Rational design of `water-soluble' bacteriorhodopsin variants

Kakoli Mitra1, Thomas A. Steitz1 and Donald M. Engelman1,3

1 Department of Molecular Biophysics and Biochemistry, 2 the Howard Hughes Medical Institute, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have explored the interchangeability of soluble and membrane proteins by attempting to render a helical membrane protein `water soluble' through mutation of its lipid-exposed residues. Using an atomic resolution structure of bacteriorhodopsin (bR), two different strategies were developed to identify lipid-exposed residues for mutation. In the first strategy all residues in trimeric bR with solvent accessibility >35% were marked for replacement. Replacement residues were chosen so as to map an average surface of helical soluble proteins onto the bR surface, resulting in the mutagenesis of 14.9% of surface residues. The second strategy took into account the observation that accessible residues can be categorized as fully or partially accessible. Consequently, three mutants were designed based on monomeric bR, all with their accessible residues changed and with varying extents of mutagenesis of partially accessible residues. 13.5–24.3% of the wild-type surface was altered in these designs. The construct for the first design was cloned into Escherichia coli. Trace amounts of the mutant protein were expressed with the concurrent overexpression of an endogenous prolyl isomerase. In contrast, all three mutant proteins of the second design expressed well and could be purified to homogeneity. Systematic refolding trials were undertaken with limited success at solubilization in aqueous media. We have discussed the feasibility of applying the `solubilization strategy' outlined here to membrane proteins.

Keywords: bacteriorhodopsin/folding/membrane protein/solubility/surface redesign


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been proposed that similarities in certain structural features of the interiors of water-soluble and membrane proteins support a unifying view of protein structure in which the structural organization of both protein classes reflect variations on the same themes (Rees et al., 1989aGo). The ultimate test of this proposal would be to render a membrane protein water soluble by mutating only surface residues, or vice versa. If such a transformation were possible, many problems associated with membrane protein manipulation might be alleviated. Due to the amphipathic nature of membrane proteins, most studies in vitro necessitate the presence of detergents and/or lipids as cosolvents to stabilize the protein in a native conformation. Rendering membrane proteins soluble in aqueous media while preserving their function might be a possible approach to facilitate the manipulation of membrane proteins for structural and biochemical studies.

`Solubilization' of a membrane protein by redesign of its exterior is contingent upon the similarities and differences in both structure and folding of soluble and membrane proteins. Structural comparisons have revealed several similarities (Cramer et al., 1992Go) in: (i) protein surface area (Israelachvili, 1985Go); (ii) packing density of buried atoms (Rees et al., 1989bGo); and (iii) the relative average hydrophobicities of buried residues (Rees et al., 1989aGo). These findings suggest that from a protein stability point of view changing the lipid-exposed residues of a membrane protein into polar and charged residues might not result in a significantly destabilized protein. However, certain differences arise when taking protein folding mechanisms into account. Some aspects of secondary structure formation, for instance, seem to be driven in membrane proteins by forced hydrogen bond formation in low dielectric environments (Engelman and Steitz, 1981Go; Popot and Engelman, 1990Go) and partly by secondary structure propensities in soluble proteins (Chakrabarty and Baldwin, 1995Go). Thus, transmembrane helices can accommodate many residues that have high ß sheet propensities (Deber and Goto, 1996Go).

In attempting to `solubilize' a membrane protein two requirements should be met by the test system. First, the lipid-exposed residues should be identifiable. Second, there should exist a simple assay to test for retention of functionality of the `solubilized' protein. The seven transmembrane helix protein bacteriorhodopsin (bR) is well suited for this study. High resolution structures make possible the identification of lipid-exposed residues (Grigorieff et al., 1996Go; Pebay-Peyroula et al., 1997Go). Moreover, native binding of retinal produces a color change that serves as a visible assay for folding (Khorana, 1988Go; Steinhoff et al., 1994Go). Finally, bR has been shown to be a very stable protein, tolerant of a wide variety of mutations/deletions (Popot et al., 1987Go; Khorana, 1988Go) and capable of being refolded from denatured states (Booth et al., 1995Go), thus being an ideal candidate for mutagenesis studies.

To generate water-soluble bR variants two distinct sets of mutant proteins were designed using different strategies to identify solvent-exposed residues. Since wild-type bR is observed to be trimeric in its native membrane lattice, the first design aimed at preserving native trimer contacts. The second set of bR variants were designed to be monomeric. Of the mutant proteins that express well in Escherichia coli, solubility varies somewhat with the extent of surface mutagenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification and replacement of solvent-accessible residues

All designs were initially based on the bR structure (2BRD) reported by Grigorieff et al. (Grigorieff et al., 1996Go). For the first design (d1tbR), which aimed to preserve native trimer contacts, interacting residues were identified initially using InsightII and GRASP (Nicholls et al., 1991Go). A PDB file for trimeric bR was generated using the symmetry operations reported in the Grigorieff et al. structure. Accessible surface area calculations were performed using ACCESS (Lee and Richards, 1971Go). The FRACtional Sidechain accessibility (FRACS) of a residue is defined as the absolute accessible surface area (Å2) of the side chain divided by the `Eisenberg area' for that side chain (Eisenberg et al., 1989Go), giving a value between 0 (not accessible) and 1 (completely accessible). The `Eisenberg area' is an average accessible surface area for residue X derived from Gly–X–Gly sequences in the Brookhaven Protein Database. In this study the side chain for glycine is defined as its C{alpha} atom. Model PDB files for the designed proteins were generated using MidasPlus (Computer Graphics Laboratory, University of California, San Francisco, CA (sup. NIHRR-01081)) by amino acid substitutions into 2BRD. In calculating soluble surface templates for d1tbR, the following {alpha} helical structures were used: interleukin 4 (1RCB) (Wlodawer et al., 1992Go), {alpha}-parvalbumin (1RTP) (McPhalen et al. 1994Go), uteroglobin (2UTG) (Bally and Delettre, 1989Go), Rop (1ROP) (Banner et al., 1987Go), Fis protein (1FIA) (Kostrewa et al., 1992Go), cytochrome c' (2CCY) (Finzel et al., 1985Go), calmodulin (1OSA) (Rao et al., 1993Go), hemerythrin (1HMD) (Holmes et al., 1991Go) and myoglobin (1MBO) (Phillips, 1980Go). The protein structures used for analysis in the design of the Design Two (d2) constructs are reported elsewhere (K.Mitra, unpublished data).

Gene synthesis

The gene for d1tbR was constructed de novo by annealing 16 80–100 bp oligonucleotides with 20–25 bp overhangs following an established procedure (Pompejus et al., 1993Go). The genes for the d2 constructs were constructed de novo by overlapping oligonucleotide PCR (Chen et al., 1994Go) using Pfu DNA polymerase (Stratagene). Subsequent errors in DNA sequence were corrected by Quick-Change site-directed mutagenesis (Stratagene). The genes for all constructs encoded residues 7–227 corresponding to wild-type bR. All oligonucleotides were synthesized at the HHMI Biopolymer and W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

Cloning and protein expression

Plasmids (Novagen) were used which contained IPTG-inducible T7 promoters. The d1tbR gene was cloned into pET15b using NdeI and BamHI (restriction enzymes from New England BioLabs) sites to obtain a thrombin cleavable N-terminally His-tagged construct. The d1tbR and d2 genes were also cloned into pET29b using NcoI and XhoI to obtain thrombin cleavable N-terminally S-tagged and C-terminally His-tagged constructs. The E.coli strains BL21(DE3), BL21(DE3)pLysS, HMS174(DE3) and Novablue(DE3) were purchased from Novagen. In some protein induction conditions the chromophore all-trans-retinal was used, which was purchased from Sigma. For western blots the Ni2+-NTA HRP-conjugated antibodies were obtained from Qiagen, the S-protein HRP conjugates from Novagen and the anti-bR antibodies were a kind gift from H.G.Khorana. Ni2+-NTA Superflow resin was purchased from Qiagen. S- and His-tagged bR variants all had 265 amino acids and molecular weights of 29.5 kDa. Cleaved variants had molecular weights of 25 kDa.

Biophysical characterization

To determine whether purified proteins are monodisperse, analysis was done on an HR-10/30 Superdex-200 size exclusion column connected to ultraviolet, multiangle laser light scattering (MALLS) and refractive index detectors. Samples were loaded at 240 µM in 2 M urea, 0.1 M NaH2PO4, 0.01 M Tris–HCl, pH 5.0 at room temperature. Light scattering and differential refractometry were carried out using the Mini-Dawn and Optilab instruments of Wyatt Technology Corp. Analysis was carried out as described by Astra software (Wyatt, 1993Go). Circular dichroism (CD) spectra were recorded at 25°C at protein concentrations of 3 µM in 2 M urea, 0.1 M NaH2PO4, 0.01 M Tris–HCl, pH 5.0. The spectrum was obtained by averaging over five scans with a step size of 1 nm and an averaging time of 4 s on an AVIV Model 62DS CD spectrometer. Measurements were performed in a Hellma quartz cuvette of path length 0.2 cm. Wild-type bR was purchased from Sigma and reconstituted into DMPC/CHAPS micelles following Booth et al. (Booth et al., 1996Go). Excess retinal was dialyzed away from both designed and wild-type bR proteins before making biophysical measurements. The CD spectrum of wild-type bR was taken at a protein concentration of 3 µM, the UV–Vis absorbance spectrum at 12 µM. The UV–Vis absorbance spectra of the designed proteins were measured at protein concentrations of 10 µM in 2 M urea, 0.1 M NaH2PO4, 0.01 M Tris–HCl, pH 5.0 using a 1 cm pathlength quartz cuvette. The protein concentration was determined by amino acid analysis performed at the W.M.Keck Foundation Biotechnology Resource Laboratory at Yale University.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trimeric bR: design rationale and strategy for `soluble' variant

Our goal was to render bR water soluble in as native-like a state as possible while introducing the smallest number of changes on the protein surface. Wild-type bR is an integral membrane protein consisting of a bundle of seven transmembrane helices. In the native purple membrane of Halobacterium halobium, bR is observed to form p3 lattices of trimers (Henderson et al., 1990Go). Indeed, bR also crystallizes by cubic phase methods in three-dimensional crystals as a stack of p3 lattices (Pebay-Peyroula et al., 1997Go). Figure 1Go shows bR as the trimer observed in purple membranes (Grigorieff et al., 1996Go) with each monomer binding one retinal molecule in its hydrophobic core. The area buried in the trimeric interface was calculated to be 1748 Å2 per monomer, a large fraction of the monomer total accessible surface area. As our initial attempt, we decided to design a `soluble' bR variant (designated d1tbR) which preserved the identity of interfacial residues as this would maximize the number of residues left unchanged and potentially preserve the oligomeric state of the protein. Unchanged (inaccessible) residues in trimeric bR were identified from the structure solved by Grigorieff et al. (Grigorieff et al., 1996Go). To a first approximation those helical residues involved in pairwise helix interactions, i.e. within 4 Å of each others' van der Waals surfaces, were identified as being inaccessible. Some residues mapped in this way were ambiguous as to their solvent exposure. To impartially assign residues as inaccessible their FRACS were calculated (see Materials and methods). Residues with FRACS <0.35 were defined as inaccessible and were left unchanged in the design along with residues in the loops connecting helices.



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Fig. 1. The design of d1tbR is based on wild-type bR. bR as seen in the plane of a bilayer is a trimer of seven transmembrane helix bundles. Diphosphatidylglycerophosphate lipid molecules are shown in red and the chromophore retinal in purple. The C{alpha} backbone of each subunit is depicted by a ribbon colored in cyan. On the subunit where transmembrane helices are labeled with Roman numerals the side chains of residues changed in d1tbR are depicted in yellow and blue. This figure was generated by InsightII using the coordinates (2brd) of the structure reported by Grigorieff et al. (Grigorieff et al., 1996Go).

 
To map an average surface of a soluble helical protein onto the surface of trimeric bR the series of steps in the left half of Table IGo was followed. The resultant surface composition of residue side chains (0.35 <= FRACS) in d1tbR consisted of 25.3% hydrophobic residues (as opposed to 50.2% in trimeric bR), 21.6% polar residues (18.5% in bR) and 39.2% charged residues (16.8% in bR). This distribution closely approximated that found for soluble protein surfaces. Overall, 14.9% of all residues were changed. Replacement residues with high helix propensity (in soluble proteins) (Chakrabarty and Baldwin, 1995Go) were chosen to maximize the probability of helix formation in the designed proteins. Wild-type serine and threonine residues were not replaced so as not to disrupt possible existing hydrogen-bonding to the backbone. Residues with charges opposite to the partial charge at helix termini were used to potentially stabilize helical macrodipoles. Those residues that were changed are shown in yellow and blue in Figure 1Go. Figure 2AGo contains the final amino acid sequence of the seven helices in d1tbR as compared to that of wild-type bR.


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Table I. Rules for replacement of residues in `water-soluble' bR variants
 


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Fig. 2. Amino acid sequences of wild-type bR and of designed bR variants. (A) The amino acid sequence for each helix is shown for wild-type bR and the designed proteins. Those residues in shaded boxes are changed relative to wild-type bR. Changed residues in d1tbR are encased in light gray. Acc residues changed in the d2 constructs are encased in dark gray, PAc residues are lowercase and encased in black, with those residues with FRACS >0.4 in bold. (B) When FRACS values are plotted against residue number for helix VII of wild-type bR it can be seen that solvent-exposed residues fall into categories of Acc (FRACS >0.5) and PAc (0.25 < FRACS <= 0.5). In the sequence Acc residues are bold and underlined, PAc residues are lowercase and those PAc residues with FRACS >0.4 are bold and lowercase.

 
Trimeric bR: expression of `soluble' variant

The d1tbR construct was made with a thrombin cleavable N-terminal His-tag and expressed in BL21(DE3) cells using an IPTG-inducible T7 promoter. Under various induction and growth conditions (temperature: 16–42°C; [IPTG]: 0.05–5 mM; media: Luria–Bertani, Terrific broth; retinal: 0–20 µM) a protein of ~25 kDa (molecular weight of cleaved d1tbR protein), as discerned on SDS–PAGE gels, was overexpressed and localized to both lysed cell pellets and supernatants (data not shown). A faint 25 kDa band was detected in western blots of the lysed cell pellets using both Ni2+-NTA-conjugated antibodies and antibodies against the C-terminal tail of wild-type bR. The protein could be purified from the supernatant and pellet fraction under 8 M urea as well as native conditions on an Ni2+ column. N-terminal amino acid sequencing identified >99% of the purified material to be an FKBP-type peptidyl-prolyl cistrans isomerase SLYD endogenous to E.coli, and 0.7% as the N-terminus of His-tag cleaved d1tbR. The expression of d1tbR was also attempted in other E.coli strains such as HMS174(DE3) and Novablue(DE3) with similar results. Finally, a d1tbR construct with a thrombin cleavable N-terminal S-tag and C-terminal His-tag was made. This construct was cloned into BL21(DE3)pLysS cells under an IPTG-inducible promoter to achieve more stringent control over protein induction and expression. No protein was expressed using this construct.

In summary, the `soluble' bR variant designed to be trimeric could in most cases not be expressed in E.coli. Trace amounts of the partially degraded variant could be poorly expressed under certain conditions (~0.1 mg/l of culture, as estimated from western blot) with concomitant overexpression of an endogenous prolyl isomerase, indicating that the bR variant might be misfolded, proteolyzed and/or aggregated. Sufficient protein could not be separated from the prolyl isomerase for biophysical characterization.

Monomeric bR: design rationale and strategy for `soluble' variants

The problems encountered with the bR variant d1tbR prompted us to reevaluate our design strategy. A concern was that the potential failure of d1tbR to trimerize would expose a large hydrophobic surface area (the trimeric interface), which could lead to aggregation/proteolysis and preclude correct folding. A second concern was the observation that wild-type bR purifies as a monomer in the absence of lipids (Lustig et al., 2000Go). These problems could be avoided by redesigning the bR monomer to be soluble. An additional concern was that the interiors of helical membrane proteins might contain a higher proportion of ß-branched residues (low helix propensities) relative to soluble helical proteins. This difference might hamper the formation of helices and subsequently of a correctly folded helical bundle in a bR variant forced to fold in an aqueous environment. To compare the interiors of both classes of proteins helical soluble proteins and helical integral membrane proteins from the Brookhaven Protein Database were analyzed for the amino acid compositions of their hydrophobic cores. One result from this study (K.Mitra, unpublished data) was that the overall amino acid composition of the interiors of both protein classes were found to be similar with differences arising as solvent accessibility of residues increases. This encouraged us to design a second set of three `water-soluble' bR variants (d2 series), based on monomeric bR, in which the extent of surface mutagenesis was varied. As shown in Figure 2BGo, residues in a given helix can be categorized according to their FRACS values. Accessible residues are either completely accessible (Acc) (FRACS > 0.5) or partially accessible (PAc) (0.25 < FRACS <= 0.5).

To introduce flexibility into our design strategy we made three mutant proteins, each with solvent inaccessible residues and loops left unchanged and all Acc residues changed. The three designs varied in the extent of mutagenesis of PAc residues. No PAc residues were altered in the protein designated d2Acc, PAc residues with FRACS > 0.4 were mutated in the protein d2PAc*, and all PAc residues were changed in the protein d2PAc. Following the rationale given in the right half of Table IGo, templates of average soluble protein surfaces, delineated at FRACS > 0.5 and 0.25 < FRACS <= 0.5, were mapped onto the corresponding surfaces of monomeric bR to give the designed proteins. The final amino acid sequences of all three d2 proteins is given in Figure 2AGo. Figure 3AGo shows models of all three mutants, where the side chains of changed residues are coded by different colors according to their solvent accessibility. In Figure 3BGo the accessible surfaces of wild-type bR, of models of d2Acc and d2PAc, and of a representative helical soluble protein (factor for inversion stimulation (Fis) protein) are colored according to residue type (e.g. polar, hydrophobic, etc.). It can be seen that the residue type distributions on the surfaces of the d2 mutants are similar to the distribution on the soluble protein surface.



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Fig. 3. The extent of surface mutagenesis varies in the d2 variants. (A) Only the side chains of changed residues are shown in model structures of each variant. Acc residues (yellow) are changed in all constructs. In d2PAc* PAc residues with FRACS >0.4 are shown in purple. For d2PAc all PAc residues are colored in red. (B) Accessible surfaces of wild-type bR, d2Acc, d2PAc and of a representative soluble {alpha} helical protein, Fis protein (1fia), are shown, where yellow represents hydrophobic, purple polar, red negatively charged and blue positively charged residues. PDB files for d2Acc, d2PAc* and d2PAc were generated by simple substitution of residues into 2brd using MidasPlus. Figures were generated using GRASP.

 
Monomeric bR: expression of `soluble' variants

All d2 constructs were made with a thrombin cleavable N-terminal S-tag and C-terminal His-tag and were cloned separately into BL21(DE3) or BL21(DE3)pLysS. When mutants were expressed in the latter, some protein was found in the supernatant of the cell lysate. These proteins, however, pelleted when centrifuged at 40000g. The identity of the mutants was verified by western blot using S-protein HRP conjugates and by MALDI–TOF mass spectrometry. When expressed in BL21(DE3), proteins were found in the cell pellet (Figure 4Go). Initially, the pellets were dissolved in 8 M urea and the proteins purified on an Ni2+ column under denaturing conditions. The proteins were then dialyzed to 2 M urea in incremental steps of 1 M urea. Further dialysis or direct transfer into aqueous, organic or surfactant solutions was attempted, where protein concentration, retinal concentration, ionic strength, osmolarity, pH, surfactant type and organic solvent content were varied. All conditions were tried with both thrombin cleaved and uncleaved constructs. Table IIGo is a representative but not exhaustive list of the range of conditions tried at each protein manipulation step (i.e. expression, cell lysis, purification and refolding) in attempts to solubilize the designed proteins. For all cases, except for dialysis into and out of SDS, the designed proteins precipitated when organic solvents or surfactants were completely withdrawn from solution. When the mutant proteins that were originally in 2 M urea were dialyzed into 11 mM SDS and then into 50 mM Tris–HCl, pH 7.6, 0.5 M NaCl, they were initially soluble at <0.1 mg/ml. These results encouraged further trials with SDS where cell pellets were washed with Triton X-100 and then taken up in 10 mM SDS. Subsequent to dialysis into aqueous buffer KCl was added to precipitate the remaining SDS. After initially appearing soluble, all proteins precipitated after a few days. In attempts to crystallize these proteins no demonstrable protein crystals were obtained.



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Fig. 4. d2PAc* is overexpressed in BL21(DE3) cells. The overexpression of d2PAc* in BL21(DE3) is representative of both d2Acc and d2PAc. Protein is found in the induced cell pellet. Protein standard sizes are given in kilodaltons. The standard and cell fractions were run on a 12.5% SDS–PAGE gel, which was Coomassie stained.

 

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Table II. Variations at protein manipulation steps in efforts to solubilize d2 designed proteinsa
 
Monomeric bR: characterization of `soluble' variants

All three mutant proteins expressed very well in E.coli., with expression levels at hundreds of milligrams per liter of culture. After purification, the proteins remained soluble in solutions of 8 M down to 2 M urea. However, decreasing the urea concentration below 2 M resulted in the depletion of protein from solution as evidenced by massive precipitation, and confirmed by western blots and absorbance at 280 nm. In general, the solubility of the three mutant proteins followed the series: d2PAc* > d2Acc > d2PAc. Table IIIGo is a summary of some biochemical and biophysical characteristics of the three mutant proteins. MALLS showed that the S- and His-tagged proteins were monodisperse in 2 M urea (Figure 5AGo). CD spectra were measured for the proteins in 2 M urea, 25%, 50% and 75% TFE. The CD signal for protein in 2 M urea demonstrated mostly ß structure, potentially a convolution of ß and helical components in 25% TFE and helical structure in 50 and 75% TFE (Figure 5BGo). Wild-type bacteriorhodopsin apoprotein takes up the chromophore all-trans-retinal into its hydrophobic core to give bR. If the retinal pocket (i.e. hydrophobic core) is correctly folded the retinal forms a Schiff base with Lys216 and lends the complex its characteristic purple color (Bayley et al., 1981Go). All-trans-retinal was added to the mutant proteins at different stages of protein expression, purification and refolding. The pH of solutions was varied from 4.5 to 8.0 to promote Schiff base formation (especially under acidic conditions). UV–Vis absorbance spectra of the mutant proteins were measured to determine whether retinal was bound and/or present in its Schiff base form. Under all conditions and combinations of retinal addition neither was a red shifted absorption band detected (Schiff base) nor could spectral characteristics of bound or unbound retinal be observed (Figure 5CGo): none of the proteins was purple in color.


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Table III. Biochemical and biophysical characteristics of d2 designed proteins
 


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Fig. 5. Biophysical characterization of d2 designed proteins. (A) d2PAc* is monodisperse as measured by MALLS. (B) CD spectra of d2PAc* in 2 M urea (black filled circles), 2 M urea diluted with TFE to 25% TFE (dark gray filled circles), 2 M urea diluted with TFE to 50% TFE (light gray filled circles), and 2 M urea diluted with TFE to 75% TFE (empty circles). The reference spectrum of wild-type bR reconstituted into DMPC/CHAPS vesicles is shown (gray triangles). (C) UV–Vis absorbance spectra of d2PAc* (black filled circles) and wild-type bR (gray triangles) as reference. The spectra for d2PAc* are representative of both d2PAc and d2Acc proteins.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several laboratories have attempted to convert bR into a functional water-soluble protein by altering the residues in contact with the lipid environment. To our knowledge, the success of these experiments has been limited and the redesign strategies have not been documented in the literature. To prevent recycling of design strategies and to stir discussion of membrane protein engineering and design we present our strategies for redesign of bR together with our results. The aim of this presentation is to discuss the basic ideas in such design efforts and to document a range of efforts.

Two classes of design were implemented in attempts to produce `water-soluble' bR. Expression of the trimeric construct (d1tbR) in E.coli induces overexpression of a cistrans prolyl isomerase, which co-purifies with His-tag cleaved fragments of d1tbR. Very little d1tbR is produced. The proteins designed to be monomeric (d2 series) express and purify well but are not soluble upon removal of urea, surfactants or organic solvents. None of the proteins is purple in color, which was one of the criteria used to assess folding of the protein core.

Poor expression of d1tbR (~0.1 mg/l of culture) may be due to the nature of the native oligomeric interface of bR, which was left unchanged. In crystal structures the trimeric interface of wild-type bR is involved in specific interactions with lipids (Essen et al., 1998Go) and trimer stability depends partly on surrounding solvation lipids (Stelzer and Gordon, 1986Go). As a result, the hydrophobic trimer interface alone may not suffice to hold d1tbR together as a trimer, resulting in unstable d1tbR molecules with exposed hydrophobic patches. Indeed, in the absence of lipids wild-type bR purifies as a monomer (Lustig et al., 2000Go). The overexpression of E.coli cistrans prolyl isomerase upon induction might be explained by the large number of prolines in the d1tbR sequence, which could serve as substrates for the isomerase.

The proteins designed to be monomeric (d2 series) express well and are moderately soluble at low concentrations of urea. The lower solubility of the d2PAc variant, in which all PAc residues are mutated, might be due to steric disturbances of residue packing in the protein core. The CD spectra of the proteins in 2 M urea indicate that they adopt mostly ß structures, i.e. are not properly folded. In urea/TFE solutions there are indications of some helical secondary structure: in 75% TFE the `soluble' variants are ~75% as helical as wild-type bR. The experiments in TFE only serve to indicate that the proteins are capable of forming helices. The designed proteins are not soluble in fully aqueous media, which may be a result of any of the following. First, the accuracy of mapping the lipid exposed surface of bR may have been compromised by the moderate resolution (3.5 Å horizontal, 4.3 Å vertical) of the structure used as the basis of the design strategies described here (Grigorieff et al., 1996Go). To address this concern the solvent accessibilities of residues in four more recent higher resolution bR structures (Kimura et al., 1997Go; Pebay-Peyroula et al., 1997Go; Luecke et al., 1998Go; Mitsuoka et al., 1999Go) were calculated for comparison. All of the residues calculated to be Acc or PAc in the Grigorieff et al. structure are also accessible in the more recent structures, suggesting that surface mapping was not compromised by using a lower resolution structure. Second, upon comparing the interiors of soluble and membrane helical proteins, differences in average hydrophobicity are found. Helical membrane protein interiors are on average 0.15 kcal/mol/residue less hydrophobic than helical soluble protein interiors (K.Mitra, unpublished data). Given that a bR monomer has 50 buried residues, the interior is ~7.5 kcal/mol less hydrophobic than the interior of an average helical soluble protein. Upon `solubilization' of a membrane protein the hydrophobic effect may not be large enough to drive folding towards a stably folded state, given that the free energy of unfolding of most soluble proteins lies between 5 and 20 kcal/mol (Privalov and Gill, 1988Go). Third, glycine and proline occur on average less frequently in soluble proteins than in bR. Since these residues are thought to be helix breakers in soluble proteins, `solublized' bR proteins may encounter problems in forming/stabilizing helices in an aqueous environment. Finally, membrane proteins experience anisotropic forces contributed by the hydrophobic bilayer core in terms of lateral pressure and by the water surrounding the protein on either side of the bilayer in terms of surface tension. A solubilized membrane protein would not experience anisotropic solvent forces and might have difficulties in forming its core structure. Effects of lateral pressure in the bilayer on protein folding and stability have been demonstrated (Booth et al., 1997Go; Cantor, 1997Go).

Chen and Gouaux (Chen and Gouaux, 1997Go) introduced five polar residues in helix D of bR and found that the enthalpy of activation for thermal unfolding of the mutant was ~3.4 kcal/mol less favorable as compared to wild-type bR. Between 30 and 54 (13.5–24.3% of all residues) bR residues have been altered to generate the constructs used in this study. Destabilization may be linked to the extent of mutagenesis and may be too large to fold the proteins properly. This is reflected in the solubility trend of the monomeric bR variants, where the more soluble mutant proteins, d2Acc and d2PAc*, have a smaller proportion of their surface altered than d2PAc, which is perhaps more destabilized. The difficulty in determining the degree of success in solubilizing a membrane protein by redesign lies in not knowing how close a designed protein is to being properly folded, since there are a large number of possible misfolded states. Some lessons can be learned, however, from our `solubilization' trials on bR. In redesigning the surface of a membrane protein it is best to avoid leaving large hydrophobic patches, as for putative oligomeric interfaces, since these may lead to poor expression, aggregation and/or proteolysis. When designing `soluble' membrane protein variants it is advisable to replace all or most Acc residues but only a few PAc residues in order to minimize interference with packing in the protein core. It is best to design a series of proteins with permutations of different PAc residues mutated so as to sample a large set of mutants for proper folding. When redesigning the surface of a `solubilized' membrane protein the stability in aqueous solution might be increased by introducing pairs of salt bridges (Strop and Mayo, 2000Go).

The strategy of `solubilization' described in this work can be applied to membrane proteins for which atomic resolution structures do not exist, since mutagenesis experiments can determine which residues are solvent exposed (Lemmon et al., 1994Go). In addition, there are several biophysical techniques, such as electron paramagnetic resonance (Altenbach et al., 1990Go) and sulfhydryl exchange of cysteine groups (Arkin et al., 1996Go), that can be used to map lipid-exposed residues. Currently, in our laboratories, the `solubilization strategy' outlined here is being applied to simpler transmembrane systems, such as homooligomeric proteins. The use of simpler systems will enable us to address some of the questions posed here about the feasibility of `solubilizing' membrane proteins.


    Notes
 
3 To whom correspondence should be addressed. E-mail: don{at}chimera.csb.yale.edu Back


    Acknowledgments
 
We thank I.Ubarretxena for scrupulous reading of the manuscript and many helpful suggestions. We thank Patrick J. Fleming for generous assistance in data processing. K.M. was supported by a PGS Fellowship granted by the Natural Science and Research Council of Canada.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received March 28, 2001; revised September 13, 2001; accepted February 8, 2002.





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