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Partially formed native tertiary interactions in the A-state of cytochrome c1
Article No. jmbi.1999.2764 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 289, 639±644
Partially Formed Native Tertiary Interactions in the A-state of Cytochrome c Daniel R. Hostetter1, Gresham T. Weatherly1, James R. Beasley1 Kara Bortone1, David S. Cohen2, Shelly A. Finger1, Philip Hardwidge1 Dionysios S. Kakouras1, Aleister J. Saunders2, Sonja K. Trojak1 Jennifer C. Waldner1 and Gary J. Pielak1* 1
Department of Chemistry
2
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state. # 1999 Academic Press
*Corresponding author
Keywords: A-state; cytochrome c; molten-globule model; saturation mutagenesis; tertiary structure
Introduction The short time-scale of protein folding makes the observation of folding intermediates a challenging task. An alternative is to study models that can be populated at equilibrium. The most thoroughly studied models, A-states (Ptitsyn, 1996; Goto et al., 1990), are non-native forms that are populated at low pH and high ionic strength. We have utilized the A-state of yeast iso-1-cytochrome c to gain insight into its tertiary structure. Here, we focus on the evolutionarily conserved interactions of the 60s helix that help to form the crucial hydrophobic heme pocket. A-states share many properties with authentic kinetic species (ColoÂn & Roder, 1996; Ptitsyn, 1996). The A-state of cytochrome c is an especially good example. Under conditions that suppress non-native heme ligands (EloÈve et al., 1994), ferricytochrome c appears to fold in four sequential steps. The fastest step detected (50 ms) corresponds to activated chain collapse (Shastry & Roder, 1998). D.R.H. and G.T.W. contributed equally to this work. E-mail address of the corresponding author: gary [email protected] 0022-2836/99/230639±6 $30.00/0
In the second step (2 ms), ¯uctuating helical structure is formed (EloÈve et al., 1994). The third step (2100 ms) is characterized by formation of, and interaction between, the N and C-terminal helices (ColoÂn et al., 1996; Roder et al., 1988). The 60s helix (Roder et al., 1988) and the Met80-iron bond (Brems & Stellwagen, 1983) are formed in the ®nal step (0.1-1 second). Which kinetic species is best modeled by the A-state? X-ray scattering data show that the Astate is compact (Kataoka et al., 1993), so only species after the fastest phase require consideration. Amide proton-deuteron exchange data show that the N-terminal, C-terminal, and 60s helices are formed (Jeng et al., 1990). However, these data alone cannot describe tertiary interactions. We showed that the native interaction between the terminal helices is present in the A-state by examining the stability of variant proteins (Marmorino & Pielak, 1995; Marmorino et al., 1998). These speci®c interactions were subsequently shown to affect the folding kinetics (ColoÂn et al., 1996). We undertook the studies presented here to determine the status of the tertiary interactions involving the 60s helix, which runs from residue 60 to 70. We concentrated on Leu68, because its # 1999 Academic Press
640
Structure in a Molten-globule Model
side-chain forms evolutionarily invariant interactions with the heme, the C-terminal helix, and the Met80 ligand (Moore & Pettigrew, 1990; Brayer & Murphy, 1996). We made all 19 missense mutations at position 68, plus a stop codon control, and examined their effects on the phenotypes of yeast harboring the mutants as their only source of cytochrome c. We then examined both the native state and the A-state stabilities of three functional variants.
Results Phenotype analysis and protein expression Eight of the 20 mutants (L68V, M, I, F, T, A, Y and C) produce the functional phenotype. Two mutants each produce the temperature-sensitive (L68Q, S) and very temperature-sensitive (L68N, W) phenotypes. The remaining eight mutants are non-functional (L68G, P, K, R, E, D, H, and Stop). Only three functional mutants (L68 M, V, and I) exhibit cytochrome c expression levels that approach the value for the wild-type protein (>0.2 mg lÿ1 culture). The phenotype and expression data on the S and M variants are consistent with the only other studies of changes at position 68 (Hampsey et al., 1986; Woods et al., 1996).
Figure 1. Top panel, Fraction denatured versus temperature plot for the wild-type protein (^), and the L68I (*), L68V ( & ), and L68 M (~) variants. Filled symbols represent the N D transition, and open symbols represent the A D transition. The curves represent the best ®ts to a two-state model. Bottom panel, ÿRTlnK versus temperature plots. The symbols are the same as in the top panel. Vertical lines are drawn at the wild-type Tm for the A D and N D transitions.
and Hm obtained at several pH values (Figure 2) to equation (2):
Determining GND Denaturation was shown to be reversible, using established criteria (Pielak et al., 1995). The data (Figure 1) were ®t to a two-state model (Allen & Pielak, 1998) to yield Tm (the temperature at which half the protein is denatured) and Hm (the van't Hoff enthalpy of denaturation at Tm). The values for the wild-type protein, and the L68V, M, and I variants are shown in Table 1. Equation (1) was used to calculate the stabilities, GD(T), at pH 4.6 and the Tm for the wild-type protein, as described (Pielak et al., 1995). T GD T Hm 1 ÿ Tm h i 1 ÿCp Tm ÿ T T ln TTm Cp, the change in heat capacity upon denaturation, for each protein was determined by ®tting Tm
Hm Cp Tm b
2
The values of Cp and GD(T) (GD(T) for the wild-type protein minus GD(T) for each variant) are shown in Table 1. The data were also analyzed by using a method that does not depend on knowing Cp (Becktel & Schellman, 1987). GD(BS) was calculated with equation (3): GD BS
Hm;wt Tm;wt ÿ Tm;var Tm;wt
3
where var and wt refer to the variant and the wildtype proteins, respectively. The results are shown in Table 1. Stability differences were also determined more directly by examining plots of ÿRT ln KD versus temperature at the Tm of the wild-type protein
Table 1. Thermodynamic parameters for the N D transition (1 cal 4.184 J) Protein WT L68I L68V L68M
Tm (K) 327.0 0.8 322.2 0.2 316.7 0.4 316.1 0.4
Hm (kcal molÿ1) 87.810.4 83.63.2 64.54.7 64.61.5
Cp (kcal molÿ1 Kÿ1)
GD(327 K) (kcal molÿ1)
GD(BS) (kcal molÿ1)
GD(ex) (kcal molÿ1)
1.47 0.12 1.84 0.30 1.21 0.08 1.18 0.11
0.00 0.05 1.49 0.07 2.42 0.08 2.68 0.06
0.0 0.3 1.3 0.3 2.8 0.4 2.9 0.4
0.0 0.2 1.2 0.1 2.3 0.1 2.2 0.2
Experiments were performed in 0.05 M sodium acetate. Tm, Hm, and GD are for pH 4.6. Uncertainties are sample standard deviations from ®ve or six experiments, except for the uncertainties in Cp, GD(327 K), and dGD(BS), which were determined as described in the text.
641
Structure in a Molten-globule Model
(Figure 1), where KD is the equilibrium constant for denaturation (Marmorino & Pielak, 1995). This stability change, GD(ex), is the vertical distance between the data for the wild-type protein and a variant at the Tm of the wild-type protein. Values of GD(ex) are shown in Table 1. The analyses give consistent results. Determining GAD The A D transitions for the wild-type protein and the three variants were also shown to be reversible. The data (Figure 1) were ®t as described above to yield the Hm and Tm values in Table 2. The A-state is stable over a narrow pH range. Therefore, equation (1) cannot be used to calculate GD(T) for the A-state, because Cp cannot be obtained by varying the pH (Marmorino & Pielak, 1995). The stabilities differences from BecktelSchellman analysis and direct examination are shown in Table 2. The analyses give consistent results.
Discussion Relationships between phenotype, stability and the amino acid residue at position 68 Despite the invariance of Leu68 among the >100 eukaryotic cytochromes c whose sequence is known (Pielak et al., 1995), only seven of the possible 19 amino acid substitutions lead to the nonfunctional phenotype. Of the nine amino acids that give the functional phenotype, only four (leucine, isoleucine, valine, and methionine) yield proteins that are expressed at levels suf®cient for further characterization. These data show that the hydrophobicity (Radzicka & Wolfenden, 1998) and, to a lesser extent, helix propensity (Myers et al., 1997) play a role in maintaining function. The amino acid substitutions lower the stability of both the native state (Table 1) and the A-state (Table 2). For the valine and isoleucine variants, some of the decrease can be attributed to lower helix propensity (Myers et al., 1997). However, methionine is predicted to increase the helix propensity. The observation that increasing the propensity decreases the stability, and the fact that the side-chains have almost identical surface areas (171 Ê 2 for methionine and leucine, respectand 168 A ively; Chothia, 1975) shows the importance of sidechain packing. The importance of packing is also highlighted by the observation that variants con-
Figure 2. Hm versus Tm plots for the wild-type and variant proteins. The lines are from linear least-squares analysis. Error bars represent the ranges of two experiments or the sample standard deviations from three to six experiments. Some bars are smaller than the symbols.
taining the amide structural analogs of leucine and valine (glutamine and asparagine) give rise to a partially functional (i.e. temperature-sensitive) phenotype. In summary, speci®c tertiary interactions involving Leu68 stabilize both the native state and the A-state. Native interactions involving position 68 are partially formed in the A-state For all three variants, GD values for the N D transitions are greater than those for the A D transitions (Tables 1 and 2). This observation shows that native interactions are stronger in the native state than in the A-state. Furthermore, the rank order of stability changes for the N D and A D transitions is different for L68V and M. This observation shows that the interactions involving position 68 that are made or broken upon denaturation are different for the N D and A D transitions. In summary, some but not all, of the native tertiary interactions involving Leu68 are present in the A-state, and different interactions in and around Leu68 are made or broken upon denaturation of the A-state and the N-state. Relationship between global and local stability Our observations are consistent with studies of local stability evaluated by using NMR-monitored
Table 2. Thermodynamic parameters for the A D transition Protein WT L68I L68V L68M
Tm (K)
Hm (kcal molÿ1)
GD(BS) (kcal molÿ1)
GD(ex) (kcal molÿ1)
311.9 0.3 306.1 2.0 301.1 2.1 303.6 1.5
52.1 3.2 38.2 1.1 34.5 4.1 34.6 3.3
0.0 0.1 1.0 0.3 1.8 0.4 1.4 0.3
0.0 0.1 0.8 0.2 1.3 0.1 1.0 0.2
Experiments were performed in 0.5 M Na2/H2SO4, pH 2.1. The uncertainties are the sample standard deviation from four experiments except GD(BS), which was calculated as described in the text.
642 amide proton-deuteron exchange experiments (Jeng et al., 1990). The protection factors for the 60s helix in the A-state are less than those for the N and C-terminal helices. The average protection factor for a residue in the 60s helix is 176 and the average values for the N and C-terminal helices are 359 and 728, respectively (Jeng et al., 1990). The maximum for a non-helix residue (Ile85) is 109, less than the average for the 60s helix. These data support the conclusion that interactions involving Leu68 are only partially present in the A-state. Summary In the A-state, the N and C-terminal helices are formed (Jeng et al., 1990), they interact to the same extent as in the native state (Marmorino & Pielak, 1995; Marmorino et al., 1998), and the 60s helix is present (Jeng et al., 1990). In fact, except for the non-native heme ligation (Jeng et al., 1990; Robinson et al., 1983) and a general structural loosening, the protein is highly native-like in the A-state. We have shown here, however, that native tertiary interactions involving Leu68 are incompletely formed. Taken together with the results of kinetic studies, these observations suggest that the A-state is most closely related to species that occur late in folding.
Materials and Methods Nomenclature Variants are denoted by the one-letter amino acid code with the wild-type residue given ®rst followed by the position number and the new residue. The C102T variant of yeast iso-1-cytochrome c is referred to as the wild-type protein, and all variants contain the C102T mutation. This mutation facilitates biophysical characterization but does not alter the structure or function of the protein (Berghuis & Brayer, 1992; Cutler et al., 1987; Gao et al., 1991). Production of mutants DNA manipulations involved the yeast-shuttle phagemid pJB750 (Beasley & Pielak, 1996). Escherichia coli strain DH5aF0 [F0 , f80dlacZM15, recA1, endA1, gyrA96, thi-1, hsdR17(rÿ k , Mk ), supE44, relA1, deoR, (lacZYA-argF) U169] (Hanahan, 1983) and yeast strain B6748 (MAT cycl-::lacZ cyc7-::CYH2 ura3-52 his31 leu2-3 leu2-112 trp1-289 can1-100 cyh2) (Holzschu et al., 1987) were used for all transformations. Competent E. coli cells were prepared by treatment with DMSO (Hanahan et al., 1995) or purchased from Gibco BRL. Gibco's protocol was used for all transformations except that Luria Broth medium was used for DMSO-treated cells. Yeast transformations were performed with lithium acetate (Ito et al., 1983; Schiestl & Gietz, 1989; Hill et al., 1991; Gietz et al., 1992). DNA sequence analysis was performed by the sequencing facility at UNC-CH or by using a Petri-plate modi®ed version of the Sequenase double-stranded DNA sequencing protocol (Ner et al., 1988). Random oligonucleotide-directed mutagenesis (Kunkel et al., 1987) was used to create 17 L68 mutants.
Structure in a Molten-globule Model Unique oligonucleotides (oligos) were used to create the L68I, L68N, and L68Stop mutants. E. coli strain CJ236 was used to prepare single-stranded uracil-containing DNA. Oligos were phosphorylated at their 50 end as described (Zoller & Smith, 1983), except the buffer provided with the phage T4 polynucleotide kinase was used. A 2:1 mole ratio of oligo to template was used for mutagenesis. The extension reaction utilized phage T4 DNA ligase, phage T7 DNA polymerase, and phage T4 gene 32 protein and was allowed to proceed for one hour at 0 C, one hour at room temperature, and then two hours at 37 C. Putative mutants were identi®ed by the elimination of a ScaI restriction enzyme site at position 3579 bp (Beasley, 1995). The sequence of the entire cytochrome c gene was con®rmed before transforming the phagemid into yeast. Phagemids were isolated from yeast with a modi®cation of the spheroplast method (Rose, 1987), transformed into DH5aF0 , and then the sequence of the entire cytochrome c gene was recon®rmed. Phenotype analysis Transformants harboring each of the 20 different mutants were examined for growth on YPG and YPL at different temperatures as described (Beasley & Pielak, 1996). The results are medium-independent. A transformant has a functional phenotype if it grows at all temperatures tested. The temperature-sensitive phenotype is assigned if a transformant grows at 25 and 30 C but not at 37 C. The very temperature-sensitive phenotype is assigned if a transformant grows at only 25 C and the non-functional phenotype is assigned if the transformant does not grow at any of the three temperatures. Protein purification Distilled and deionized water was used throughout this procedure. Yeast cultures (1 l) harboring the functional L68 mutants were used to evaluate protein expression by using a scaled-down version of our puri®cation scheme. The three mutants with the highest expression were puri®ed according to a modi®ed version of the protocol described by Sherman et al. (1968). Four 5-ml YPS liquid cultures were inoculated with single colonies from YPG plates and incubated at 30 C with shaking for 36 hours. Three 1 l YPG cultures were each inoculated with a 5 ml culture and incubated at 30 C with shaking for three days. A 1 l culture was used to inoculate 11 l of medium in a New Brunswick Scienti®c Microferm fermentor (series MF-2000). The medium comprised YPG, 180 g of sucrose, and 300 ml of antifoam (Rug-Doctor1). After inoculation, 0.75 g of penicillin and 0.45 g of streptomycin were added. The antibiotics had been dissolved in 5 to 10 ml of water and sterile-®ltered with a 0.2 mm Acrodisc1 ®lter (Gelman Sciences). The 12 l culture was incubated at 25 C overnight and then at 30 C for three days. Approximately 10 l were harvested, and 10 l of sterile medium were added to the remaining 2 l. This fermentation procedure was repeated before harvesting the entire culture. After centrifugation, cells were re-suspended in a minimal amount of 1 M NaCl, 50 mM EDTA, 1 mM phenylmethylsulfonyl ¯uoride. One half-volume of ethyl acetate was added and the mixture was stirred at 4 C for 24 hours. The solution was diluted ®ve- to tenfold with water and 130-230 ml of wet CG-50 cationexchange resin was added. The slurry was stirred at 4 C
643
Structure in a Molten-globule Model for two hours, and then the resin was allowed to settle for 0.5 hour. The supernatant was discarded, and the resin was washed with water until the supernatant was clear. The pink resin was transferred to a 2.5 cm 30 cm column and washed with LSB (20 mM sodium phosphate buffer, pH 7). The cytochrome c was eluted with HSB (1 M NaCl, 20 mM sodium phosphate, pH 7). The protein was then dialyzed against LSB and subsequently puri®ed with an HR 10/10 S-Sepharose cation-exchange column on a Pharmacia FPLC with a linear gradient. Red fractions were pooled, concentrated, and further puri®ed with an HSB-equilibrated G-50 gel ®ltration column (2.5 cm 50 cm) ®tted with a peristaltic pump. SDSPAGE with Coomassie-blue staining was used to identify the pure fractions, which were pooled.
The uncertainty in GD(BS) was calculated using the following equation: sGD BS v #2 u 2 " u Tm;var T H m;var m u ÿ 1 sHm sTm;wt u u Tm;wt Tm;wt 2 u u 2 u t Hm sT Tm;wt m;wt For Cp, the uncertainty is the standard deviation of the slope as determined from linear least-squares analysis of the data in Figure 2.
Sample preparation Cytochrome c was oxidized as described by Betz & Pielak (1992), except that LSB was used to equilibrate the CG-50 resin and HSB was used to elute the protein. Variants were desalted by dialysis against water or exchanged into 0.05 M sodium phosphate buffer (pH 7.0) by multiple concentrations with a Centricon-10 microconcentrator (Amicon). The fraction of ferri protein was checked as described (Marmorino et al., 1998). All protein samples were 595 % oxidized. Concentration was determined by averaging the values obtained from the following isobestic points and molar absorptivities: 556.5 nm, 7800 Mÿ1 cmÿ1; 541.8 nm, 9900 Mÿ1 cmÿ1; 526.5 nm, 11,000 Mÿ1 cmÿ1; 504 nm, 600 Mÿ1 cmÿ1; and 434 nm, 22700 Mÿ1 cmÿ1 (Margoliash & Frohwirt, 1959). Protein solutions were then concentrated to >1 mM. Samples were exchanged into the appropriate buffer by either diluting an aliquot of the protein stock solution or by multiple concentrations with a Centricon-10 microcentrator. If <40-fold dilution was required, the latter method was employed. Thermal denaturation An Aviv model 62DS spectropolarimeter ®tted with a thermostatted ®ve-position sample changer was used. Ellipticity was monitored at 222 nm with a 3 nm bandwidth. Data were collected every 1 deg. C at a protein concentration of 30 mM. The N D transition (0.05 M acetate, pH 3.8-5.0) was monitored from 5 C to 85 C following a 15 minute equilibration at 40 C. The A D transition (0.50 M Na2/H2SO4 pH 2.1.) was monitored from ÿ1 to 60 C following a 15 minute equilibration at 30 C. Analysis of uncertainties Uncertainties are the sample standard deviation for all values except GD(T), GD(BS), and Cp. The uncertainty in GD(T) was calculated using the following equation (Cohen & Pielak, 1994): sGD T v 2 2 u u T T u 1ÿ s s T ÿ T ÿ T ln H m C m p u Tm Tm u u 2 u Cp T Hm T t ÿ Cp sTm 2 Tm Tm
Acknowledgements We thank the Pielak group and Chris Dobson for helpful discussions. This work was supported by N.I.H grant GM42501, P.H. was part of the N.S.F.-sponsored S.U.R.E. program, J.C.W. was partially supported by a G.A.A.N.N. fellowship from the U.S. Department of Education, and G.T.W. was partially supported by a U.N.C. Board of Governors fellowship.
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Edited by A. R. Fersht (Received 15 February 1999; received in revised form 7 April 1999; accepted 7 April 1999)
J. Mol. Biol. (1999) 289, 639±644
Partially Formed Native Tertiary Interactions in the A-state of Cytochrome c Daniel R. Hostetter1, Gresham T. Weatherly1, James R. Beasley1 Kara Bortone1, David S. Cohen2, Shelly A. Finger1, Philip Hardwidge1 Dionysios S. Kakouras1, Aleister J. Saunders2, Sonja K. Trojak1 Jennifer C. Waldner1 and Gary J. Pielak1* 1
Department of Chemistry
2
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state. # 1999 Academic Press
*Corresponding author
Keywords: A-state; cytochrome c; molten-globule model; saturation mutagenesis; tertiary structure
Introduction The short time-scale of protein folding makes the observation of folding intermediates a challenging task. An alternative is to study models that can be populated at equilibrium. The most thoroughly studied models, A-states (Ptitsyn, 1996; Goto et al., 1990), are non-native forms that are populated at low pH and high ionic strength. We have utilized the A-state of yeast iso-1-cytochrome c to gain insight into its tertiary structure. Here, we focus on the evolutionarily conserved interactions of the 60s helix that help to form the crucial hydrophobic heme pocket. A-states share many properties with authentic kinetic species (ColoÂn & Roder, 1996; Ptitsyn, 1996). The A-state of cytochrome c is an especially good example. Under conditions that suppress non-native heme ligands (EloÈve et al., 1994), ferricytochrome c appears to fold in four sequential steps. The fastest step detected (50 ms) corresponds to activated chain collapse (Shastry & Roder, 1998). D.R.H. and G.T.W. contributed equally to this work. E-mail address of the corresponding author: gary [email protected] 0022-2836/99/230639±6 $30.00/0
In the second step (2 ms), ¯uctuating helical structure is formed (EloÈve et al., 1994). The third step (2100 ms) is characterized by formation of, and interaction between, the N and C-terminal helices (ColoÂn et al., 1996; Roder et al., 1988). The 60s helix (Roder et al., 1988) and the Met80-iron bond (Brems & Stellwagen, 1983) are formed in the ®nal step (0.1-1 second). Which kinetic species is best modeled by the A-state? X-ray scattering data show that the Astate is compact (Kataoka et al., 1993), so only species after the fastest phase require consideration. Amide proton-deuteron exchange data show that the N-terminal, C-terminal, and 60s helices are formed (Jeng et al., 1990). However, these data alone cannot describe tertiary interactions. We showed that the native interaction between the terminal helices is present in the A-state by examining the stability of variant proteins (Marmorino & Pielak, 1995; Marmorino et al., 1998). These speci®c interactions were subsequently shown to affect the folding kinetics (ColoÂn et al., 1996). We undertook the studies presented here to determine the status of the tertiary interactions involving the 60s helix, which runs from residue 60 to 70. We concentrated on Leu68, because its # 1999 Academic Press
640
Structure in a Molten-globule Model
side-chain forms evolutionarily invariant interactions with the heme, the C-terminal helix, and the Met80 ligand (Moore & Pettigrew, 1990; Brayer & Murphy, 1996). We made all 19 missense mutations at position 68, plus a stop codon control, and examined their effects on the phenotypes of yeast harboring the mutants as their only source of cytochrome c. We then examined both the native state and the A-state stabilities of three functional variants.
Results Phenotype analysis and protein expression Eight of the 20 mutants (L68V, M, I, F, T, A, Y and C) produce the functional phenotype. Two mutants each produce the temperature-sensitive (L68Q, S) and very temperature-sensitive (L68N, W) phenotypes. The remaining eight mutants are non-functional (L68G, P, K, R, E, D, H, and Stop). Only three functional mutants (L68 M, V, and I) exhibit cytochrome c expression levels that approach the value for the wild-type protein (>0.2 mg lÿ1 culture). The phenotype and expression data on the S and M variants are consistent with the only other studies of changes at position 68 (Hampsey et al., 1986; Woods et al., 1996).
Figure 1. Top panel, Fraction denatured versus temperature plot for the wild-type protein (^), and the L68I (*), L68V ( & ), and L68 M (~) variants. Filled symbols represent the N D transition, and open symbols represent the A D transition. The curves represent the best ®ts to a two-state model. Bottom panel, ÿRTlnK versus temperature plots. The symbols are the same as in the top panel. Vertical lines are drawn at the wild-type Tm for the A D and N D transitions.
and Hm obtained at several pH values (Figure 2) to equation (2):
Determining GND Denaturation was shown to be reversible, using established criteria (Pielak et al., 1995). The data (Figure 1) were ®t to a two-state model (Allen & Pielak, 1998) to yield Tm (the temperature at which half the protein is denatured) and Hm (the van't Hoff enthalpy of denaturation at Tm). The values for the wild-type protein, and the L68V, M, and I variants are shown in Table 1. Equation (1) was used to calculate the stabilities, GD(T), at pH 4.6 and the Tm for the wild-type protein, as described (Pielak et al., 1995). T GD T Hm 1 ÿ Tm h i 1 ÿCp Tm ÿ T T ln TTm Cp, the change in heat capacity upon denaturation, for each protein was determined by ®tting Tm
Hm Cp Tm b
2
The values of Cp and GD(T) (GD(T) for the wild-type protein minus GD(T) for each variant) are shown in Table 1. The data were also analyzed by using a method that does not depend on knowing Cp (Becktel & Schellman, 1987). GD(BS) was calculated with equation (3): GD BS
Hm;wt Tm;wt ÿ Tm;var Tm;wt
3
where var and wt refer to the variant and the wildtype proteins, respectively. The results are shown in Table 1. Stability differences were also determined more directly by examining plots of ÿRT ln KD versus temperature at the Tm of the wild-type protein
Table 1. Thermodynamic parameters for the N D transition (1 cal 4.184 J) Protein WT L68I L68V L68M
Tm (K) 327.0 0.8 322.2 0.2 316.7 0.4 316.1 0.4
Hm (kcal molÿ1) 87.810.4 83.63.2 64.54.7 64.61.5
Cp (kcal molÿ1 Kÿ1)
GD(327 K) (kcal molÿ1)
GD(BS) (kcal molÿ1)
GD(ex) (kcal molÿ1)
1.47 0.12 1.84 0.30 1.21 0.08 1.18 0.11
0.00 0.05 1.49 0.07 2.42 0.08 2.68 0.06
0.0 0.3 1.3 0.3 2.8 0.4 2.9 0.4
0.0 0.2 1.2 0.1 2.3 0.1 2.2 0.2
Experiments were performed in 0.05 M sodium acetate. Tm, Hm, and GD are for pH 4.6. Uncertainties are sample standard deviations from ®ve or six experiments, except for the uncertainties in Cp, GD(327 K), and dGD(BS), which were determined as described in the text.
641
Structure in a Molten-globule Model
(Figure 1), where KD is the equilibrium constant for denaturation (Marmorino & Pielak, 1995). This stability change, GD(ex), is the vertical distance between the data for the wild-type protein and a variant at the Tm of the wild-type protein. Values of GD(ex) are shown in Table 1. The analyses give consistent results. Determining GAD The A D transitions for the wild-type protein and the three variants were also shown to be reversible. The data (Figure 1) were ®t as described above to yield the Hm and Tm values in Table 2. The A-state is stable over a narrow pH range. Therefore, equation (1) cannot be used to calculate GD(T) for the A-state, because Cp cannot be obtained by varying the pH (Marmorino & Pielak, 1995). The stabilities differences from BecktelSchellman analysis and direct examination are shown in Table 2. The analyses give consistent results.
Discussion Relationships between phenotype, stability and the amino acid residue at position 68 Despite the invariance of Leu68 among the >100 eukaryotic cytochromes c whose sequence is known (Pielak et al., 1995), only seven of the possible 19 amino acid substitutions lead to the nonfunctional phenotype. Of the nine amino acids that give the functional phenotype, only four (leucine, isoleucine, valine, and methionine) yield proteins that are expressed at levels suf®cient for further characterization. These data show that the hydrophobicity (Radzicka & Wolfenden, 1998) and, to a lesser extent, helix propensity (Myers et al., 1997) play a role in maintaining function. The amino acid substitutions lower the stability of both the native state (Table 1) and the A-state (Table 2). For the valine and isoleucine variants, some of the decrease can be attributed to lower helix propensity (Myers et al., 1997). However, methionine is predicted to increase the helix propensity. The observation that increasing the propensity decreases the stability, and the fact that the side-chains have almost identical surface areas (171 Ê 2 for methionine and leucine, respectand 168 A ively; Chothia, 1975) shows the importance of sidechain packing. The importance of packing is also highlighted by the observation that variants con-
Figure 2. Hm versus Tm plots for the wild-type and variant proteins. The lines are from linear least-squares analysis. Error bars represent the ranges of two experiments or the sample standard deviations from three to six experiments. Some bars are smaller than the symbols.
taining the amide structural analogs of leucine and valine (glutamine and asparagine) give rise to a partially functional (i.e. temperature-sensitive) phenotype. In summary, speci®c tertiary interactions involving Leu68 stabilize both the native state and the A-state. Native interactions involving position 68 are partially formed in the A-state For all three variants, GD values for the N D transitions are greater than those for the A D transitions (Tables 1 and 2). This observation shows that native interactions are stronger in the native state than in the A-state. Furthermore, the rank order of stability changes for the N D and A D transitions is different for L68V and M. This observation shows that the interactions involving position 68 that are made or broken upon denaturation are different for the N D and A D transitions. In summary, some but not all, of the native tertiary interactions involving Leu68 are present in the A-state, and different interactions in and around Leu68 are made or broken upon denaturation of the A-state and the N-state. Relationship between global and local stability Our observations are consistent with studies of local stability evaluated by using NMR-monitored
Table 2. Thermodynamic parameters for the A D transition Protein WT L68I L68V L68M
Tm (K)
Hm (kcal molÿ1)
GD(BS) (kcal molÿ1)
GD(ex) (kcal molÿ1)
311.9 0.3 306.1 2.0 301.1 2.1 303.6 1.5
52.1 3.2 38.2 1.1 34.5 4.1 34.6 3.3
0.0 0.1 1.0 0.3 1.8 0.4 1.4 0.3
0.0 0.1 0.8 0.2 1.3 0.1 1.0 0.2
Experiments were performed in 0.5 M Na2/H2SO4, pH 2.1. The uncertainties are the sample standard deviation from four experiments except GD(BS), which was calculated as described in the text.
642 amide proton-deuteron exchange experiments (Jeng et al., 1990). The protection factors for the 60s helix in the A-state are less than those for the N and C-terminal helices. The average protection factor for a residue in the 60s helix is 176 and the average values for the N and C-terminal helices are 359 and 728, respectively (Jeng et al., 1990). The maximum for a non-helix residue (Ile85) is 109, less than the average for the 60s helix. These data support the conclusion that interactions involving Leu68 are only partially present in the A-state. Summary In the A-state, the N and C-terminal helices are formed (Jeng et al., 1990), they interact to the same extent as in the native state (Marmorino & Pielak, 1995; Marmorino et al., 1998), and the 60s helix is present (Jeng et al., 1990). In fact, except for the non-native heme ligation (Jeng et al., 1990; Robinson et al., 1983) and a general structural loosening, the protein is highly native-like in the A-state. We have shown here, however, that native tertiary interactions involving Leu68 are incompletely formed. Taken together with the results of kinetic studies, these observations suggest that the A-state is most closely related to species that occur late in folding.
Materials and Methods Nomenclature Variants are denoted by the one-letter amino acid code with the wild-type residue given ®rst followed by the position number and the new residue. The C102T variant of yeast iso-1-cytochrome c is referred to as the wild-type protein, and all variants contain the C102T mutation. This mutation facilitates biophysical characterization but does not alter the structure or function of the protein (Berghuis & Brayer, 1992; Cutler et al., 1987; Gao et al., 1991). Production of mutants DNA manipulations involved the yeast-shuttle phagemid pJB750 (Beasley & Pielak, 1996). Escherichia coli strain DH5aF0 [F0 , f80dlacZM15, recA1, endA1, gyrA96, thi-1, hsdR17(rÿ k , Mk ), supE44, relA1, deoR, (lacZYA-argF) U169] (Hanahan, 1983) and yeast strain B6748 (MAT cycl-::lacZ cyc7-::CYH2 ura3-52 his31 leu2-3 leu2-112 trp1-289 can1-100 cyh2) (Holzschu et al., 1987) were used for all transformations. Competent E. coli cells were prepared by treatment with DMSO (Hanahan et al., 1995) or purchased from Gibco BRL. Gibco's protocol was used for all transformations except that Luria Broth medium was used for DMSO-treated cells. Yeast transformations were performed with lithium acetate (Ito et al., 1983; Schiestl & Gietz, 1989; Hill et al., 1991; Gietz et al., 1992). DNA sequence analysis was performed by the sequencing facility at UNC-CH or by using a Petri-plate modi®ed version of the Sequenase double-stranded DNA sequencing protocol (Ner et al., 1988). Random oligonucleotide-directed mutagenesis (Kunkel et al., 1987) was used to create 17 L68 mutants.
Structure in a Molten-globule Model Unique oligonucleotides (oligos) were used to create the L68I, L68N, and L68Stop mutants. E. coli strain CJ236 was used to prepare single-stranded uracil-containing DNA. Oligos were phosphorylated at their 50 end as described (Zoller & Smith, 1983), except the buffer provided with the phage T4 polynucleotide kinase was used. A 2:1 mole ratio of oligo to template was used for mutagenesis. The extension reaction utilized phage T4 DNA ligase, phage T7 DNA polymerase, and phage T4 gene 32 protein and was allowed to proceed for one hour at 0 C, one hour at room temperature, and then two hours at 37 C. Putative mutants were identi®ed by the elimination of a ScaI restriction enzyme site at position 3579 bp (Beasley, 1995). The sequence of the entire cytochrome c gene was con®rmed before transforming the phagemid into yeast. Phagemids were isolated from yeast with a modi®cation of the spheroplast method (Rose, 1987), transformed into DH5aF0 , and then the sequence of the entire cytochrome c gene was recon®rmed. Phenotype analysis Transformants harboring each of the 20 different mutants were examined for growth on YPG and YPL at different temperatures as described (Beasley & Pielak, 1996). The results are medium-independent. A transformant has a functional phenotype if it grows at all temperatures tested. The temperature-sensitive phenotype is assigned if a transformant grows at 25 and 30 C but not at 37 C. The very temperature-sensitive phenotype is assigned if a transformant grows at only 25 C and the non-functional phenotype is assigned if the transformant does not grow at any of the three temperatures. Protein purification Distilled and deionized water was used throughout this procedure. Yeast cultures (1 l) harboring the functional L68 mutants were used to evaluate protein expression by using a scaled-down version of our puri®cation scheme. The three mutants with the highest expression were puri®ed according to a modi®ed version of the protocol described by Sherman et al. (1968). Four 5-ml YPS liquid cultures were inoculated with single colonies from YPG plates and incubated at 30 C with shaking for 36 hours. Three 1 l YPG cultures were each inoculated with a 5 ml culture and incubated at 30 C with shaking for three days. A 1 l culture was used to inoculate 11 l of medium in a New Brunswick Scienti®c Microferm fermentor (series MF-2000). The medium comprised YPG, 180 g of sucrose, and 300 ml of antifoam (Rug-Doctor1). After inoculation, 0.75 g of penicillin and 0.45 g of streptomycin were added. The antibiotics had been dissolved in 5 to 10 ml of water and sterile-®ltered with a 0.2 mm Acrodisc1 ®lter (Gelman Sciences). The 12 l culture was incubated at 25 C overnight and then at 30 C for three days. Approximately 10 l were harvested, and 10 l of sterile medium were added to the remaining 2 l. This fermentation procedure was repeated before harvesting the entire culture. After centrifugation, cells were re-suspended in a minimal amount of 1 M NaCl, 50 mM EDTA, 1 mM phenylmethylsulfonyl ¯uoride. One half-volume of ethyl acetate was added and the mixture was stirred at 4 C for 24 hours. The solution was diluted ®ve- to tenfold with water and 130-230 ml of wet CG-50 cationexchange resin was added. The slurry was stirred at 4 C
643
Structure in a Molten-globule Model for two hours, and then the resin was allowed to settle for 0.5 hour. The supernatant was discarded, and the resin was washed with water until the supernatant was clear. The pink resin was transferred to a 2.5 cm 30 cm column and washed with LSB (20 mM sodium phosphate buffer, pH 7). The cytochrome c was eluted with HSB (1 M NaCl, 20 mM sodium phosphate, pH 7). The protein was then dialyzed against LSB and subsequently puri®ed with an HR 10/10 S-Sepharose cation-exchange column on a Pharmacia FPLC with a linear gradient. Red fractions were pooled, concentrated, and further puri®ed with an HSB-equilibrated G-50 gel ®ltration column (2.5 cm 50 cm) ®tted with a peristaltic pump. SDSPAGE with Coomassie-blue staining was used to identify the pure fractions, which were pooled.
The uncertainty in GD(BS) was calculated using the following equation: sGD BS v #2 u 2 " u Tm;var T H m;var m u ÿ 1 sHm sTm;wt u u Tm;wt Tm;wt 2 u u 2 u t Hm sT Tm;wt m;wt For Cp, the uncertainty is the standard deviation of the slope as determined from linear least-squares analysis of the data in Figure 2.
Sample preparation Cytochrome c was oxidized as described by Betz & Pielak (1992), except that LSB was used to equilibrate the CG-50 resin and HSB was used to elute the protein. Variants were desalted by dialysis against water or exchanged into 0.05 M sodium phosphate buffer (pH 7.0) by multiple concentrations with a Centricon-10 microconcentrator (Amicon). The fraction of ferri protein was checked as described (Marmorino et al., 1998). All protein samples were 595 % oxidized. Concentration was determined by averaging the values obtained from the following isobestic points and molar absorptivities: 556.5 nm, 7800 Mÿ1 cmÿ1; 541.8 nm, 9900 Mÿ1 cmÿ1; 526.5 nm, 11,000 Mÿ1 cmÿ1; 504 nm, 600 Mÿ1 cmÿ1; and 434 nm, 22700 Mÿ1 cmÿ1 (Margoliash & Frohwirt, 1959). Protein solutions were then concentrated to >1 mM. Samples were exchanged into the appropriate buffer by either diluting an aliquot of the protein stock solution or by multiple concentrations with a Centricon-10 microcentrator. If <40-fold dilution was required, the latter method was employed. Thermal denaturation An Aviv model 62DS spectropolarimeter ®tted with a thermostatted ®ve-position sample changer was used. Ellipticity was monitored at 222 nm with a 3 nm bandwidth. Data were collected every 1 deg. C at a protein concentration of 30 mM. The N D transition (0.05 M acetate, pH 3.8-5.0) was monitored from 5 C to 85 C following a 15 minute equilibration at 40 C. The A D transition (0.50 M Na2/H2SO4 pH 2.1.) was monitored from ÿ1 to 60 C following a 15 minute equilibration at 30 C. Analysis of uncertainties Uncertainties are the sample standard deviation for all values except GD(T), GD(BS), and Cp. The uncertainty in GD(T) was calculated using the following equation (Cohen & Pielak, 1994): sGD T v 2 2 u u T T u 1ÿ s s T ÿ T ÿ T ln H m C m p u Tm Tm u u 2 u Cp T Hm T t ÿ Cp sTm 2 Tm Tm
Acknowledgements We thank the Pielak group and Chris Dobson for helpful discussions. This work was supported by N.I.H grant GM42501, P.H. was part of the N.S.F.-sponsored S.U.R.E. program, J.C.W. was partially supported by a G.A.A.N.N. fellowship from the U.S. Department of Education, and G.T.W. was partially supported by a U.N.C. Board of Governors fellowship.
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Edited by A. R. Fersht (Received 15 February 1999; received in revised form 7 April 1999; accepted 7 April 1999)