Detergent-Associated Solution Conformations of Helical and �-Barrel Membrane Proteins

Yiming Mo,† Byung-Kwon Lee,‡ John F. Ankner,§ Jeffrey M. Becker,*,‡ andWilliam T. Heller*,†

Center for Structural Molecular Biology and Chemical Sciences DiVision, Oak Ridge National Laboratory, OakRidge, Tennessee 37831, Department of Microbiology, UniVersity of Tennessee, KnoxVille, Tennessee 37996,and Neutron Scattering Sciences DiVision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

ReceiVed: February 12, 2008; ReVised Manuscript ReceiVed: August 20, 2008

Membrane proteins present major challenges for structural biology. In particular, the production of suitablecrystals for high-resolution structural determination continues to be a significant roadblock for developing anatomic-level understanding of these vital cellular systems. The use of detergents for extracting membraneproteins from the native membrane for either crystallization or reconstitution into model lipid membranes forfurther study is assumed to leave the protein with the proper fold with a belt of detergent encompassing themembrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2pG-protein coupled receptor from Saccharomyces cereVisiae, and the Escherichia coli porin OmpF. The resultsdemonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helicalproteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles,while the �-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparisonprovided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggeststhat the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR,like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallizationfrom an initially detergent-associated state.

Membrane proteins comprise roughly 30% of most genomessequenced to date, and serve a broad array of functions,including membrane transport and transmembrane signaling. Yetdespite intense interest and study, membrane proteins continueto pose significant scientific and technical challenges, particu-larly for high-resolution structure determination. The numberof atomic-resolution membrane protein structures makes up avery small fraction of the total number of structures depositedin the Protein Data Bank.1 Membrane protein purificationinvolves the use of detergents to extract the protein from thenative membrane. Care must be taken to ensure that thedetergent is efficient at recovering the membrane protein, whichis often difficult to express in quantities comparable to solubleproteins. Further, the detergent must not irreversibly denaturethe membrane protein, thereby precluding refolding for furtherstructural or functional studies.

It is generally assumed that a detergent-solubilized membraneprotein remains properly folded with the detergent encompassingthe trans-membrane portions of the structure,2 as has beenobserved for the detergent visible in high-resolution structuresof membrane proteins. For example, the crystal structure of theEscherichia coli porin OmpF shows a one molecule thick beltof surfactant molecules around the membrane-spanning face ofthe protein.3 Similarly, high-resolution crystal structures ofbacteriorhodopsin (BR) show a limited number of detergent4

and lipid5 molecules surrounding the protein. Crystal structuresof rhodopsin, a G-protein coupled receptor (GPCR), also havedetergent molecules.6-8 The detergent is not always well-ordered, leading to its exclusion from the reported structure.Prince and co-workers utilized neutron diffraction to visualizethe low-resolution structure of the detergent around the lightharvesting complex LH2 complex from Rhodopseudomonasacidophila, again showing a clear belt of detergent around thehydrophobic portion of the protein.9 While these structuresprovide important insight into the interaction between membrane-proteins and detergent or lipids in the native membrane, theresults provide little insight into the solution behavior of thedetergent-solubilized proteins.

Detergent interactions with proteins have been studied bysmall-angle scattering techniques, most commonly using solubleproteins. The detergent micelles bind to hydrophobic portionsof the protein sequence, disrupting the hydrophobic interactionsthat maintain the properly folded tertiary structure of the protein.This structure, in which micelles decorate the unfolded protein,is often referred to as “beads-on-a-string” or a necklacemodel.10,11 The small-angle scattering signal from such astructure can be described analytically by treating it as fractalscattering. Little work has been done to study membraneproteins,12,13 as they tend to be much harder to produce insufficient quantities for study by small-angle scattering tech-niques, and detergent is required to dissolve the membraneprotein in solution. Characterization of the structures ofdetergent-associated membrane proteins would validate assump-tions using in functional studies and provide insight into thechallenges encountered in crystallization of membrane proteins.

Here, a study of the solution structures of three differentmembrane protein-detergent complexes using circular dichro-

* Corresponding authors. (J.M.B.) Telephone: 865-974-3006. Fax: 865-974-4007. E-mail: jbecker@utk.edu. (W.T.H.) Telephone: 865-241-5694.Fax: 865-574-6268. E-mail: hellerwt@ornl.gov.

† Center for Structural Molecular Biology and Chemical SciencesDivision, Oak Ridge National Laboratory.

‡ Department of Microbiology, University of Tennessee.§ Neutron Scattering Sciences Division, Oak Ridge National Laboratory.

J. Phys. Chem. B 2008, 112, 13349–13354 13349

10.1021/jp801266r CCC: $40.75 2008 American Chemical SocietyPublished on Web 09/25/2008

ism (CD) spectroscopy and small-angle X-ray scattering (SAXS)is presented. The porin OmpF was studied in complex with thedetergent octyl polyoxyethylene (OPOE), the detergent used incrystallization.3 Bacteriorhodopsin, a 7TM protein, was studiedin octyl glucoside (OG), also used for crystallization.4 As a thirdmodel system, the Ste2p GPCR from Saccharomyces cereVisiae,another member of the 7TM family of proteins, was studied incomplex with dodecyl maltoside (DM). Unlike the other twoproteins, the high-resolution structure of Ste2p has not beendetermined. The results demonstrate that detergent associateswith OmpF in solution in the expected, belt-like state. Incontrast, the two 7TM proteins are found to exist in partiallyunfolded states. The results have important implications forbiophysical characterization of detergent-solubilized membraneproteins and for membrane protein crystallization from adetergent-solubilized state.

Materials and Methods

Octyl polyoxyethylene (OPOE) was purchased from AxxoraLLC (San Diego, CA). Dodecyl maltoside (DM) and octylglucoside (OG) were from Anatrace Inc. (Maumee, OH). Bac-teriorhodopsin (BR) from H. salinarum was bought from Sigma(St. Louis, MO). Other chemicals were obtained from VWR(West Chester, PA) or Sigma (St. Louis, MO). All chemicalswere used without further purification.

Purification and analysis of the S. cereVisiae Ste2p GPCRwere recently described.14 Briefly, Ste2p with a rhodopsinaffinity tag, consisting of a nine amino acid residue sequenceof rhodopsin, was constructed and used for purification. Yeastcells were homogenized and membranes were collected fromsoluble fraction using ultracentrifugation. Resulting membraneswere solubilized in the presence of DM and the insolublefraction was removed by ultracentrifugation. Solubilized mem-branes were mixed with monoclonal antibody (rhodopsin) 1D4-coupled Sepharose 4B and the resin slurry was batch-loadedonto a column. After extensive washing, the antibody boundreceptor was eluted by excessive competition with the 9-aminoacid rhopsin peptide.

The AD102/pNLF10 strain for wild type OmpF was agenerous gift from Dr. Anne H. Delcour of the University ofHouston. OmpF was expressed and purified as describedpreviously15,16 with slight modifications. Briefly, the cells weregrown in LB broth and induced with 1 mM isopropyl-�-thiogalactose (IPTG). After being washed with cold 10 mMHepes (pH 7.6), the cells were resuspended in 10 mM Hepes(pH 7.6). The cells were broken by French Press and the DNAwas sheared by sonication. Unbroken cells were removed bylow-speed centrifugation and the membrane pelleted at 200 000× g for 2 h at 20 °C. The membrane pellet was resuspended in20 mM sodium phosphate buffer (pH 7.6) containing 10 mMNaCl and 1% OPOE with bath sonication. The suspension wasthen centrifuged at 200 000 × g for 60 min at 20 °C. Asubsequent extraction used 1% OPOE and was followed by threeadditional extractions at 3% OPOE. OmpF was specificallyextracted at 3% OPOE. After OPOE concentration was reducedto 0.5% by dialysis, the preparation was applied to an anionexchange column (Mono Q HR5/50; Pharmacia Corp.) equili-brated with 10 mM sodium phosphate buffer (pH 7.6), 10 mMNaCl, 0.5% OPOE. Proteins was eluted with a salt gradientbetween 250 mM to 500 mM NaCl.

Circular Dichroism Spectroscopy. CD spectra were re-corded on a JASCO J-810 Spectropolarimeter (JASCO Inc.,Easton, MD) at ambient temperature. A quartz cuvette with apath length of 0.2 cm was employed. Purified proteins recon-

stituted into detergent micelles were measured. OmpF wasmeasured at a concentration of 0.2 mg/mL in phosphate bufferwith 0.5% OPOE, and Ste2p was ∼0.1 mg/mL with 0.05% DM,while the BR concentration was 0.1 mg/mL with 1% OG.Protein secondary structure content was evaluated using basisspectra from poly lysine17 and basis spectra determined fromproteins of known structure,18 which were obtained from theK2D19 webpages (http://www.embl-heidelberg.de/∼andrade/k2d.html).

Small-Angle X-ray Scattering. SAXS experiments wereperformed in the Center for Structural Molecular Biology 4mSAXS facility.20 In addition to collecting data for samples withprotein, data were collected for the associated detergent-containing buffer solutions. The detergent-containing buffersolutions were reduced with respect to a detergent-free bufferand found to have very low signal relative to the protein-containing samples, indicating that using the detergent-contain-ing buffer solutions as the background for the protein-detergentcomplex samples was reasonable. Protein concentrations were9.0 mg/mL for OmpF-OPOE and 20.0 mg/mL for BR-OG, whilethe Ste2p-DM was collected at 1.0 mg/mL. Additional data setswere collected at 4.5 mg/mL for OmpF-OPOE and 4.6 mg/mLfor BR-OG. No concentration-dependent effects were observed.Data were reduced and azimuthally averaged according topreviously published procedures20 to the 1D intensity profileI(q)vsq. Multipledatasetswerecollectedfor theprotein-detergentcomplexes to check for artifacts due to radiation damage andno artifacts were observed in the data.

Small-Angle X-ray Scattering Analysis and Modeling.When dealing with an isotropic solution of particles, the one-dimensional scattering intensity can be written as follows.

I(q))Np(∆FV)2|F(q)|2S(q) (1)

where q ) (4π sin θ)/λ, the momentum transfer, in which 2θis the scattering angle from the incident beam and λ is thewavelength of the incident radiation (1.542 Å). Np is the numberdensity of particles and the product ∆FV is the total excessscattering length of the particle relative to the solvent. Thefunction F(q), called the form factor, results from the particleshape, while the function S(q), called the structure factor, resultsfrom interparticle interactions. When the sample is an isotropic,dilute solution of noninteracting particles, S(q) ) 1 for all valuesof q. The time and ensemble average structure that exists withinthe sample volume is sampled during the measurement.

Data were fit either according to Guiner methods21 or by usinga power-law, as appropriate. Structural models were producedfrom the SAXS data using different approaches. When data wereconsistent with a dilute solution of identical particles, modelswere constructed using an existing high-resolution structureembedded in a detergent micelle with two regions of differentdensities representing the hydrophobic core and a shell torepresent the polar groups of the surfactants, as shown in Figure1, using software developed at Oak Ridge National Laboratory.The parameters for the modeling were the surfactant disk radiusR, the half-thickness T and the core half-thickness C. Disk radiiand the thicknesses of the core and shell were tested using aMonte Carlo search procedure to find a model having a SAXSprofile consistent with the data. The intensity profiles werecalculated from the models using the software ORNL_SAS.22

The 25 best models found by the search, as judged by the �2

parameter23 of the model intensity profile against the data, wereretained in a sorted list for use in assessing the reproducibilityand variability of the models.24 If such a model was inappropri-ate, triaxial ellipsoid and triaxial core-shell ellipsoid modelswere generated using the software ELLSTAT.24

13350 J. Phys. Chem. B, Vol. 112, No. 42, 2008 Mo et al.

The final modeling approach considered the possibility thatthe detergent had denatured the protein, resulting in a ”beads-on-a-string” structure displaying fractal scattering.10,11 In the“beads-on-a-string” model the polypeptide chain is in anunfolded, random state in solution with detergent micellesdistributed along the polypeptide chain. The intensity wasmodeled as described in eq 1. The F(q) of the micelles wasthat of a core-shell ellipsoid of revolution, which has ananalytical form.25 In the ”beads-on-a-string” model, S(q) ac-counts for interparticle correlations that exist between themicelles. Previous workers derived an analytical function forS(q), which is given in eq 2.10,11

S(Q)) 1+ 1

(Qr0)D

DΓ(D- 1)

[1+ 1 ⁄ (Q2�2)](D-1)/2×

sin[(D- 1)tan-1(Q�)] (2)

D is the fractal dimension of the packing of the micelles. If D) 3, then the particles are in a compact arrangement. For valuesof D less than 3, the micelles are in a more open structure. � isthe correlation length of the fractal system. The term r0 is theequivalent micelle radius. In the model, the interaction betweenthe micelles is assumed to have a finite range and thereforeprovides a measure of the extent of unfolding of the polypeptidechain. Γ(x) is the gamma function. The free parameters for themodel fitting were the ellipsoidal semiaxes and the D and �parameters of S(q). Again, the quality of the fit was evaluatedusing the reduced �2 parameter.23 The micelle sizes and polarshell thicknesses for BR and OG were allowed to range aroundthe values previously determined for free micelles of thesurfactants26

The electron density of the proteins for the modeling wasassigned to an average value for proteins (∼0.44 electrons/Å3),while the solvent was 0.34 electrons/Å3. The detergent electrondensities were divided into two regions corresponding to thehydrocarbon chains and the polar region corresponding to therest of the molecules. For DM, the electron density of the chainsis 0.276 electrons/Å3 while that of the dry polar region is 0.527electrons/Å3. In the case of OG, the electron density of thechains is 0.267 electrons/Å3 while that of the dry polar regionis 0.538 electrons/Å3. Wet densities for the polar regions of DMand OG were taken from a previously published study26 ofprotein-free detergent micelles, being 0.520 electrons/Å3 forDM. A value of 0.500 electrons/Å3, being within the range of

reported values,26 was used for OG. OPOE is actually a mixtureof different polar groups attached to the hydrocarbon chain withthe same electron density as the chain of OG. The electrondensity of the OPOE polar group was estimated at 0.37electrons/Å3.

Results

The SDS-PAGE analysis and Western blot indicated thatSte2p was purified to near homogeneity (Figure 2a). Themajority of the Ste2p exists in a monomeric state, although thegel shows a weak band near 100 kDa, suggestive of a smallpopulation of Ste2p oligomers. The higher molecular weightband, which is consistent with dimers of Ste2p, is estimated tobe ∼11% of the population. SDS-PAGE gel (Figure 2b) showsthat OmpF proteins in a detergent OPOE solution weredominated by a single band at 64 kDa. This band representsthe trimeric OmpF proteins15 with certain secondary structures.No monomeric or dimeric OmpF can be observed from the SDS-PAGE gel.

The CD scans of OmpF, BR and Ste2p are shown in Figure3. The results indicate that the trimeric OmpF proteins in 0.5%OPOE solution are rich in �-structure, similar to previouslypublished spectra,27,28 although the proteins appear to be lessfolded than the native trimeric OmpF based on the zero-crossingof the spectrum.27,29 The spectrum was fit far better by the setof basis spectra determined from proteins of known structure,yielding 19% R-helix, 74% �-structure and 7% random coil.The CD spectrum of the BR-OG complex is suggestive of a

Figure 1. Schematic of the structure used for modeling a properlyfolded membrane protein in a membrane-like detergent micelle. Theupper view shows the detergent disk (dark gray) of radius R penetratedby the structure of the membrane protein (gray) with a water channelthrough the structure (white). The lower figure shows a cut throughthe disk with the polar headgroup region of the detergent (dark gray)and nonpolar core of the bilayer (light gray), with thickness 2C. Themicelle thickness is 2T.

Figure 2. A. Purification of a yeast GPCR, Ste2p. (1) Ste2p waspurified and analyzed in SDS-PAGE. Purified sample was stained withCoomassie Blue after SDS-PAGE. A tear in the gel that occurred duringdrying is visible near 30 kDa. (2) Western blot analysis of the purifiedreceptor using 1D4 antibody is also shown. B. SDS-PAGE of purifiedOmpF in 4-20% gel. Lane 1 is the mass marker and lane 2 is purifi-ed OmpF after FPLC.

Figure 3. CD spectra of OmpF-OPOE (s), BR-OG (- -) and Ste2p-DM (---). The OmpF-OPOE spectra is consistent with a primarily�-sheet structure, while the BR-OG suggests that the structure ispredominantly R-helical. The Ste2p-DM spectra suggests that there isa mixture of secondary structures.

Structure of Membrane Protein-Detergent Complexes J. Phys. Chem. B, Vol. 112, No. 42, 2008 13351

predominantly R-helical structure, having two minima near 208and 222 nm. Fitting using the poly lysine basis spectra producedbetter results and show the protein secondary structure to consistof 84% R-helical structures and 16% �-structures. The CDspectrum of Ste2p in 0.5% DM solution indicates that thestructure is not strongly R-helical. Instead, the data, which wasbest fit by the basis spectra determined from known proteins,indicates that the structure is 41% R-helix, 39% �-structuresand 20% random coil, roughly consistent in helical contentaccording to a secondary structure prediction using PSIPRED30,31

(49% R-helix, 9% �-structures and 42% random coils).Small-angle X-ray scattering profiles for the three systems

studied are shown in Figure 4. The OmpF-OPOE profile has aclearly different character than the other two systems studied.The particle is compact with a radius of gyration of 50.2 ( 2.2Å. In contrast, the scattering profiles for the BR-OG and Ste2p-DM samples display power-law behavior, with slopes of -2.4and -2.5, respectively, over a q-range of 0.02 to 0.08 Å-1. Aslope of this magnitude is consistent with mass fractals, andthe q-range over which the behavior is displayed includes lengthscales consistent with the size of the native trimeric form ofBR.4,5 Neither of the helical protein data sets contain a regionsuitable for Guinier fitting (qRg < 1.3). If the data were theresult of aggregation of the properly folded monomeric or nativeoligomer of either protein encompassed by a detergent micellebelt, one would expect to see evidence of the scatteringcharacteristic of the detergent-associated trimeric form in thecase of BR, or the minimal oligomer in the case of the Ste2p,with higher order aggregates. The data would have a charactersimilar to that of the OmpF-OPOE shown in Figure 4. A smallpopulation of lower-order oligomers (i.e., dimers or trimers),such as is indicated by the SDS-PAGE gel (Figure 2), wouldhave a limited impact on the data, being most noticeable asslightly increased intensity over the minimal structure at thelowest q-values. The data at larger q-values of slightly ag-gregated samples would tend toward the intensity profileexpected for the minimal oligomer. The fractal character of thedata precludes reliable estimation of the amount of oligomersin the sample from the SAXS data.

The model fits to the measured scattering profiles are alsoshown in Figure 4. In the case of the OmpF-OPOE complex,the best-fit model intensity profile, which has a �2 of 0.798,

results from a relatively small detergent micelle surroundingthe intact trimer structure, having a radius R of 57.0 Å. Thebest fit model had T ) 26.6 Å and C ) 15.5 Å. If one looks atthe set of best-fit models with �2 values within 5% of the bestvalue, the structural parameters R, T, and C average, 58.0 Å,26.6 Å and 15.4 Å, with standard deviations of 0.6 Å, 0.6 Å,and 0.2 Å, respectively.

In the case of the BR-OG complex, both the beads-on-a-stringmodels and the core-shell ellipsoidal models produced reason-able fits to the data, as can be seen in Figure 4. The beads-on-a-string model fit the BR-OG data reasonably well, having a �2

of 2.08, although there are clear deviations present near 0.07Å-1, where the local minima in model profile is located. Forthis best-fit model, D was found to be 1.04, � was 177.5 Å andthe ellipsoidal semiaxes were (15.0 Å, 15.0 Å, 41.0 Å) with ashell thickness of 2.5 Å. Models having �2 within 5% of thevalue of the best-fit model have structural parameters that areconsistent with the best fit model. The core-shell ellipsoidmodel, in which the core and shell densities were assigned thatof the protein-free detergent micelles, fits the data even better,having a �2 of 0.89. The ellipsoid is large and flat, havingsemiaxes of (227.4 Å, 215.0 Å, 17.3 Å) and a shell thicknessof 5.9 Å. An attempt to fit the data using a solid, triaxial ellipsoid(not shown) did not produce acceptable fits to the data.

Both modeling approaches also provided acceptable fits tothe Ste2p-DM SAXS data (Figure 4). The beads-on-a-stringmodel fit the BR-OG data reasonably well, having a �2 of 1.02,with the strongest deviations near the minima in the modelprofile near 0.1 Å-1. For this best-fit model, D was found to be1.41, � was 92.5 Å and the ellipsoidal semiaxes were (19.9 Å,32.9 Å, 32.9 Å) with a shell thickness of 4.5 Å. Models having�2 within 5% of the value of the best-fit model have structuralparameters that are consistent with the best fit model. Again,the core-shell ellipsoid model having core and shell densitiesfrom the protein-free detergent micelles, fits the data very well,having a �2 of 0.48. The ellipsoid is large and flat, havingsemiaxes of (249.0 Å, 127.5 Å, 20.8 Å) and a shell thicknessof 5.9 Å. An attempt to fit the data using a solid, triaxial ellipsoid(not shown) did not fit the Ste2p-DM SAXS data as well as thecore-shell model or the beads-on-a-string model.

Discussion

The results presented here demonstrate that a detergent-solubilized membrane protein is not necessarily properly foldedwith a belt of detergent encompassing the membrane-spanningportions of the structure. The �-barrel membrane protein OmpF,which is unrelated to the other proteins studied, in complex withthe detergent OPOE forms a compact particle that is consistentwith a native trimer3 embedded in a membrane-like structure.The situation was considerably different for the two helicalmembrane proteins studied. While the CD spectroscopy suggeststhat the secondary structures are consistent with expectationsof helical content, the SAXS data sets are not consistent with anatively folded protein surrounded by a belt of detergent.Previous studies found that BR in OG detergent solution canadopt a different conformation than both native and vesicle-associated states of the protein based on UV-vis absorptionand CD spectroscopic analysis.32 Both an unfolded beads-on-a-string model10,11 and a large core-shell ellipsoid model werecapable of fitting the data well, but the core-shell modelsconsistently provided better fits to the data. Large, solidellipsoidal models were not capable of fitting the data as wellas either of these two approaches. Yet, the large core-shellmodel is not particularly physically realistic due to the need

Figure 4. Small-angle X-ray scattering profiles for the three membraneprotein-detergent complexes studied: BR-OG (0), Ste2p-DM (O) andOmpF-OPOE (4). The black curves shown with the data are the bestmodel fits for each of the complexes, being the beads-on-a-stringmodel10,11 for BR-OG and Ste2p-DM and the OmpF crystal structure3

embedded in a membrane-like detergent micelle for the OmpF-OPOEcomplex. The dashed curves are triaxial core-shell ellipsoidal modelbest fits generated using ELLSTAT.24

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for a strong density difference between the core and shell. Theprotein-detergent ratio for both helical membrane proteins wasroughly 1:50 (per monomer), meaning that such a large assemblyshould have a more uniform density than was required to obtainsuch good fits to the data due to the penetration of the helicesacross the ellipsoid along the narrow axis. An alternativepossibility is that the helices are adsorbed into the polar regionof the bilayer, lying parallel to the surface. Such a configurationwould maintain the large scattering length density differencebetween the core and shell. The nature of the transmembranestructure of natively folded BR makes such a configurationunlikely, as would be the case for Ste2p. The low detergentcontent of the complexes may also explain why the data lack astrong minima. A muted minima has been observed for bovineserum albumin complexes with sodium dodecyl sulfate at lowconcentrations33 and may be due to incomplete denaturing ofthe protein. The population of oligomers indicated in the Ste2psamples by the SDS-PAGE analyses (Figure 2a) would onlyflatten the minima significantly if the oligomers were properlyfolded. The data cannot exclude this possibility. Unfolded Ste2poligomers would not have a significant impact on the data inthis q-range, which is dominated by the micelle scattering. Astate in which the individual helices of each polypeptide chainin BR and Ste2p are solubilized by detergent is the most likelymodel for the system, but no such model for scattering datahas been developed. The results demonstrate that the solutionstate of a detergent-solubilized membrane protein cannot alwaysbe assumed to be properly folded and that relatively simplecharacterizations can be employed to verify the conformationof the protein-detergent complex before continuing on tofunctional studies or crystallization trials.

Other studies of membrane protein-detergent complexessuggest that both properly folded and denatured states ofmembrane proteins result from the interaction. An early SAXSwith contrast variation study of rhodopsin in complex withdodecyldimethylamine oxide found compact, monomeric rhodop-sin associated with detergent shielding the hydrophobic segmentsof the protein that suggested the detergent formed a belt aroundthe membrane spanning portions of the structure.13 Neutronscattering studies found there to be a significant distance betweenthe centers of mass of detergent and rhodopsin within thecomplexes studied, but this separation was attributed to dif-ferential detergent association resulting from the amphiphilicnature of the protein.34,35 A similarly compact and well-definedstructure was observed for the light-harvesting complex LH2from Rhodobacter spheroides 2.4.1.36 In contrast, another studyof the light-harvesting complex LH2 from R. acidophila suggeststhe possibility that the protein-detergent complex does not havethe expected conformation.37 While the small-angle scatteringdata from the LH2 of R. acidophila were analyzed assumingthat the complex was correctly folded, the data displays a clear,broad peak near 0.14 Å-1, which can result from smallellipsoidal structures. Similar results were observed for the Wzzprotein.38 While the data in both studies were interpretedassuming that the particle was properly folded, the presence ofthe wide angle peak, suggestive of a micelle structure, raisesthe possibility that the two different protein-detergent com-plexes studied were also at least partially unfolded.

A recent study of detergent-solubilized photosystem-1 mem-brane protein complex from spinach suggested that the entiremembrane protein complex may not be completely unfoldedby the detergent used.39 Instead, the results of the studysuggested that a properly folded core of protein was surroundedby a cloud of unfolded polypeptide associated with detergent

micelles. The photosystem-1 complex differs significantly fromany of the proteins studied in the current work, being a greatdeal larger and composed of multiple, nonidentical subunits.A considerable portion of the photosystem-1 complex is notexposed to the hydrophobic core of the membrane, makingit possible that association of those portions of the complexwith detergent is unfavorable relative to the association ofany available detergent with the membrane-exposed portionsof the structure. It is unlikely that such an arrangement existsfor the much smaller helical proteins studied here becausemuch more of the structure is normally exposed to thenonpolar core of the membrane.

Conclusions

This work demonstrates that detergent-associated membraneproteins need not fold properly with their hydrophobic segmentsencapsulated in a belt of detergent when in solution. Instead, adetergent, even one expected to be suitably gentle to retainprotein functionality, can produce a non-native conformation.Small-angle scattering can play an important role in thepreliminary characterization of protein-detergent complexes,particularly those that will be used as foundation material forcrystallization work. The results from Ste2p further suggest thatone of the causes of difficulties in obtaining crystals for high-resolution structure determination from detergent-solubilizedmembrane proteins may in fact be that the protein is toodenatured to refold when in complex with detergent. The recenthigh-resolution structures of the engineered40,41 and antibody-bound42 �2-adrenergic GPCR demonstrate the importance oflipidic systems, rather than traditional detergent preparations,for crystallization of GPCRs that should also be applicable tocrystallization of other membrane proteins because they bettermimic the native membrane environment.

Acknowledgment. The authors would like to thanks Dr. AnneH. Delcour of the University of Houston for the generous giftof the OmpF clone. This work was supported by the LaboratoryDirected Research and Development Program of Oak RidgeNational Laboratory, managed and operated by UT-Battelle,LLC, for the U.S. Department of Energy under Contract No.DE-AC05-00OR22725, the Joint Directed Laboratory Develop-ment Program from the University of Tennessee, and NIH GM-022087 from the National Institute of General Medical Sciences.

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