine protein, which has an acid denaturation transition centered near pH 2.5 in HCl at 25 C (Fink et al., 1994), the yeast wt protein has a transition centered near pH 2.2 in H2SO4 at 1 C (Figure 1). The far-UV CD spectra of our proteins are also very sensitive to sulfate concentration (Figure 2). These strong pH and sulfate dependencies prompted our efforts to test more rigorously our hypothesis that native 3 interactions stabilize the A-state.

Figure 2. Far-UV CD spectra for the native and A-states of the F10W (a), L94A (b), L94 T (c) and Y97A (d) variants. Native states (ÐÐ); A-states in 0.33 M Na2SO4/ H2SO4 (. . . .), 0.67 M Na2SO4/H2SO4 ( ± ± ± ) and 1 M Na2SO4/H2SO4 ( ±

± ). For F10W, L94A and Y97A variants data were acquired at pH 1.9 to 2.0. For the L94 T variant the data were acquired at pH 1.8.

A>D transition From 15 to 60 mM protein there is no concentration dependence to the thermodynamic parameters, so data from different proteins can be compared without conversion to [222]. Unless otherwise noted, A>D transitions were examined in 0.33 M Na2SO4/H2SO4. Typical fraction denatured versus T plots are shown in Figure 3 for the wt protein, the interface variants (a) and the Ala7 variants (b). Data were ®t to a two-state model to obtain Tm and
Hm (Tables 2 and 3) and
GA>D was calculated as described below. The A-state is stable over a narrow pH range, so
Cp cannot be obtained by varying pH. Therefore,
GA>D must be calculated differently than
GN>D. Two methods were used; ®rst,
GA>D,EX was obtained by examining ÿRTlnKD versus T plots at Tm for the wt protein under the same conditions, where KD is the equilibrium constant for A-state denaturation. Values of
GA > D,EX rely solely on the two-state assumption but can be obtained only for proteins that exhibit complete transitions. Second,
GA>D,BS was calculated using equation (2) (Becktel & Schellman, 1987):
GA!D;BS ˆ …
Hm;wt =Tm;wt † …Tm;variant ÿ Tm;wt † …2† where Tm,wt and
Hm,wt refer to values for the wt protein under the same conditions (i.e. same day, same buffer, etc.). Values of
GA>D,BS rely on more assumptions than do
GA>D,EX, but
GA>D,BS can be estimated for variants with incomplete or unobservable A>D transitions. Furthermore, we have shown that equations (1) and (2) give the same results for N>D transitions (Pielak et al., 1995).

382

Figure 3. Fraction denatured versus temperature plots for the wt protein, interface and Ala7-variants. a, wt (*), Y97F (&), L94V (!), F10Y (~), F10W (}), and L94A (*), all performed at pH 1.9 on the same day except for L94A (pH 2.0, different day). b, wt (*), A7Y (&), A7F (~), A7L (*), and A7G (}), all performed at pH 1.9 on the same day.

Two-state analysis is reasonable because the wt protein exhibits similar A>D transitions monitored with different probes, 222 and 282 (Marmorino & Pielak, 1995), and because the transitions in 0.33 M sulfate are reversible for the wt protein and the variants. The transitions in 0.33 M sulfate included a 15 minute pre-incubation at 30 C. Reversibility for the experiments listed in Tables 2 and 3 is 595% based on return of A-state signal. For most experiments, values of Tm and
Hm for the ®rst denaturation and the reversibility tests agree to within the measured uncertainties from repetition of the experiment. These observations indicate that denaturation is structurally and thermodynamically reversible. Experiments performed at pH 2.1, 2.0 and 1.8 without pre-incubation give similar thermodynamic parameters, but denaturation is less reversible and the plots have bumpier A-state baselines. Near-UV and visible CD spectra obtained before and after pre-incubation are the same within experimental uncertainty, indicating that large structural changes do not occur upon pre-incubation. Under

Helix-Helix Packing in Cytochrome c

conditions different from those for the standard A>D experiment, there is evidence for irreversible behavior in the wt protein. Two separate near-UV 282-monitored A>D transitions of 60 mM protein in 0.33 M sulfate were 580% reversible based on return of A-state signal, but resulted in some protein aggregation. In summary, for 30 mM protein samples in 0.33 M sulfate, the wt and variant A>D transitions are reversible and pre-incubation does not change the thermodynamic parameters. Inspection of Tables 2 and 3 reveals pH changes do not change the relative stabilities of the proteins. Additional experiments were performed in an attempt to observe transitions that are incomplete or undetectable in 0.33 M sulfate above ÿ1 C (L94 T, L94A and Y97A). Fraction denatured versus T plots for >0.33 M sulfate experiments are shown in Figure 4 and the resulting parameters are summarized in Table 4. As shown in Figure 2, increasing the sulfate concentration beyond 0.33 M induces helix at 1 C in the L94A, L94 T and Y97A variants, but the A-state of the F10W variant is nearly fully formed in 0.33 M sulfate. A>D transitions in 0.67 to 0.68 M Na2SO4/H2SO4 were monitored for the F10W and the L94 T variants and in 1.0 M Na2SO4/H2SO4 for the F10W, L94 T, L94A and Y97A variants. Experiments in 0.68 M sulfate yield reversible Tm values within 1.3 K but experiments in 1 M sulfate are irreversible and, for the Y97A variant, give insuf®cient native baseline for precise ®tting.

Discussion Based on our initial results (Marmorino & Pielak, 1995), we examined several variants in more detail. We found that A-state and aciddenatured state CD spectra for the less stable variants depend strongly on sulfate concentration and pH. To determine the sulfate sensitivity, we examined A-state stability at different sulfate concentrations. To examine the pH sensitivity, we studied A-state stability over a range close to the uncertainty for pH measurement. To further control our experiments, A-state stabilities are compared to the wt value obtained under the same conditions and at the same time rather than to averaged values. We also included three new variants, L94V, A7F and A7G. Values of
GA>D,EX and
GA>D,BS are in reasonable agreement from pH 2.1 (Marmorino & Pielak, 1995) to 1.8 (Tables 2 and 3) in 0.33 M sulfate. Inspection of Tables 2 and 4 shows that the stability differences are independent of sulfate concentration and pH. Figure 5 shows a
GA>D versus
GN>D plot for the interface variants. As in our original report, the slope is near unity. Examination of far-UV CD spectra also supports the idea that the helix-helix interaction stabilizes the A-state. As shown in Figure 2, the helix content follows the trend in native-state stabilities. For example, in 0.67 M salt, the F10W contains a

383

Helix-Helix Packing in Cytochrome c

Table 2. Thermodynamic parameters for the A>D transitions of the interface variants in 0.33 M Na2SO4/H2SO4 buffer pH Y97F L94V F10Y F10W L94T L94A

2.0 1.9 1.8 2.0 1.9 1.8 2.0 1.9 1.8 2.0 1.9 1.8 2.0 1.8 2.0

Tm (K)


Hm (kcal molÿ1) a

303.2 (303.2) 304.5 (303.6) 300.6 (300.4) 304.1 (303.4) 300.4 299.0 (298.9) 298.7 (299.2) 298.0 (297.8) 296.4 (295.2) 295.9 (297.4) 295.5 (293.4) 293.6 (290.5) <283b <272d <272d

31.0 (30.0) 30.3 (36.0) 28.3 (34.8) 39.3 (38.2) 35.6 30.6 (33.6) 33.3 (31.6) 30.8 (33.1) 32.3 (27.6) 26.1 (32.2) 28.2 (24.8) 24.9 (22.0) Ð Ð Ð

Tm,wt (K) 306.1 307.6 303.6 308.0 304.6 304.4 305.9 305.4 304.0 306.2 304.0 304.0 307.0 303.6 306.1


Hm,et

(305.4) (306.9) (303.7) (308.2) (302.9) (306.0) (305.1) (303.8) (306.1) (304.6) (303.8) (305.4)

38.9 37.5 35.7 37.5 35.2 35.1 37.7 38.6 35.5 36.9 30.4 35.5 38.0 35.7 38.9

(41.3) (36.1) (36.9) (35.8) (36.3) (36.6) (40.1) (34.4) (36.3) (38.6) (34.4) (41.3)


GEX (kcal molÿ1)


GBS

ÿ0.27 ÿ0.20 ÿ0.24 ÿ0.49 ÿ0.46 ÿ0.54 ÿ0.84 ÿ0.73 ÿ0.92 ÿ0.88 ÿ0.72 ÿ0.77 Ð Ð Ð

ÿ0.37  0.20 ÿ0.38  0.21 ÿ0.35  0.25 ÿ0.47  0.19 ÿ0.49  0.20 ÿ0.62  0.25 ÿ0.89  0.19 ÿ0.94  0.22 ÿ0.89  0.25 ÿ1.24  0.19 ÿ0.85  0.19 ÿ1.21  0.26 <ÿ3.0c <ÿ3.7c <ÿ4.3c

a

Values in parentheses are from reversibility tests. Partial transition. Estimated value. d No transition. b c

native-like amount of helix but the less stable L94A variant has less helix content. In 1.0 M salt, the L94 T and L94A variants contain higher percentages of helix than the less stable Y97A variant. In summary, the new data strengthen our conclusion that the 3 interaction between the N and C-terminal helices stabilizes the A-state of cytochrome c. The Ala7 variants test the effects of non-interface helix substitutions on A-state stability changes. The destabilization of the interface variants can be attributed speci®cally to interface disruption because the substitutions in the controls have little effect on stability compared to their effect on the stability of the interface variants. For example, the introduction of a residue with low helix propensity near the interface (A7G) decreases A-state stability by 1.0 kcal molÿ1, but substitutions that are expected to increase helix (MunÄoz & Serrano, 1994; Myers et al., 1997) have a large destabilizing effect when the substitution is at the interface (e.g. L94A and Y97A). Therefore, the speci®c helix-helix interaction, and not helix propensity alone, determines the A-state stability.

The A-state of equine ferricytochrome c has native-like compactness (Kataoka et al., 1993), native-like helix content, a native helix-helix interaction and one native heme ligand (Hamada et al., 1996). The native hydrophobic core, however, is most likely altered, as indicated by examination of near-UV and visible CD spectra. Determining
Cp for the A-state may help explain core packing because of its relation to surface area changes upon denaturation (Myers et al., 1995). An estimate of
Cp for the A-state formed in 0.33 M Na2SO4/H2SO4 can be obtained from the slope of a
Hm versus Tm plot for variants exhibiting complete transitions. The
Cp value for the A>D transition,
Cp,A>D, is 0.78 (0.09) kcal molÿ1 Kÿ1 (Figure 6a). A second ®t was performed to check for bias toward the region of the plot containing the most data points. Replacing the 20 wt points with an average value at each pH (Figure 6b) increases
Cp,A>D slightly to 0.87 (0.12) kcal molÿ1 Kÿ1. Estimating
Cp,A>D in this manner is reasonable because individual
Cp,N>D values are similar

Table 3. Parameters for the A>D transition of the non-interface variants in 0.33 M Na2SO4/H2SO4 buffer pH A7Y A7F A7L A7G

a

2.0 1.9 1.8 2.0 1.9 1.8 2.0 1.9 1.8 2.0 1.9 1.8

Tm (K) 306.7 (306.7)a 305.2 (305.0) 304.7 (304.5) 303.1 (303.9) 304.0 (302.6) 300.9 (299.2) 306.1 (305.4) 301.8 (302.2) 302.8 (302.1) 295.8 (296.9) 296.9 (296.5) 291.4 (293.0)


Hm (kcal molÿ1) 37.1 39.0 36.8 39.3 36.5 33.5 41.8 38.1 35.8 29.3 31.1 27.1

(35.1) (35.7) (31.3) (32.8) (35.1) (30.8) (42.3) (37.6) (34.8) (32.4) (33.2) (30.5)

Values in parentheses are from reversibility tests.

Tm,wt (K) 305.9 305.0 303.8 304.8 305.0 301.5 308.0 304.0 305.0 304.5 305.0 300.6

(305.9) (304.2) (304.0) (304.6) (304.2) (301.4) (308.2) (304.1) (304.2) (305.3) (304.2) (301.0)


Hm,wt 37.2 36.2 34.7 35.8 36.2 39.8 37.5 31.6 36.2 34.9 36.2 31.9

(36.3) (34.9) (33.3) (38.9) (34.9) (32.5) (35.8) (38.2) (34.9) (34.0) (34.9) (31.9)


GEX (kcal molÿ1)


GBS

‡0.06 ‡0.06 ‡0.02 ÿ0.39 ÿ0.12 ÿ0.06 ÿ0.27 ÿ0.32 ÿ0.26 ÿ0.83 ÿ0.85 ÿ0.83

‡0.10  0.19 ‡0.02  0.20 ‡0.10  0.24 ÿ0.20  0.18 ÿ0.12  0.20 ÿ0.08  0.28 ÿ0.23  0.19 ÿ0.23  0.18 ÿ0.26  0.20 ÿ1.00  0.18 ÿ0.96  0.22 ÿ0.98  0.23

384

Helix-Helix Packing in Cytochrome c

Figure 5.
GA>D,BS (*) and
GA>D,EX (}) versus
GN>D for the wt protein and the interface variants in 0.33 M Na2SO4/H2SO4, pH 1.8 to 2.0. A line with a slope of unity and an intercept of zero is shown for comparison. Filled symbols represent data acquired in 1 M Na2SO4/H2SO4. The points are, from left to right, the Y97A, L94A, L94 T, F10W, L94V and F10Y variants, the wt protein, and the Y97F variant. Horizontal error bars are from Pielak et al. (1995). With three exceptions, vertical error bars indicate the range of values from pH 1.8 to 2.0 for the
GA>D values (Tables 2 and 4). Vertical error bars for F10W in 1 M sulfate are the range from two pH 1.9 experiments. The error bars for the L94A variant in 1 M sulfate and the Y97A variant are estimates.

Figure 4. Fraction denatured versus temperature plots for the wt protein, and the F10W, L94A, L94 T and Y97A variants in >0.33 M Na2SO4/H2SO4. a, the wt protein (*) and the F10W variant (}) in 1 M Na2SO4/ H2SO4 at pH 1.9, and the Y97A variant (&) in 1 M Na2SO4/H2SO4 at pH 2.0. b, The wt protein (*) and the L94A variant (*) in 1 M Na2SO4/H2SO4 at pH 1.9. c, The wt protein (*) and the F10W variant (}) in 0.68 M Na2SO4/H2SO4 at pH 1.9. d, The wt protein (*) and the L94 T (!) variant in 0.67 M Na2SO4/H2SO4 at pH 2.0.

(Pielak et al., 1995). Our
Cp,A>D compares well to that for equine ferricytochrome c; Kuroda et al. (1992) ®nd that for the most highly populated equine A-state
Cp,A>D is 0.9 to 1 kcal molÿ1 Kÿ1 and Hamada et al. (1994) estimate that
Cp,A>D is 0.5 kcal molÿ1 Kÿ1 for the Na2SO4-induced equine A-state. The results suggest that signi®cant non-polar surface area is buried. Speci®cally, the ratio of
Cp,A>D to
Cp,N>D (0.8 kcal molÿ1 Kÿ1/ 1.4 kcal molÿ1 Kÿ1) shows that approximately 60% of the buried non-polar surface area in the native state is also buried in the A-state of the yeast protein. Since publication of our preliminary report, ColoÂn & Roder (1996) con®rmed our results with equine ferricytochrome c, Wu et al. (1995) and Schulman et al. (1997) have shown that the A-state of a-lactalbumin has a native-like fold, and Kay &

Baldwin (1996) have shown that helix-helix interactions stabilize the apomyoglobin intermediate. ColoÂn and Roder (1996) also studied the folding kinetics and showed that the A-state of the wildtype protein is a good model for a late intermediate and that the A-state of the L94A variant is a good model for a less structured early intermediate. Their conclusion is consistent with our observation that the CD spectra of less stable variants possess less helix. Also, the 60% decrease in solvent exposure described above agrees with values from kinetic studies of other proteins (Roder & ColoÂn, 1997). Taken together, these results explain why the A-state of equine cytochrome c folds to the native state in one fast kinetic phase (Sosnick et al., 1994).

Table 4. Parameters for the A>D transitions of the interface variants in >0.33 M Na2SO4/H2SO4 Protein Y97A L94A L94T F10W L94T a b

[SO24] (M)

pH

Tm (K)

1.00 1.00 1.00 1.00 1.00 1.00 0.68 0.67

2.0 1.9 1.9 2.0 1.8 1.9 1.9 2.0

4298a 4298a 298.6 299.4 295.1 318.1 311.9 287.7


Hm (kcal molÿ1)

34.5 35.2 30.1 40.1 37.3 25.7

Tm,wt (K) 325.5 325.4 323.6 321.7 320.0 325.4 319.0 317.4


Hm,wt


GEX (kcal molÿ1)

52.1 54.3 61.3 48.5 51.8 54.3 46.4 41.5

Ð Ð Ð Ð Ð ÿ1.23 ÿ1.01 Ð

From inspection. Uncertainty is estimated with sTm ˆ 3.4 and s
Hm ˆ 5.7, the standard deviations from the ®t.


GaBS 44.4 44.6 ÿ4.7  1.0b ÿ3.3 ÿ4.0 ÿ1.2 ÿ1.0 ÿ3.9

385

Helix-Helix Packing in Cytochrome c

Figure 6.
Hm versus Tm plot for complete A>D transitions in 0.33 M Na2SO4/H2SO4. a, All repetitions of the wt protein. b, average values for the wt protein at each pH value. The slopes and intercepts of the best ®t lines in a and b are: 0.78 (0.09) kcal molÿ1 Kÿ1, ÿ202 ( 28) kcal molÿ1 (r2 ˆ 0.61, Pr < 0.5%) and 0.87 (0.12) kcal molÿ1 Kÿ1, ÿ228 (35) kcal molÿ1, (r2 ˆ 0.66, Pr < 0.05%), respectively.

Materials and Methods Nomenclature The term mutant refers to DNA with nucleotide substitutions and the term variant refers to proteins with amino acid substitutions. Variants are named using the one-letter amino acid code with the wt residue given ®rst, followed by the position number, and the substituting residue. The C102 T variant is referred to as the wt protein. This variant (Cutler et al., 1987) is structurally identical with the true wt protein (Gao et al., 1991; Berghius & Brayer, 1992) but is more amenable to biophysical studies (Betz & Pielak, 1992). All variants also contain the C102T mutation. The mammalian numbering system is used (Moore & Pettigrew, 1990). Mutants and variants Yeast strains harboring the genes for the A7F, A7G, A7L and A7Y variants were produced as described (Hilgen & Pielak, 1991) with the following exceptions. Escherichia coli strain DH5aF0 [F0 ,f80dlacZ
M15, recA1, endA1, gyrA96, thi-1, hsdR17 (rK ÿ ,MK ‡ ), supE44, relA1, deoR,
(lacZYA-argF) U169 (Hanahan, 1983)] was used in place of JM101, transformations were performed using electroporation (Dower et al., 1988), and a Petri plate modi®ed (Ner et al., 1988) version of the Sequenase (USB) double-stranded DNA sequencing technique was used. As described by Hilgen & Pielak (1991), each mutant gene sequence was determined to con®rm the absence of unwanted mutations.

A modi®ed version of the procedure described by Sherman et al. (1968) was used to purify proteins. The Y97A, L94 T, F10W, F10Y, L94V and Ala7 variants were isolated and puri®ed as described below. The L94A and Y97F variants, gifts from Dr Zoey Fredericks, were repuri®ed as described below. Yeast cultures used for protein isolation were from a single colony on CM-Leu medium. CM-Leu cultures (5 ml) were usually grown for one day and then inoculated into one litre of YPG medium. After two to three days, the one litre culture was added to a New Brunswick Scienti®c Microform fermentor (series MF-2000) containing a mixture of YPG, antifoam (Sigma or Rug Doctor) and 270 g of sucrose. Antibiotics (0.4 g of streptomycin, 0.6 g of penicillin and 0.4 g of kanamycin) were then added. After one to two days at saturation (108 cells mlÿ1), cells were harvested, suspended in sodium phosphate buffer (pH 6.5 to 7.2), and NaCl and EDTA were added to a ®nal concentration of 0.8 M and 0.05 M, respectively. One-half volume of ethyl acetate was added and the solution was stirred for 1.5 to 2 hours at 4 C. This solution was diluted tenfold with water and 100 ml of the cation-exchange resin CG-50 was added to bind the protein. After washing the resin with water, the protein was eluted with 1.2 M NaCl and dialyzed against a solution of 10 mM phosphate buffer (pH 6.5 to 7.2), 5 mM EDTA, 10% (v/v) glycerol. The desalted protein was puri®ed with an HR-10 Sepharose ion-exchange column on a Pharmacia FPLC. SDS-PAGE with Coomassie staining was used to ®nd the purest fractions. Some of the wt protein and all of the variants were further puri®ed on a gravity-run G-50 gel-®ltration column equilibrated and eluted with 1 M NaCl, 50 mM phosphate buffer (pH 6.5 to 7.0). Purity was again checked by using Coomassie-stained SDS-PAGE. The protein was desalted 25-fold and oxidized by rinsing a protein-bound CG-50 column with a saturated NH4[Co(dipicolinate)2] solution (Mauk et al., 1979). Oxidized proteins were eluted with 1 M NaCl, 50 mM phosphate buffer (pH 6.5 to 7.0). Proteins were shown to be 595% oxidized by examining an aliquot in the oxidized and sodium dithionite-reduced forms using at least one of the following equations: f‰…A415 nm;ox =A410 nm; ox †

314:8Š ÿ 280:1g 100 ˆ % reduced

‰
A550 nm =…A550 nm; red ÿ …A550 nm; red

0:325††Š 100 ˆ % oxidized

where
A550 nm is the A550 nm after sodium dithionite addition minus the value before addition. Proteins were desalted >500-fold in HPLC-grade water and stored at ÿ70 C either lyophilized or frozen in HPLC-grade water. All the variant protein samples used to obtain CD spectra and A>D thermal denaturation data were puri®ed using both FPLC and gel-®ltration. The following N>D thermal denaturation experiments, however, were performed on protein samples puri®ed using only FPLC: the ®ve A7L experiments, two of the 11 A7G experiments, and three of the 11 A7F experiments. Samples were judged to be pure by inspection of Coomassiestained gels. Sample preparation Protein concentrations for thermal denaturation were 30 mM and concentrations for obtaining spectra were

386

Helix-Helix Packing in Cytochrome c

18 to 35 mM. Initially, samples were prepared by adding buffer to weighed, lyophilized protein, checking the concentration, and then diluting at least tenfold with buffer. Later, to simplify the procedure and to obtain more accurate concentrations, protein solutions were either concentrated to 3 mM in HPLC-grade water or lyophilized and brought to 3 mM in HPLC-grade water. Samples were then diluted 5100-fold with the appropriate buffer. No signi®cant difference was found when the preparation methods were compared. Accurate protein concentrations are required to compare CD spectra. To this end, an aliquot of protein stock solution was diluted and its concentration was de®ned as the average of the concentrations determined using the extinction coef®cients e550 nm ˆ 2.77  104 Mÿ1 cmÿ1 for the reduced protein and e410 nm ˆ 109.4 mMÿ1 cmÿ1 for the oxidized protein (Hilgen-Willis, 1993). For thermal denaturation experiments and CD spectra, the proteins were 595% oxidized.

deviations of Tm and
Hm are 0.3 K and 4.9 kcal molÿ1 from seven repetitions on the wt protein from different preparations. These values are different than those described by Cohen & Pielak (1994), who analyzed data for the wt protein obtained by different people on different protein batches at different times; for ten repetitions in 100 mM sodium acetate buffer, the uncertainties are 1.1 K and 3.9 kcal molÿ1. To be conservative, uncertainties in Tm and
Hm from Cohen & Pielak (1994) were used to construct Table 1 because they give larger
GN>D uncertainties. Uncertainties in
GA>D,BS (s
GA>D,BS) were estimated by applying propagation of error analysis to equation (2):

Circular dichroism

Uncertainties in Tm and
Hm (sTm,wt, sTm,var and s
Hm) are the standard deviations of six or seven repetitions of the wt experiment at each pH value. Values of sTm,wt and s
Hm are 1.5 K, 2.5 kcal molÿ1; 1.2 K, 3.1 kcal molÿ1; and 1.1 K, 1.3 kcal molÿ1 at pH 1.8, 1,9 and 2.0, respectively.

Data were acquired using an Aviv Model 62DS spectropolarimeter equipped with a thermostated, ®ve-position sample changer. Spectra were acquired at 1 C: 1 mm quartz cells were used between 200 and 240 nm, and 10 mm quartz cells were used between 240 and 600 nm. The A-state buffer was 0.33 M Na2SO4/H2SO4 (pH 1.8 to 2.0), but higher sulfate concentrations were used for additional experiments. Native-state buffers used to acquire CD spectra were 50 mM or 10 mM sodium phosphate (pH 7.0). Salt has an insigni®cant effect on the native-state spectrum. Buffers were made using HPLCgrade water and were sterile ®ltered before use. All pH measurements were made at room temperature. pH titration Values of [222 nm] used in Figure 1 come from farUV CD spectra acquired as described above. All values at 1 C were obtained on the same day. Values at 30 C and 85 C were obtained on a different day. Because concentration calculations are sensitive to day-to-day changes in pipetting and cuvettes, the outer limits of a range of possible [222 nm] values are shown for the 30 C and 85 C experiments. Thermal denaturations Ellipticity at 222 nm was followed from ÿ1 to 60 C for the A>D transition and from 2 to 85 C for the N>D transition. A-state samples were pre-incubated for 15 minutes in a 30 C incubator. Data were acquired at 1 deg. C intervals. The time between successive points was about six minutes. Reversibility was checked by returning the heated samples to the initial temperature and revisiting two to ®ve points or by repeating the entire experiment on the same sample. Buffers used for nativestate thermal denaturations were 50 mM sodium acetate (pH 3.2 to 5.0). Error analysis The uncertainty in
GN>D was estimated by applying propagation of error analysis to equation (1) (Cohen & Pielak, 1994). Uncertainties in
Cp are the standard deviations of the ®ts of
Hm versus Tm plots. The standard

s
GA!D;BS f‰…Tm;var =Tm;wt ÿ 1†s
Hm Š2 ‡ ‰…Tm;wt †ÿ2 …Tm;var † …
Hm;wt † …sTm;wt †Š2 ‡ ‰…
Hm;wt =Tm;wt † …sTm;var †Š2 g0:5

…3†

Acknowledgments We thank the Pielak group and Terry Oas for helpful discussions, and Jack Aviv for help with the spectropolarimeter. This work was supported by the N. I. H. (GM42501). M. L. was part of the U. N. C. Summer Undergraduate Research Experience program supported by the N. S. F. J. L. M. was partially supported by an N. I. H. training grant (GM08332).

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Edited by P. E. Wright (Received 17 July 1997; received in revised form 26 September 1997; accepted 29 September 1997)