Non-ionic detergent affects the conformation of a functionally active mutant of Bcl-XL

Yee-Joo Tan,1 and Anthony E. Ting

Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We found that a mutant, Bcl-XL(F131V), which was previously reported to have impaired binding capacity, can bind to Bax almost as strongly as wild-type Bcl-XL. In the absence of detergent, the Bcl-XL(F131V) mutant adopts the same conformation as wild-type Bcl-XL, as determined by circular dichroism spectroscopy and size-exclusion chromatography. However, non-ionic detergent induces a conformational change in the Bcl-XL(F131V) mutant and causes it to lose Bax-binding capacity. Wild-type Bcl-XL, on the other hand, is more resistant to detergent-induced effects and retains its ability to bind Bax in the presence of detergent. Since it has been shown that the Bcl-XL(F131V) mutant has nearly the same anti-apoptotic activity as wild-type Bcl-XL, it would be likely that the Bcl-XL(F131V) mutant can adopt the wild-type conformation, rather than the detergent-induced conformational state and can bind to Bax in vivo. Therefore, our data demonstrated that non-ionic detergent can have unpredicted effects on protein conformation, differential effects on wild-type and mutant Bcl-XL proteins in this case and may cause complications in the interpretation of in vitro binding studies.

Keywords: Bcl-2 family/conformation change/non-ionic detergent/protein–protein interactions


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein–protein interactions play important roles in many biological systems and protein complexes are implicated as essential components of many diverse processes. Therefore, it is not surprising that many in vitro assays have been designed to identify as well as characterize protein–protein interactions (Phizicky and Fields,1995Go; Bagby et al., 1998Go; Farmer and Caprioli, 1998Go). The use of in vitro binding assays, which are usually fast and simple, in combination with site-directed mutagenesis often allows the determination of the relationship between the binding properties of proteins and their functional/enzymatic activities, as well the structural details of the site of interactions (see review by Di Cera, 1998).

In the development of in vitro assays for studying protein–protein interactions, it has become common to use mild non-ionic detergents to assist in the solubilization of proteins and to reduce non-specific interactions. However, non-ionic detergents can cause changes in protein conformation. For example, recent studies have shown that Bax, a member of the Bcl-2 family, can undergo detergent-induced conformation changes (Hsu and Youle, 1997Go, 1998Go).

The Bcl-2 family of proteins constitutes one of the biologically important gene products for the process of apoptosis (also called programmed cell death). Some members of this family are anti-apoptotic (e.g. Bcl-2, Bcl-XL) as the over-expression of these proteins inhibits apoptosis induced by many different stimuli, whereas other members are pro-apoptotic (e.g. Bax, Bak) with over-expression promoting apoptosis (Reed 1994Go, 1997Go; Boise et al., 1995Go; White 1996Go; Yang and Korsmeyer 1996Go; Gross et al., 1999Go). Protein–protein interactions between members of the Bcl-2 family are believed to play a role in the regulation process as homodimerization of the pro-apoptotic proteins promotes apoptosis, whereas heterodimerization between pro-apoptotic and anti-apoptotic proteins enhances cell survival (Oltvai et al., 1993Go; Sato et al., 1994Go; Yin et al., 1994Go, 1995Go; Sedlak et al., 1995Go; Zha et al., 1996Go). However, other studies have indicated that pro- and anti-apoptotic Bcl-2 family members can function independently under certain circumstances (Cheng et al., 1996Go; Simonian et al., 1996Go, 1997Go; Knudson and Korsmeyer, 1997Go; Zha and Reed, 1997Go).

We initiated a study to characterize the kinetics and equilibrium for the interaction between Bcl-XL and Bax proteins. A mutant created by a single substitution of Phe131 to Val, Bcl-XL(F131V), was intended as a control as it was previously found to have impaired binding capacity (Cheng et al., 1996Go), but to our surprise, we find that this mutant can bind to Bax as strongly as wild-type Bcl-XL in the absence of detergent. Further investigations revealed differential effects of non-ionic detergent on the conformations of Bcl-XL wild-type and Bcl-XL(F131V) mutant. These findings allow us to re-assess the binding properties of this mutant and may have implications on the relationship between the binding properties and anti-apoptotic activities of Bcl-XL proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Purification of bacterially expressed proteins

Bacterially expressed glutathione S-transferase (GST)-fusion Bax({Delta}21) was purified using GSH-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). The mouse Bax protein used in this study lacks the 21 amino acids at the COOH terminus. Bacterially expressed wild-type 6His-N-terminal tag full-length human Bcl-XL (amino acid 1–234) protein was purified with NTA-Ni-Sepharose beads (Amersham Pharmacia Biotech) followed by FPLC using a mono Q column. The Bcl-XL(F131V) mutant was expressed and purified in the same manner. Protein concentrations were determined using a Coomassie Plus assay kit (Pierce, Rockford, IL).

BIAcore experiments

The experiments were run on a BIAcore 2000 (BIAcore, Uppsala, Sweden) and the ligands [GST and GST-Bax({Delta}21)] were immobilized on two different flow cells of a CM5 chip using the amine-coupling kit according to the manufacturer's instructions (BIAcore). The running buffer contained 10 mM HEPES (pH 7.2) and 150 mM NaCl. No detergent was added to the running buffer. The analyte (6His-Bcl-XL wild-type or mutant) was injected over the flow cells in a sequential mode at a flow-rate of 1 µl/min. A pulse (1 min) of 50 mM NaOH was used to regenerate the surface between each run. The RU(eq) reading after the binding had reached a plateau was taken for each analyte concentration (C), after the subtraction of any non-specific binding of the analyte to the GST flow cell and the equilibrium dissociation constant, KD, calculated from a plot of RU(eq)/C versus RU(eq) (BIAcore AB, 1992Go).

Pull-down binding assay

GST-Bax({Delta}21) (0.1–4 µM) was captured on GSH-Sepharose beads, washed with reaction buffer [10 mM HEPES (pH 7.2), 150 mM NaCl, with or without non-ionic detergent, Nonidet P-40 (NP40; BDH Laboratory Supplies, Poole, UK)] and equilibrated for 1 h at room temperature with in vitro translated [35S]methionine-labeled Bcl-XL proteins, which were prepared using a coupled transcription–translation system (Promega, Madison, WI). The beads were washed three times with reaction buffer, heated with 50 µl of Laemmli's buffer, subjected to electrophoresis on a 15% SDS–polyacrylamide gel, fixed and autoradiographed. A densitometer was used for quantification. Equilibrium dissociation constants, KD, were calculated as previously described (Tan et al., 1999Go). Briefly, KD is obtained from a plot of 1/[AB] versus 1/[A0], where [AB] is the concentration of Bax–Bcl-XL complex and [A0] is the concentration of GST-Bax immobilized on GSH-Sepharose beads.

Co-immunoprecipitation experiments

Some 293 cells were transiently transfected with Myc-tag Bax({Delta}21) and HA-tag Bcl-XL wild-type or mutants [Bcl-XL(F131V) and Bcl-XL(V135A/N136I/W137L)] using superfect transfection reagent (Qiagen, Hilden, Germany). After 24–48 h, the cells were harvested and lysed in reaction buffer [10 mM HEPES (pH 7.2), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and 0.2% NP40 or reduced Triton-X-100 (Sigma Chemical, St. Louis, MO)]. The lysate was then incubated with a polyclonal anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Myc antibody (Santa Cruz Biotechnology) for 2 h at 4°C. Immuo complexes were collected by adsorption on 10 µl of protein A-Sepharose (Roche Molecular Biochemicals, Indianapolis, IN), washed three times with reaction buffer, separated on a 15% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk, incubated with a primary antibody for 3 h, washed and incubated with a secondary antibody (Pierce) for 1 h, followed by detection using an enhanced chemiluminescence method (Pierce). The primary antibodies used were monoclonal anti-HA antibody (Roche Molecular Biochemicals) or anti-Myc antibody (Santa Cruz Biotechnology).

Circular dichroism measurements and mass spectrometry

Circular dichroism (CD) measurements were carried out using a JASCO-J715 spectropolarimeter (JASCO, Tokyo, Japan). Spectra were taken using cells of 1 mm pathlength and recorded in 0.2 nm wavelength increments with a slit width of 2 nm, a scan speed of 50 nm/min and a 4 s response time. Each spectrum (200–260 nm) is an average of three scans corrected for background solvent effects. The Bcl-XL proteins were dialyzed and diluted into reaction buffer [10 mM HEPES (pH 7.2), 150 mM NaCl, with or without reduced Triton-X]. The protein concentration used was about 3 µM. The mean residue molar ellipicity ([{theta}]) was calculated using the molecular weights of proteins, which were determined on a Qstar mass spectrometer according to the manufacturer's instructions (Perkin-Elmer SCIEX, Thornhill, ON, Canada).

Size-exclusion chromatography

A 30 µl volume of purified protein (~10–15 µM) was run on a Superdex 200 PC 3.2/30 column using a SMART system (Amersham Pharmacia Biotech) at a flow-rate of 30 µl/min at room temperature. Molecular weight standards were purchased from Sigma or Amersham Pharmacia Biotech. The buffers used were as in CD measurements.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dissociation constant for the binding of Bcl-XL wild-type and Bcl-XL(F131V) mutant to Bax in the absence of detergent

BIAtechnology allows real-time measurement of macromolecular interactions through the use of surface plasmon resonance technology (Jönsson et al., 1991Go; Malmqvist, 1993Go). In this study, GST-Bax (ligand) was immobilized on the surface and the analyte, 6His-Bcl-XL, was flowed over the surface. The binding profiles (also called sensograms) in Figure 1Go show that the binding of Bcl-XL(F131V) mutant to Bax appears to be similar to that for Bcl-XL wild-type. A control protein, GST, did not show much binding to GST-Bax (Figure 1Go) and there was also little non-specific binding of analyte to GST protein immobilized in another flow cell (data not shown). The equilibrium dissociation constants, KD, for the binding of wild-type Bcl-XL or Bcl-XL(F131V) mutant to Bax were determined on the BIAcore by obtaining the equilibrium binding RUs at various concentrations of analyte (BIAcore AB, 1992Go) and the results are shown in Table IGo. The KD value for binding of the wild-type Bcl-XL to Bax is in fair agreement with published data (Xie et al., 1998Go).



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Fig. 1. Sensograms showing real-time binding of Bcl-XL wild-type (dashed line) and Bcl-XL(F131V) mutant (thin solid line) to GST-Bax({Delta}21) immobilized on a CM5 chip. A control protein, GST (thick solid line), did not bind to GST-Bax({Delta}21). The flow-rate was 1 µl/min. The concentrations of proteins used were Bcl-XL wild-type 14 µM, Bcl-XL(F131V) mutant 10 µM and GST 20 µM.

 

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Table I. Dissociation constant, KD, for the binding of Bcl-XL wild-type and Bcl-XL(F131V) mutant to Bax determined by two independent equilibrium methods
 
A second independent pull-down assay was also used to determine the KD values for the interactions between Bax and Bcl-XL wild-type or Bcl-XL(F131V) mutant. Here, various amounts of GST-Bax were immobilized on GSH beads and the immobilized GST-Bax was then used to capture in vitro translated radioactive-labeled Bcl-XL proteins. The amount of Bcl-XL proteins pulled down was determined to give KD values (Tan et al., 1999, Table IGo).

The dissociation constants obtained using both methods are in good agreement and showed that the Bcl-XL(F131V) mutant can bind to Bax as strongly as Bcl-XL wild-type (Table IGo). This result is in contrast to a previous study, where it was found by co-immunoprecipitation that Bcl-XL(F131V) mutant showed strongly impaired binding to Bax but yet retained similar anti-apoptotic activity as Bcl-XL wild-type (Cheng et al., 1996Go). We note that those authors used non-ionic detergent, whereas our BIAcore and pull-down experiments were carried out without detergent, suggesting that detergent may be the cause of the discrepancies. Therefore, pull-down assays were repeated in presence of detergent to compare the binding of wild-type and mutant Bcl-XL to Bax.

Differential effects of non-ionic detergent on the binding of in vitro translated Bcl-XL proteins to Bax

In this experiment, GST-Bax was immobilized on GSH-Sepharose beads and then used to pull-down in vitro translated 35S-labeled Bcl-XL wild-type or mutants. The binding was carried out in reaction buffer containing different amounts of detergent. From Figure 2aGo, it is clear that increasing the amount of non-ionic detergent (NP40) in the reaction buffer reduces significantly the binding of Bcl-XL(F131V) mutant to GST-Bax. In contrast, the presence of detergent had little effect on the binding of Bcl-XL wild-type to GST-Bax (Figure 2aGo). When the amount of Bcl-XL mutants bound to Bax is expressed as a percentage of the amount of wild-type Bcl-XL bound, it can be seen that the binding of Bcl-XL(F131V) mutant reduces from 60% of wild-type value to only 25% when the amount of NP40 is increased from 0 to 0.2% (Figure 2bGo). For another mutant, Bcl-XL(V135A/N136I/W137L), the corresponding reduction in binding is from 23 to 0% (Figure 2bGo).



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Fig. 2. (a) In vitro translated 35S-labeled Bcl-XL wild-type and mutants were bound by GST-Bax({Delta}21) (labeled `Bax') or GST alone (labeled `GST') immobilized on GSH-Sepharose beads. Buffers with different amount of detergent (NP40) were used for the interaction and washing steps. `INPUT' represents 10% of the amount of in vitro translated protein used in the pull-down assays. Bcl-XL wild-type abbreviated to `wild-type', Bcl-XL(F131V) mutant abbreviated to `F131V' and Bcl-XL(V135A/N136I/W137L) mutant abbreviated to `VNW->AIL'. (b) For each detergent concentration, the amount of Bcl-XL mutants bound to GST-Bax({Delta}21), after subtraction of non-specific binding to GST alone, was expressed as a percentage of the value for Bcl-XL wild-type.

 
Co-immunoprecipation results depend on antibodies used for immuno-adsorptions

Co-immunoprecipation experiments were performed to understand further the effects of detergent on the binding properties of Bcl-XL(F131V). Firstly, mammalian cells were transiently transfected with Myc-tag Bax({Delta}21) (abbreviated to Myc-Bax) and HA-tag Bcl-XL (abbreviated to HA-Bcl-XL) wild-type or mutants. Following lysis in reaction buffer containing 0.2% NP40, either an anti-HA antibody is used to immuno-adsorb the HA-Bcl-XL proteins or an anti-Myc antibody is used to immuno-adsorb Myc-Bax.

In the first instance where HA-tag Bcl-XL proteins were immuno-absorbed on protein A-agarose beads, both Bcl-XL wild-type and Bcl-XL(F131V) mutant can bind to Bax with similar affinity (Figure 3aGo). In contrast, another mutant, Bcl-XL(V135A/N136I/W137L), does not bind Bax at all (Figure 3aGo). Interestingly, the presence of 0.2% NP40 does not have an effect on the binding of Bcl-XL(F131V) mutant to Bax in this experiment. However, when the experiment is reversed such that Myc-Bax is immuno-absorbed instead, it is clear that the binding of Bcl-XL(F131V) mutant to Bax is significantly lower than that for wild-type Bcl-XL (Figure 3bGo). Consistently, the second experiment reproduced the binding results of Cheng et al. (1996), where the immuno-adsorbed Bax protein was not capable of binding Bcl-XL(F131V) mutant.



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Fig. 3. Co-immunoprecipation experiments where (a) HA-Bcl-XL proteins were immuno-adsorbed on protein A beads and any Myc-Bax proteins bound to Bcl-XL were detected with an anti-Myc antibody. (b) In the reverse experiment, Myc-Bax proteins were immuno-adsorbed on protein A beads and any HA-Bcl-XL proteins bound to Bax were detected with an anti-HA antibody. The same abbreviations for Bcl-XL proteins as in Figure 2Go are used. `Before IP' represents 5% of the cell lysate used for immunoprecipation experiments and `after IP' represents the protein complex adsorbed on protein A beads. No non-specific binding of any of the tagged proteins to protein A beads was observed (data not shown).

 
In summary, our BIAcore experiments, pull-down assays and co-immunoprecipation experiments consistently suggest that Bcl-XL(F131V) mutant can bind to Bax in the absence of detergent. However, the presence of non-ionic detergent significantly reduced the binding capacity of Bcl-XL(F131V) mutant. Only in the case where HA-Bcl-XL(F131V) was immobilized on protein A-Sepharose beads did the presence of detergent not affect the binding of Bcl-XL(F131V) to Bax, probably because immobilization on a solid support made the Bcl-XL(F131V) mutant more resistant to detergent-induced unfolding or conformation changes.

Identical co-immunoprecipation results were obtained when a structurally similar non-ionic detergent, reduced Triton-X, replaced NP40 (data not shown). Reduced Triton-X has a low absorbance in the ultraviolet (UV) region and is suitable for spectroscopic studies. Therefore, reduced Triton-X was used subsequently for CD and size-exclusion experiments.

Conformation changes monitored using far-UV CD spectrum

The far-UV CD spectrum of a protein reflects the secondary structures of a protein (Alder et al., 1972Go; Greenfield, 1996Go). The spectra of Bcl-XL wild-type and Bcl-XL(F131V) mutant in buffer in the absence of detergent are similar (Figure 4a and bGo). However, in the presence of a non-ionic detergent (Triton-X), the spectrum of Bcl-XL(F131V) mutant changes significantly with a negative peak at 208 nm increasing in magnitude as the detergent concentration increases to 0.2% (Figure 4bGo). In contrast, the spectrum of wild-type Bcl-XL showed only slight changes in the presence of 0.2% detergent (Figure 4aGo). We noticed slight differences between our spectrum of Bcl-XL wild-type and that from another laboratory (Xie et al., 1998Go), which could be due to the differences in buffer system or protein constructions, as we used full-length Bcl-XL whereas a C-terminal truncated form is used by that laboratory.



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Fig. 4. Far-UV CD spectra of (a) 6His-Bcl-XL wild-type and (b) 6His-Bcl-XL(F131V) mutant in the absence or presence of non-ionic detergent (reduced Triton-X). No detergent (dotted line), 0.04% detergent (dashed line) and 0.2% detergent (solid line). Mean residue molar ellipicities ([{theta}]) were calculated using molecular weights determined by mass spectrometry. Molecular weights of 6His-Bcl-XL wild-type and 6His-Bcl-XL(F131V) mutant are 26 991.85 and 26 943.44 Da, respectively.

 
Together with the above binding data, it appears that non-ionic detergent induces a conformation change in Bcl-XL(F131V) mutant, resulting in its failure to bind to Bax in in vitro assays. On the other hand, Bcl-XL wild-type is resistant to detergent-induced effects and maintained its ability to bind Bax even in the presence of non-ionic detergent (0.2%).

Bcl-XL(F131V) mutant changes mobility in the presence of detergent

Analysis by size-exclusion chromatography showed that both wild-type Bcl-XL and Bcl-XL(F131V) mutant are monomeric at neutral pH (10 mM HEPES, pH 7.2, 150 mM NaCl) (Figure 5aGo). In the presence of 0.2% reduced Triton-X, the elution time of wild-type Bcl-XL did not change but that of Bcl-XL(F131V) mutant decreased significantly, coinciding with the molecular weight of a dimer instead (Figure 5bGo). The change in elution time of Bcl-XL(F131V) in the presence of detergent can be caused by the formation of non-covalent homodimer or, alternatively, may be a result of a conformational change. Indeed, it was shown previously that wild-type Bcl-XL can undergo a conformation change at low pH (and in the presence of detergent) and it was suggested that this state may be a membrane-insertion competent structure that enhances the ion-channel activity of Bcl-XL at low pH (Xie et al., 1998Go). Therefore, in support of the CD data, size-exclusion experiments showed that Bcl-XL(F131V) mutant undergoes a conformation change in the presence of non-ionic detergent. In contrast, non-ionic detergent (0.2%) has a minimum effect on the conformation of wild-type Bcl-XL.



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Fig. 5. Chromatograms showing the elution times of 6His-Bcl-XL wild-type (thin solid lines) and Bcl-XL(F131V) mutant (dashed/thick lines) in the absence (a) or presence (b) of non-ionic detergent (0.2% reduced Triton-X) from a gel filtration column (Superdex 200). The elution times of 6His-Bcl-XL wild-type corresponded to the molecular weight standard of 29 000 in both cases, showing that the wild-type is monomeric. For Bcl-XL(F131V) mutant, the elution time corresponded to a molecular weight of 29 kDa in the absence of detergent, but to 66 kDa in the presence of 0.2% reduced Triton X. Arrows mark the elution times of molecular weight standards in the respective buffers.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Differential effects of detergent on the conformation of Bcl-XL wild-type and mutant

Analysis by two independent in vitro binding assays (BIAcore and pull-down experiments) showed that a mutant, Bcl-XL(F131V), which was previously shown to have impaired binding capacity, can bind to Bax almost as strongly as wild-type Bcl-XL in the absence of detergent (Figures 1 and 2GoGo and Table IGo). However, in the presence of non-ionic detergent, the binding capacity of this mutant is strongly reduced (Figure 2Go).

Co-immunoprecipation experiments showed that if Bcl-XL(F131V) mutant is immobilized on a solid support, then the presence of detergent does not affect its binding capacity (Figure 3aGo). In solution containing non-ionic detergent, however, the mutant underwent conformation change, as illustrated by CD and size-exclusion chromatography and, as a result, lost its capacity to bind Bax (Figures 2, 3b, 4 and 5GoGoGoGo). Non-ionic detergent, on the other hand, does not significantly affect the conformation and binding capacity of Bcl-XL wild-type.

Although, it is not known what the physiological environment under which the interaction between Bcl-XL and Bax occurs in vivo is, it may be the case that the ability of Bcl-XL(F131V) mutant to interact with Bax in the absence of detergent is correlated with its anti-apoptotic activities (Cheng et al., 1996Go). Therefore, the Bcl-XL(F131V) mutant should be re-classified into the class of Bcl-XL mutants whose Bax-binding capacities and anti-apoptotic activities are similar to those of the wild-type Bcl-XL protein.

Changes in hydrophobic energetics as a result of a single Phe to Val mutation

The residue Phe131 is positioned in the BH1 domain of Bcl-XL and forms part of the hydrophobic surface of amphipathic helix 4 in the three-dimensional structure (Muchmore et al., 1996Go). Phe131 has a fairly low solvent accessibility area of 24.3 Å2 [calculated using the software ASC provided by F.Eisenhaber at EMBL, Heidelberg (Eisenhaber and Argos, 1993Go; Eisenhaber et al., 1995Go)], which suggests that Phe131 is involved in many hydrophobic contacts and that this region of the protein is tightly packed. In most of the hydrophobicity scales that were determined by transfer energies of amino acid analogs, the hydrophobicity of a Phe residue is found to be higher than a Val [see Cornette et al. (1987) for a comparison of hydrophobicity scales]. Therefore, we can expect that the mutation of Phe131 to a Val [Bcl-XL(F131V)] would result in a decrease in energy penalty for the hydration of this residue (i.e. a smaller change in the free energies of solvation of side chain, {Delta}Gsolv). In addition, the replacement of a bulky hydrophobic residue in a protein core with a smaller one would result in the loss of hydrophobic contacts and thus hydrophobic energies (Kellis et al., 1988Go, 1989Go; Hurley et al., 1992Go; Lim et al., 1992Go; Jackson et al., 1993Go; Tan et al., 1997Go).

Non-ionic detergents are commonly used in the preparation of membrane proteins because they can increase the hydrophobicity of the solvent environment and thus help to stabilize the highly hydrophobic membrane proteins (see review by Garavito et al., 1996). The addition of non-ionic detergent (0.2%) would be expected to cause a substantial increase in solvent hydrophobicity such that it is no longer energetically unfavorable for the Bcl-XL(F131V) mutant to expose the Val131 residue. Under the same conditions, it would still be energetically unfavorable for the more hydrophobic Phe131 in the Bcl-XL wild-type to become hydrated. Therefore, the mutation of Phe131 to Val131 may have changed the overall solvation/packing energetics of Bcl-XL protein, resulting in increased sensitivity to detergent-induced conformation change. Since residue 131 is located on helix 4, which is an integrated part of the binding site for Bax binding (Muchmore et al., 1996Go; Sattler et al., 1997Go), it would be logical that the (increased) hydration of Val131 in the presence of detergent will affect Bax binding.

Conclusions

We have shown that a single substitution mutation in Bcl-XL may cause changes in the solvation/packing energetics of the protein and result in an increased sensitivity to non-ionic detergent. Our data illustrated that the common use of non-ionic detergent in in vitro binding assays can sometimes introduce artefacts and complicate structure–function analysis. It is not known if other Bcl-XL mutants in published papers may show similar sensitivity to non-ionic detergent as Bcl-XL(F131V) mutant. It may be necessary to re-evaluate the ability of these mutants to bind Bax and the correlation between their binding capacities and anti-apoptotic activities.


    Notes
 
1 To whom correspondence should be addressed. E-mail: mcbtanyj{at}imcb.nus.edu.sg Back


    Acknowledgments
 
We thank S.J.Korsmeyer, C.B.Thompson, K.O.Tan and V.C.Yu for the gifts of Bax, Bcl-XL (wild-type) and Bcl-XL (mutants) genes. We also thank R.Qi for performing the mass spectrometry experiments.


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 Introduction
 Materials and methods
 Results
 Discussion
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Received December 31, 1999; accepted November 9, 2000.





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