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Published online before print December 29, 2005, 10.1110/ps.051807706
Protein Science (2006), 15:248-257. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Acid destabilization of the solution conformation of Bcl-XL does not drive its pH-dependent insertion into membranes

Guruvasuthevan R. Thuduppathy and R. Blake Hill

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA

Reprint requests to: R. Blake Hill, Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA; e-mail: hill{at}jhu.edu; fax: (702) 441-2490.

(RECEIVED August 24, 2005; FINAL REVISION October 22, 2005; ACCEPTED October 22, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xL{Delta}TM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xL{Delta}TM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xL{Delta}TM ({Delta}G°) was determined to be 15.8 kcal·mol–1. Surprisingly, under conditions that favor a membrane conformation (pH 4.9), {Delta}G° was 14.6 kcal·mol–1. These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity.

Keywords: Bcl-2 family; Bcl-xL; solution to membrane conformational change; diphtheria toxin; pH-dependence; pore-forming toxins; protein folding

Abbreviations: TM, transmembrane anchor • NMR, nuclear magnetic resonance • CD, circular dichroism • GdnHCl, guanidine hydrochloride • HSQC, heteronuclear single quantum coherence

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051807706.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
The Bcl-2 proteins regulate programmed cell death by acting in the cytosol and organellar membranes (Adams and Cory 1998; Chao and Korsmeyer 1998; Green and Reed 1998; Ng and Shore 1998; Harris and Thompson 2000; Hengartner 2000). Some Bcl-2 proteins act by adopting at least two different structural conformations: a solution conformation and an integral membrane conformation. For example, pro-apoptotic Bax is a monomeric, helical bundle protein localized in the cytosol until an apoptotic signal causes translocation to the mitochondrial outer membrane (Suzuki et al. 2000). At the mitochondrial outer membrane, Bax inserts and folds into a large, multimeric integral membrane protein that is thought to regulate the release of cytochrome c (Hsu et al. 1997; Wolter et al. 1997; Shimizu et al. 1999; Antonsson et al. 2000, 2001; Saito et al. 2000).

Similar to Bax, the anti-apoptotic protein Bcl-xL is a soluble, primarily monomeric, helical bundle protein local-izedinparttothecytosol (Muchmore et al. 1996). However, in contrast to Bax, Bcl-xL inserts into the mitochondrial outer membrane and folds into a small, integral membrane protein (Hsu et al. 1997; Minn et al. 1997). For both proteins, the solution-to-membrane conformational change has been reconstituted in vitro with only recombinant proteins and vesicles from synthetic lipids, suggesting that this conformational change might not be receptor-mediated (Minn et al. 1997; Basanez et al. 2001).

The dual structural nature of the Bcl-2 proteins allows for dual mechanisms for their biological activity. In the case of Bcl-xL, the solution conformation acts by sequestering pro-apoptotic factors in the cytosol as a water-soluble helical bundle (Sedlak et al. 1995; Sattler et al. 1997). By contrast, the membrane conformation acts as a small, moderately selective cationic channel in the mitochondrial outer membrane (Minn et al. 1997; Vander Heiden et al. 2001). The exact mechanism of Bcl-xL activity in the membrane is still under debate, but mutants of Bcl-xL that possess altered ion channel properties also have altered apoptotic activities, confirming a biological role for the membrane conformation (Minn et al. 1997, 1999; Xie et al. 1998; Losonczi et al. 2000; Basanez et al. 2001; Vander Heiden et al. 2001). These properties of Bcl-xL were actually demonstrated with Bcl-xL{Delta}TM1 that lacks the C-terminal hydrophobic anchor, which is not required for biological activity or ion channel activity (Muchmore et al. 1996; Minn et al. 1997).

The dual mechanisms for the anti-apoptotic activity of Bcl-xL{Delta}TM in the cytosol and in the membrane raise the question of how a water-soluble protein undergoes a conformational change to become an integral membrane protein. This conformational change appears to have two requirements: acidic pH and presence of lipid vesicles (Basanez et al. 2001). In fact, Bcl-xL{Delta}TM is only weakly associated with lipid vesicles at pH 7.0 but is fully associated at pH 4.5 as measured by a sedimentation assay (Basanez et al. 2001). The ion channel activity of Bcl-xL{Delta}TM also shows a similar pH dependence with conductance occurring readily under acidic conditions but not readily at pH 7.0 (Minn et al. 1997). Because these experiments were performed in vitro with recombinant protein and vesicles derived from synthetic lipids, they suggest that the amino acid sequence alone can specify both solution and membrane conformations.

Many bacterial toxins also undergo a pH-dependent conformational change from solution to membrane conformations including the translocation domain from diphtheria toxin and the colicin family of pore-forming toxins (Parker et al. 1990; Lakey et al. 1992; London 1992; Lacy and Stevens 1998; Zakharov and Cramer 2002a). The solution structures of many of these proteins are known and they share a common helical bundle topology (Parker et al. 1989, 1992; Choe et al. 1992; Elkins et al. 1997). In fact, the motivation to explore ion channel properties of Bcl-xL{Delta}TM arose, in part, from the structural similarity it shares with the translocation domain of diphtheria toxin (Fig. 1Go), which also binds to lipid vesicles in a pH-dependent manner (Sandvig and Olsnes 1980; Muchmore et al. 1996). Some of these helical bundles retain a helical conformation in the membrane (Oh et al. 1996; Lindeberg et al. 2000; Chenal et al. 2002; Zakharov and Cramer 2002b), but no high-resolution structure of a membrane conformation of these proteins has been determined. The topology of this helical membrane conformation must be quite different from the solution conformation, because the polar or charged residues on the surface of the solution conformation would need to be sequestered from the hydrophobic milieu of the membrane bilayer. Based on these considerations, the solution to membrane conformational change has been referred to folding inside-out (Lesieur et al. 1997).


Figure 1
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  		Figure 1. Structural similarity between Bcl-xL (left, from 1MAZ.pdb) and the translocation domain of diphtheria toxin (right, from 1DDT.pdb). The hydrophobic helical hairpin motifs, helices 5,6 of Bcl-xL and helices 8,9 of diphtheria toxin T-domain are represented by a darker shade of gray. These figures were generated using MOLSCRIPT (Kraulis 1991).

 
A change in the solution conformation in the absence of lipid vesicles is known for many proteins to lower the activation energy for the solution to membrane conformational change (Lesieur et al. 1997; Zakharov and Cramer 2002a). This change in the solution conformation can be large, such as a change in quaternary structure, or it can be small, such as a change in the tertiary structure commonly referred to as a molten globule conformation (Bychkova et al. 1996). A molten globule conformation is characterized by native-like secondary structure without the well-packed hydrophobic core found in native-like proteins (Ptitsyn et al. 1990; van der Goot et al. 1991). For example, an acid-induced conformational change in the pore-forming toxin Colicin A, even in the absence of lipid vesicles, results in formation of a molten globule conformation that more readily associates with lipid vesicles than the solution conformation that predominates at pH 7.4 (van der Goot et al. 1991). In fact, an acid-induced molten globule formation is the dominant mechanism for membrane insertion of many other proteins that undergo a solution to membrane conformational change including annexin 6, TRAIL, StAR, diphtheria toxin, and other toxins (Blewitt et al. 1985; van der Goot et al. 1991; Bychkova et al. 1996; Song et al. 2001; Chenal et al. 2002; Nam and Choi 2002).

For Bcl-xL{Delta}TM, a mechanism for the solution to membrane conformational change is not known beyond the requirement for lipid vesicles and acidic conditions. Therefore, we first asked what changes are occurring to this protein under acidic conditions in the absence of lipid vesicles. Specifically we tested whether lowering the pH induces a change in the tertiary or quaternary structure by examining changes in the thermodynamic stability and structural properties of Bcl-xL{Delta}TM.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Thermodynamic stability of Bcl-xL{Delta}TM upon acidification is slightly lowered
Bcl-xL{Delta}TM is able to bind to lipid vesicles upon acidification (Minn et al. 1997; Basanez et al. 2001). This process could be facilitated by a change in structure, or dynamics, of the protein upon acidification even in the absence of lipid vesicles, which will be reflected in the thermodynamic stability of the protein. Therefore, the free energy of unfolding of Bcl-xL{Delta}TM was determined at pH 7.4 and 4.9 by chemical denaturation monitoring the circular dichroism signal at 222 nm as a function of increasing GdnHCl concentration (Fig. 2Go). The data fit well to a two-state model for the unfolding reaction. At pH 7.4, the change in free energy of unfolding ({Delta}G°) for Bcl-xL{Delta}TM was determined to be 15.8 ± 1.2 kcal · mol–1, while the corresponding value at pH 4.9 was 14.6 ± 1.0 kcal · mol–1. Chemical denaturation of Bcl-xL{Delta}TM monitored by the change in intrinsic tryptophan fluorescence confirmed these results (data not shown).


Figure 2
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  		Figure 2. The thermodynamic stability of Bcl-xL{Delta}TM is slightly reduced upon acidification. The circular dichroic signal at 222 nm of Bcl-xL{Delta}TM was monitored as a function of GdnHCl and the resulting data fit as described in the text. The free energy of folding, {Delta}G° (H2O), under conditions that do not favor membrane insertion (pH 7.4 [•]) (A) is 15.8 ± 1.2 kcal · mol–1. Under conditions that favor membrane insertion (pH 4.9 [{circ}]) (B), {Delta}G° (H2O) is 14.6 ± 1.0 kcal·mol–1. The mG value decreases from 4.3 ± 0.3 kcal·mol–1·M–1 at pH 7.4 to 3.5 ± 0.2 kcal · mol–1 · M–1 at pH 4.9.

 
While no pH-dependent change is observed in the thermodynamic stability for Bcl-xL{Delta}TM, the relative enthalpic and entropic contributions to the free energy of folding might exhibit a pH-dependence that would inform on the mechanism of the solution to membrane conformational change. Presumably, the enthalpy of folding does have a pH-dependence because certain residues will become protonated from pH 7.4 to 5.0, and the enthalpy of protonation for these residues will contribute to the enthalpic contributions to the free energy of folding (Pfeil and Privalov 1976; Petrosian and Makhatadze 2000). To detect such enthalpy–entropy compensation to the overall free energy, we performed differential scanning calorimetry experiments at pH 7.4 and 4.9. The midpoint for the unfolding transition (Tm) is reduced upon acidification from 76°C to 71°C (data not shown). Unfortunately, the thermal unfolding transition was irreversible under these and other conditions; no further thermodynamic analysis was possible.

A decrease in pH does not induce a change in the quaternary structure
Given that the thermodynamic stability of Bcl-xL{Delta}TM at pH 4.9 is still quite high (14.6 kcal · mol–1), we postulated that this protein might undergo a pH-dependent oligomerization as an intermediary step toward membrane insertion. We hypothesized that a decrease in pH might destabilize the monomer and favor oligomer formation providing the necessary free energy to achieve a membrane-insertion competent state and lead to the insertion of Bcl-xL{Delta}TM into the membrane. Previously, Bcl-xL{Delta}TM was reported to undergo dimerization although only in the presence of nonionic detergents (Xie et al. 1998). Additionally, an oligomeric insertion mechanism is common for many beta-barrel toxins (Gouaux 1997; Heuck et al. 2001) and the annexin family of proteins (Beermann et al. 1998).

To test for acid-induced formation of oligomers in solution, we used sedimentation equilibrium experiments. Six data sets collected using protein at two different concentrations and equilibrated at three different rotor speeds were globally fit to a modified Lamm-Svedberg equation (supplemental data). Analysis performed at pH 7.4 and 4.9 illustrated that there was no significant change in the oligomeric state of the protein as a function of pH (Fig. 3Go). The data at both pH values were well described by a fit to a single monomeric species. The molecular weight estimates were 25.4 ± 0.2 kDa at pH 7.4, and 27.3 ± 0.2 kDa at pH 4.9. The actual molecular weight of Bcl-xL{Delta}TM is 23.8 kDa. The slight discrepancies in the molecular weights most likely are due to the uncertainty in the estimate for the partial specific volume (Kharakoz 1997), because fits of the data to a monomer–dimer equilibrium gave unreasonable values for KD (102 M). Therefore, Bcl-xL{Delta}TM is monomeric at both conditions; however, we cannot exclude the possibility of oligomerization in the presence of the membrane.


Figure 3
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  		Figure 3. Bcl-xL{Delta}TM is monomeric in solution upon acidification. Representative sedimentation equilibrium data are presented for 25 µM Bcl-xL{Delta}TM at a XLI rotor speed of 19,000 rpm in solution at 25°C collected at pH 7.4 (A) and 4.9 (B). The data are well described by fitting to a single species (—) that is the molecular weight of monomeric Bcl-xL{Delta}TM within experimental uncertainty. Every second data point is displayed ({circ}) but all data were globally fit. The fit to the molecular weight of the dimer of Bcl-xL{Delta}TM (- - -) and the residuals to the fits are also displayed. The global fit of the data from three concentrations and XLI rotor speeds of 17,000, 19,000, and 22,000 rpm was carried out using a modified Lamm-Svedberg equation as described in the text. The global fit to six data sets is presented as supplemental data.

 
Backbone structure of Bcl-xL{Delta}TM upon acidification is retained
To test for any pH-dependent conformational change of the backbone of Bcl-xL{Delta}TM, we recorded the far-UV circular dichroism spectrum as a function of pH (Fig. 4Go). The far-UV CD spectrum reports primarily on the secondary structure of a protein and for Bcl-xL{Delta}TM at pH 7.4 is typical of a well-folded helical bundle protein with ~36% {alpha}-helicity. At pH 4.9, the spectrum was very similar to pH 7.4 (33% {alpha}-helicity), indicating no significant changes in the backbone structure occur upon acidification.


Figure 4
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  		Figure 4. Secondary structure of Bcl-xL{Delta}TM is conserved upon acidification. The far-UV circular dichroism spectra were collected at pH 7.4 (•) and pH 4.9 ({circ}).

 
The circular dichroism spectrum measures mean residue ellipticity that is unable to report on site-specific changes in the backbone structure of Bcl-xL{Delta}TM as a function of pH, whereas NMR spectroscopy can provide more detailed information. Therefore, to gain such information and to determine that the CD results did not arise from a concomitant loss and gain of helicity in the protein upon acidification, we examined the 1H-15N HSQC NMR spectrum as a function of pH (Fig. 5AGo). 1H-15N HSQC experiments provide residue-specific information primarily of the backbone structure of a protein. Upon lowering the pH, no significant change in the chemical shifts of the backbone amides of Bcl-xL{Delta}TM were observed in either the 1H (Fig. 5BGo–D) or 15N data (not shown), which is consistent with the CD results. These results suggest that no significant structural changes, local or global, occur in the backbone structure of Bcl-xL{Delta}TM from pH 7.4 to 4.9.


Figure 5
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  		Figure 5. The backbone structure of Bcl-xL{Delta}TM does not undergo significant conformational change upon acidification. 1H-15N HSQC spectra of Bcl-xL{Delta}TM were collected on a 0.6-mM sample of uniformly 15N labeled protein as a function of pH. (A) A representative 1H-15N HSQC spectrum for Bcl-xL{Delta}TM at pH 7.4 is shown. The differences in amide proton chemical shift between pH 7.4 and pH 5.9 (B), pH 5.3 (C), and pH 4.9 (D) are displayed. Note that some minor differences are expected due to the salt dependence of the chemical shift (Schaller and Robertson 1995). Differences in amide nitrogen chemical shift also showed no significant changes as a function of pH (data not shown).

 
Bcl-xL{Delta}TM upon acidification does not form a molten globule
In the case of many bacterial toxins, membrane insertion upon acidification is aided by the formation of a molten globule intermediate. In a molten globule, the secondary structure of the protein is similar to the native state and the general tertiary fold is retained (Ptitsyn et al. 1990; Creighton 1992). However, the structural integrity of the hydrophobic core of a molten globule is compromised with the apolar side chains adopting more rotameric positions than in the native state (Creighton 1992). Thus, the hydrophobic core of a molten globule is more fluid than that of a native protein.

Therefore, we tested whether the ability of Bcl-xL{Delta}TM to insert into membranes upon acidification arises from the formation of a molten globule that could be detected by observing changes in the hydrophobic core. The near-UV CD signal can report on the structural integrity of the aromatic residues in the hydrophobic core of the protein. In the case of a molten globule state, the aromatic residues are no longer well packed in an asymmetric environment, resulting in a loss of signal in this region of the spectrum. However, at pH 4.9, we observed no change in the near-UV CD signal from that observed at pH 7.4 (Fig. 6Go), suggesting the lack of an acid-induced molten globule state of Bcl-xL{Delta}TM.


Figure 6
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  		Figure 6. The integrity of the hydrophobic core packing in Bcl-xL{Delta}TM is conserved upon acidification. Near-UV spectra were collected at pH 7.4 (•) and pH 4.9 ({circ}).

 
A hallmark of a molten globule state is the ability to bind hydrophobic dyes such as 8-anilino-1-naphthalene sulfonic acid (ANS). In fact, Bcl-xL{Delta}TM has been reported in the past to bind ANS (Xie et al. 1998). However, the solution conformation of Bcl-xL{Delta}TM reveals a hydrophobic cleft that would be expected to bind ANS without molten globule formation. For this reason, we measured the pH dependence of the methyl carbon chemical shifts, which are a more direct measure of the integrity of the hydrophobic core. These chemical shifts arise from methyl side chains of residues found primarily in the hydrophobic core of a protein. The spectrum collected at pH 7.4 showed well-dispersed resonances in the methyl region indicative of a well-packed hydrophobic core and typical of a native protein (Fig. 7AGo). Upon lowering the pH to 4.9, these methyl resonances were still well dispersed, indicating that the protein retained a well-packed hydrophobic core consistent with the absence of a molten globule state (Fig. 7BGo). The absence of any gross structural change in Bcl-xL{Delta}TM between pH 7.4 and 4.9 argues for either subtle structural or dynamical changes in solution that we were unable to detect by these CD or NMR experiments, or the requirement of lipids for its pH-dependent solution-to-membrane conformational change.


Figure 7
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  		Figure 7. No indication of molten–globule formation upon acidification of Bcl-xL{Delta}TM. 1H-13C HSQC spectra of Bcl-xL{Delta}TM were collected on a 0.8-mM unlabeled protein sample (natural abundance 13C) as a function of pH. Data shown only for pH 7.4 (A) and pH 4.9 (B).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Electronic supplemental material
 References
 
Here, we present data that suggest the driving force for the solution to membrane conformational change of Bcl-xL is different than the translocation domain from diphtheria toxin by examining the thermodynamic and structural properties of Bcl-xL{Delta}TM as a function of pH in the absence of lipid vesicles. In contrast to diphtheria toxin translocation domain, we find little change in the thermodynamic stability or structure of Bcl-xL{Delta}TM from pH 7.4 to 4.9, while in the presence of lipid vesicles this pH change results in complete association of Bcl-xL{Delta}TM with lipid vesicles (data not shown). The results presented here suggest that this protein does not insert through an obligatory molten globule intermediate that has been observed in the absence of membrane vesicles for other proteins such as cytochrome c, TRAIL, StAR, diphtheria toxin, and other toxins including colicin A, exotoxin A, and equinatoxin (Blewitt et al. 1985; van der Goot et al. 1991; Bychkova et al. 1996; Song et al. 2001; Chenal et al. 2002; Nam and Choi 2002).

The difference in the free energy of folding ({Delta}{Delta}G°) of Bcl-xL{Delta}TM between pH 7.4 and 4.9 in the absence of lipid vesicles is only 1.2 kcal · mol–1, which is within the uncertainty in the measurements and is small relative to its thermodynamic stability (14.6 kcal · mol–1 at pH 4.9). To confirm these observations, we repeated these measurements several times on different preparations of the protein. Therefore, even at a pH that favors membrane insertion, the solution conformation of Bcl-xL{Delta}TM is still quite stable. We conclude that acid-induced destabilization of the solution conformation does not contribute to the energetics of the solution to membrane conformational change. Therefore, membrane insertion of Bcl-xL{Delta}TM must derive from an increase in the number of acidic side chains that partition into the membrane upon protonation at lower pH.

This result is notable because it differs from many proteins with solution conformations and acidic isoelectric points similar to Bcl-xL{Delta}TM (calculated pI of 4.4), such as diphtheria toxin translocation domain, colicin A, and colicin B. In these cases, the primary driving force is the acid-induced destabilization of the solution conformation and not simply the acidification of side chains that favors the membrane conformation as we find for Bcl-xL{Delta}TM (Ramsay et al. 1989; London 1992; Schendel and Cramer 1994; Lesieur et al. 1997; Sathish et al. 2002; Zakharov and Cramer 2002a)). For example, the rate-limiting step for the solution to membrane conformational change of colicin A is the acid-induced unfolding rate of the solution conformation in the absence of lipid vesicles, and not membrane binding (van der Goot et al. 1991). While the driving forces behind membrane insertion differ between Bcl-xL{Delta}TM and many other toxins, our data do not exclude the possibility that the structural conformations involved for these proteins are similar. These toxins are thought to initially insert into membranes via a hydrophobic helical hairpin (Fig. 1Go) and then form an umbrella-like intermediate before becoming fully inserted. Our data do not address this issue and, given the structural similarity of the proteins, we expect that the Bcl-xL{Delta}TM follows a similar pathway.

While the {Delta}{Delta}G° is neglible, we did observe a pH-dependence to the denaturant dependence to the free energy of folding as reflected by the mG value. The mG value decreases from 4.3 ± 0.3 kcal · mol–1 · M–1 at pH 7.4 to 3.5 ± 0.2 kcal · mol–1 · M–1 at pH 4.9. Such a decrease in mG can be interpreted as the presence of an intermediate that is stabilized at acidic pH conditions, leading to a decrease in two-state character and a lower mG value (Whitten et al. 2001). This result might reflect an ensemble-based description of protein structure (Frauenfelder and Leeson 1998; Frauenfelder and McMahon 1998; Whitten et al. 2005). In this description, the macroscopic thermodynamic state of a protein is comprised of an ensemble of microstates that is quite heterogeneous. In this sense, protonation does not lead to changes in the macroscopic free energy of unfolding but can affect the distribution of microstates that could be reflected in a difference in the mG value that we observe.

Our results with Bcl-xL{Delta}TM have implications for the full-length molecule. The requirement for acidic pH conditions in vitro for the solution to membrane conformational change of Bcl-xL{Delta}TM in the presence of lipid vesicles is to potentially increase the likelihood that the protein lacking the C-terminal transmembrane anchor will associate with the membrane (Schendel et al. 1998). This acidic pH requirement might not be necessary for the full-length molecule. Or perhaps a lower decrease in pH is necessary for the full-length molecule to drive the equilibrium from solution to the membrane conformations in vivo. Interestingly, a slight decrease in the pH of the cytosol from ~7.4 to 6.8 during the initial phases of apoptosis has been reported (Matsuyama et al. 2000). Also, the anionic lipids on the surface of a membrane, like the mitochondrial outer membrane, cause a negative surface potential, increasing proton concentration near the surface and effectively lowering the local pH near the membrane surface (McLaughlin 1989; Menestrina et al. 1989; van der Goot et al. 1991; Murray et al. 1999).

Perhaps the reason for a difference between diphtheria toxin and Bcl-xL relates to the biological process that triggers these conformational changes. While the exact trigger for Bcl-xL is unknown, diphtheria toxin enters the cell via a clathrin-coated endosome that becomes acidic during maturation of the endosome (Draper and Simon 1980; Sandvig and Olsnes 1980). By contrast, Bcl-xL is exposed to a cytosolic environment that is more susceptible to intracellular proteases than the endosome. Therefore, the avoidance of a molten globule intermediate might have evolved to prevent unregulated degradation of Bcl-xL by intracellular proteases that would cleave a molten globule intermediate state more efficiently than the native state of Bcl-xL. Such proteases would not be present in the endosome and might have allowed the evolution of a diphtheria toxin that capitalizes on the acidic nature of the endosome. Based upon the results presented here, Bcl-xL evolved a different driving force for membrane insertion.

The significance of these results extends beyond the Bcl-2 and toxin field because many proteins link their protonation state to functional transitions such as hemoglobin (Ackers 1998). Typically, such changes in protonation state are accompanied by conformational changes; however, as demonstrated here, such conformational changes may not derive from protonation alone. Indeed, pH dependent cooperativity can be quantitatively described without invoking conformational changes for cases when strong electrostatic interactions exist between ligand binding sites and protonation sites (Spassov and Bashford 1998). It appears the Bcl-xL may be an example of this phenomenon.

In summary, to gain a clearer understanding of the pH-dependent solution to membrane conformational change of Bcl-xL{Delta}TM we first analyzed the thermodynamic stability and solution conformation of Bcl-xL{Delta}TM upon acidification in the absence of membranes. Contrary to many other membrane-insertable proteins, we find no evidence for acid-induced unfolding of the Bcl-xL solution conformation. Therefore, the main driving force for membrane insertion must derive from the free energy of binding to the membrane that only occurs upon protonation of acidic residues.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Electronic supplemental material
 References
 
Protein expression and purification
The gene encoding for human Bcl-xL(1–209), which lacks the C-terminal hydrophobic 24 amino acids, was subcloned into pHis-GB vector using EcoRI and SalI restriction sites by standard procedures (Sambrook and Russell 2001). This construct is an N-terminal fusion of the B1 domain from streptococcal protein G (GB1), and was selected to improve expression and solubility of Bcl-xL (Amezcua et al. 2002). The GB1 domain itself contains an N-terminal 6xHis tagfor purificationbyNi2+ affinitychromatography. 6xHis-GB1 is separated from Bcl-xL{Delta}TM by a 15-residue linker that contains a recognition site for cleavage by TEV protease, which allows for the liberation of Bcl-xL{Delta}TM from the fusion construct upon incubation with 6xHis-TEV protease. Success of the subcloning procedure was confirmed by nucleotide sequencing, and the resulting plasmid transformed into Escherichia coli Rosetta cells (Novagen) for protein overexpression.

To increase the yield of protein in the soluble fraction, cells were grown at 37°C in 2 L of LB media to an OD600 of ~0.7, collected by centrifugation, and gently resuspended in 0.5 L of M9 minimal media and continued to grow at 37°C (Marley et al. 2001). After 1 h, protein expression was induced by the addition of 0.5 mM IPTG. The cells were harvested by centrifugation after 5–6 h at 37°C and resuspended in Buffer A (20 mM Tris, 0.5 M NaCl at pH 8.0), lysed by three passes through a French press. The protein present in the soluble fraction was then purified by affinity purification on a Ni2+ affinity column (His-Trap, GE Healthcare), followed by dialysis into TEV protease cleavage buffer (50 mM Tris, 50 mM NaCl, 5 mM beta-mercaptoethanol at pH 8.0). The fusion protein was then treated overnight at 4°C with recombinant 6xHis-TEV protease (1:50 w/w ratio) to release Bcl-xL{Delta}TM from the GB1 domain. The reaction mixture was loaded onto the His-Trap column again and the flow-through fraction containing Bcl-xL{Delta}TM was collected, concentrated, and quantitated using UV-absorbance ({varepsilon}280 = 41,820 M–1cm–1 in 6 M GdnHCl). The yield of pure protein was ~10 mg/L. The purity was >95%, as judged by Coomassie-stained gel electrophoresis, and the identity of the protein was further confirmed by MALDI-TOF mass spectrometry. 15N-labeled samples were prepared in a similar manner in the presence of 15NH4Cl in the M9 minimal medium. All the proteins were stored at 4°C until used.

Chemical denaturation studies
The free energy of unfolding of Bcl-xL{Delta}TM was determined by chemical denaturation titration experiments using GdnHCl. The unfolding reaction was monitored by observing the changes in the ellipticity at 222 nm using a Jasco J-810 spectropolarimeter. The temperature was maintained at 25°C and the scan speed was 10 nm/min. Each data point represents the average of five accumulations with a response time of 2 sec. The data of {varepsilon}222 versus [GdnHCl] were fit to a two-state model using a nonlinear least-squares fit to the following equation using a script written in Igor Pro 4.04:


Formula

where bN, mN, and bD, mD represent the ordinate and slope of the native and the denatured baselines, respectively (Santoro and Bolen 1988). Values for the native and the denatured state baselines were estimated by multiple fits to the raw data to minimize the {chi}2 of the fit. The global folding stability extrapolated to standard conditions is given by {Delta}G°H2O, and mG is the slope of the linear extrapolation curve that represents the denaturant dependence of the Gibbs free energy.

Analytical ultracentrifugation
Sedimentation equilibrium experiments were performed on Bcl-xL{Delta}TM at 25°C in a Beckman XL-I analytical ultracentrifuge; 10 µM, 25 µM, and 50 µM Bcl-xL{Delta}TM samples in 20 mM sodium phosphate buffer at pH 7.4 and in 20 mM sodium acetate buffer at pH 4.9 were subjected to centrifugation at the following speeds: 17,000, 19,000 and 22,000 rpm. Equilibrium was achieved after 12 h, and the resulting data sets were globally fit to a modified Lamm-Svedberg equation as described (Hill et al. 2000). Excellent fits were obtained by fitting to a single species using the Marquardt-Levenberg nonlinear least-square algorithm (Levenberg 1944; Marquardt 1963). The partial specific volume was estimated to be 0.7217 from the amino acid composition using the method of Cohn and Edsall (1943). The density of the buffers were estimated to be 0.99971 (phosphate) and 0.999723 (acetate) using the program Sednterp (www.jphilo.mailway.com).

Circular dichroism spectroscopy
The far-UV circular dichroism spectrum was collected on a 7-µM sample of Bcl-xL{Delta}TM in 20 mM sodium phosphate at pH 7.4 or 20 mM sodium acetate at pH 4.9 using a JASCO Model 710 CD Spectropolarimeter. The far-UV spectra were collected from 260 nm to 190 nm at a scan rate of 10 nm/min. The near-UV spectra from 320 nm to 250 nm were collected at a scan rate of 10 nm/min, with a protein concentration of 40 µM. All the spectra were collected at 25°C, corrected for buffer ellipticity, and represented the signal average of five accumulations. The percent {alpha}-helicity was estimated by the method of Luo and Baldwin (1997).

NMR spectroscopy
Unlabeled or 15N-labeled NMR samples of Bcl-xL{Delta}TM (0.5 mM) were prepared in 20 mM sodium phosphate buffer plus 10% D2O at pH 7.4 (meter reading) and titrated down to pH 4.9 by the addition of small amounts of 0.1 N hydrochloric acid. The NMR experiments were performed at 25°C either on a Varian INOVA 500 MHz or a Bruker AVANCE 600 MHz spectrometer equipped with a triple resonance probe with triple axis gradients. The 1H-15N heteronuclear single quantum coherence (HSQC) spectra were collected with 1024 x 128 complex points (acquisition times of 64 msec and 32 msec in the direct and the indirect detected dimension, respectively) with the 15N carrier offset at 117.5 ppm. The 1H-13C HSQC was collected at natural abundance (2048 transients) with 1024 x 40 complex points with acquisition times of 64 and 7.4 msec in 1H and 13C, respectively, with 13C carrier offset placed at 17 ppm. The data were processed with nmrPipe (Delaglio et al. 1995) and displayed using NMRView (Johnson and Blevins 1994; Johnson 2004).


   Electronic supplemental material
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
References
 
Global fits of six sets of equilibrium sedimentation data from Bcl-xL{Delta}TM (two concentrations at three speeds each) to a single species of monomer molecular weight.


   Footnotes
 
Supplemental material: see www.proteinscience.org


   Acknowledgments
 
We thank Bertrand Garcia-Moreno and Kevin R. MacKenzie for helpful comments, and Frederick J. Tan and Corina E. Rogge for reading the manuscript. We also thank Kevin H. Gardner for generously providing the pHis-GB plasmid. This work was supported in part by the NIH GM067180 and American Cancer Society Award IRG-58-005-41, and funds generously provided by JHU.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
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