The material elastic properties of biological membranes such

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1 Elastic cupling f integral membrane prtein stability t lipid bilayer frces Heedek Hng and Lukas K. Tamm* Department f Mlecular Physilgy and Bilgical Physics, and Biphysics Prgram, University f Virginia, Charlttesville, VA Cmmunicated by Duglas C. Rees, Califrnia Institute f Technlgy, Pasadena, CA, January 15, 2004 (received fr review Nvember 3, 2003) It has been traditinally difficult t measure the thermdynamic stability f membrane prteins because fully reversible prtcls fr cmplete flding these prteins were nt available. Knwledge f the thermdynamic stability f membrane prteins is desirable nt nly frm a fundamental theretical standpint, but is als f enrmus practical interest fr the ratinal design f membrane prteins and fr ptimizing cnditins fr their structure determinatin by crystallgraphy r NMR. Here, we describe the design f a fully reversible system t study equilibrium flding f the uter membrane prtein A frm Escherichia cli in lipid bilayers. Flding is shwn t be tw-state under apprpriate cnditins permitting data analysis with a classical flding mdel develped fr sluble prteins. The resulting free energy and m value, i.e., a measure f cperativity, f unflding are G u,h2 O 3.4 kcal ml and m 1.1 kcal ml M 1, respectively, in a reference bilayer cmpsed f palmityl-leyl-phsphatidylchline (C 16:0 C 18:1 PC) and palmitylleyl-phsphatidylglycerl (C 16:0 C 18:1 PG). These values are strng functins f the lipid bilayer envirnment. By systematic variatin f lipid headgrup and chain cmpsitin, we shw that elastic bilayer frces such as curvature stress and hydrphbic mismatch mdulate the free energy and cperativity f flding f this and perhaps many ther membrane prteins. The material elastic prperties f bilgical membranes such as curvature stress and bilayer defrmatin frm hydrphbic mismatch have emerged as imprtant functinal mdulatrs f in channels, receptrs, and ther integral membrane prteins (1 5). These prperties are als expected t mdulate the thermdynamic stability f membrane prteins because the membrane envirnment impses very different mechanical cnstraints (6, 7) n membrane prteins than water des n sluble prteins (8). A thrugh understanding f the factrs that determine the stability f membrane prteins is f fundamental theretical interest t understand the frces that shape these prteins (9). Accurate assessments f their thermdynamic stability will als aid in the design f mre stable membrane prteins fr therapeutic applicatins and fr structural studies f this structurally underrepresented class f prteins. Fr example, vercming cnfrmatinal dynamics has been a majr breakthrugh in the recent determinatin f the structure f lactse permease (10) and superstable mutants f diacylglycerlkinase have dramatically imprved crystallizatin and NMR cnditins (11, 12). Unfrtunately, quantitative studies f membrane prtein stability have been hampered by difficulties t cmpletely and reversibly unfld these prteins (13, 14). We have nw designed a fully reversible system t study equilibrium flding f membrane prteins in lipid bilayers, by using the uter membrane prtein A (OmpA) frm Escherichia cli as a mdel. OmpA is an abundant prtein f the uter membranes f Gram-negative bacteria, where it serves a structural rle and als functins as a phage and clicin receptr. The eight-stranded -barrel structure f the N-terminal transmembrane dmain (residues 1 177) has been slved by x-ray crystallgraphy (15) and NMR (16). Denatured OmpA in slutin spntaneusly reflds int lipid bilayer membranes after dilutin f denaturants (17), and the kinetics f reflding f OmpA have been shwn t depend n the cmpsitin, thickness, and verall curvature f the lipid bilayer (18). We demnstrate here that OmpA flding int lipid bilayers is a reversible tw-state prcess that can be quantitatively described by the free energy f unflding and the cperativity parameter m, which bth depend n the mechanical prperties f the hst lipid bilayer. We als discuss hw ur apprach using a -barrel mdel prtein may be generalized t analyze the thermdynamic stability f -helical membrane prteins. Methds Prtein Expressin and Purificatin. OmpA was islated and purified in the fully unflded frm in 8 M urea frm the uter membranes f E. cli as described (17). Small Unilamellar Vesicles (SUVs). Lipid stcks (Avanti Plar Lipids) disslved in chlrfrm were mixed t yield the desired cmpsitins. Ttal lipid (12 ml) was dried n the bttm f a glass test tube under a stream f nitrgen and further in a desiccatr under a high vacuum vernight. The lipid films were then dispersed in 10 mm glycine buffer (ph 10.0) cntaining 2 mm EDTA t a final lipid cncentratin f 10 mm. The lipid dispersins were snicated fr 50 min by using a Bransn ultrasnifier micrtip at 50% duty cycle. SUVs were equilibrated ver night befre use. The average diameter f the SUVs determined by dynamic light scattering (Prtein Slutins DynaPr) was 30 nm. Equilibrium Unflding and Reflding f OmpA. Cncentrated OmpA in urea was diluted 100-fld int SUVs and incubated fr 3 h at 37.5 C fr reflding. The final OmpA cncentratin and lipid-t-prtein mlar ratis were 12 M and 800, respectively, in all experiments. The reflded prtein lipid cmplex was divided int aliquts. Apprpriate amunts f a freshly made 10 M urea stck slutin and a buffer slutin were added t the aliquts, which were further diluted 10-fld fr flurescence r 2.5-fld fr SDS PAGE equilibrium unflding experiments. These reactins were incubated vernight at 37.5 C. Fr equilibrium reflding measurements by SDS PAGE, aliquts f 4 mm SUVs were preequilibrated at apprpriate urea cncentratins and small amunts f OmpA were added t each sample t yield the same cncentratins as in the unflding experiments. The reflding reactins were incubated vernight at 37.5 C. SDS PAGE. The equilibrated samples were laded n SDS 12.5% PAGE withut biling. The reactins were terminated by 1:1 mixing f the samples with the treatment buffer at rm temperature (17). Flurescence and CD Spectrscpy. Flurescence spectra were cllected in SPEX Flurmax r Flurlg spectrflurmeters. Abbreviatins: OmpA, uter membrane prtein A; LUV, large unilamellar vesicles; NBD, [7-nitr-2 1,3-benzxadiazl-4-yl]; PC, phsphatidylchline; PE, phsphatidylethanlamine; PG, phsphatidylglycerl; SUV, small unilamellar vesicles. See Cmmentary n page *T whm crrespndence shuld be addressed. lkt2e@virginia.edu by The Natinal Academy f Sciences f the USA BIOPHYSICS SEE COMMENTARY cgi di pnas PNAS March 23, 2004 vl. 101 n

2 The excitatin wavelength was 290 nm and flurescence was measured in the nm wavelength range at a scan rate f 0.15 nm s using 6-nm slits. Far-UV CD spectrscpy was perfrmed n an Aviv 215 spectrplarimeter. The prtein and lipid cncentratins were the same as in the SDS PAGE experiments. All spectra were crrected fr light scattering with reference samples f identical cmpsitin and cncentratin, but withut prtein. Airfuge Fltatin Assay. Discntinuus sucrse gradients with 30% (30 l), 25% (40 l), 20% (40 l), 10% (40 l) in ph 10.0 glycine buffer cntaining 6 M urea were carefully prepared in Airfuge tubes frm bttm t tp. Three different 50- l samples in 6 M urea and 30% sucrse were placed at the bttm: 2.4 mm SUVs with 0.1 ml% [7-nitr-2 1,3-benzxadiazl-4-yl] (NBD)- dic 18:1 PE, 3 M OmpA, and 3 M OmpA that had been reflded in 2.4 mm SUVs. The tubes were spun in a Beckman Airfuge at 25 psi fr 50 min. Thirty-tw-micrliter fractins were taken frm each tube and diluted with 93 l f 8 M urea. Each fractin was analyzed fr NBD flurescence by the excitatin at 460 nm and emissin at 532 nm and fr OmpA flurescence at 290 nm and 352 nm, respectively. Fitting f Unflding Curves. Flurescence spectra were parameterized by calculating an average emissin wavelength, defined as (F i i ) (F i ) (19). i and F i are the wavelength and the crrespnding flurescence intensity at the ith measuring step in the spectrum. The unflding curves, vs. [urea], were fitted t the fllwing frm f the tw-state mdel 1 F U exp m denaturant C Q m RT R 1 1 exp m denaturant C Q m RT R [1] using Wavemetrics IGORPRO sftware. F and U are the average emissin wavelengths f the flded and unflded states, respectively, determined frm linear extraplatins t 0 M urea f the plateau values f the tw states. C m is the urea cncentratin where the fractins f flded and unflded states are equal. Q R is the relative rati f the ttal flurescence intensity f the native state t that f the unflded state and is needed fr nrmalizatin when ne uses s t represent species cncentratins (19). The free energy f unflding is btained frm the fitted values f C m and m: G u,h2 O mc m [2] Results and Discussin Previus kinetic studies shwed that urea-denatured OmpA can be reflded in bilayers f small and large unilamellar vesicles cmpsed f a large variety f lipids and that flding int SUVs is faster than int large unilamellar vesicles (LUVs) (18). Fr thick bilayers, the insertin kinetics becme prhibitively slw in LUVs, but are still experimentally accessible in SUVs. Therefre, we are using here equilibrated SUVs t assess equilibrium flding and t study effects f bilayer mechanics n the thermdynamic stability f OmpA. T test the reversibility f flding, OmpA was unflded and reflded in SUVs cmpsed f 92.5% palmityl-leyl-phsphatidylchline (C 16:0 C 18:1 PC) and 7.5% palmityl-leyl-phsphatidylglycerl (C 16:0 C 18:1 PG) by variatin f the urea cncentratin in ph 10 buffer at 37.5 C. T estimate the fractin f flded OmpA, we used the fact that withut sample biling, native OmpA migrates in SDS gels as a 30-kDa frm, but as a 35-kDa frm when fully r partially denatured (20). Fig. 1A shws that unflding and reflding reactins prduce the same gel migratin patterns and, tgether Fig. 1. Reversibility and tw-state behavir f OmpA flding in lipid bilayer membranes cmpsed f 92.5% C 16:0 C 18:1 PC and 7.5% C 16:0 C 18:1 PG at ph 10 and 37.5 C. (A) Cmparisn f equilibrium unflding and reflding f OmpA mnitred by SDS PAGE withut sample biling. The 30-kDa frm represents the native state, and the 35-kDa frm represents denatured states. Transitin midpints are indicated with arrws. (B) Cmparisn f the urea-induced equilibrium unflding f OmpA in lipid bilayers mnitred by SDS PAGE (pen circles) and Trp flurescence (filled circles). (C) Same as B, but with increasing ml fractins f the shrt-chain lipid dic 12 PC. Fr clarity, successive curves are each shifted n the abscissa by 1 M urea. with many similar experiments under different cnditins, indicates that the reactin is at equilibrium. The midpint f the transitin ccurs at 3.2 M urea. The denatured state in the presence f lipid vesicles is indistinguishable frm the denatured state in 6 8 M urea as shwn by Trp flurescence (Fig. 2A) and CD spectrscpy (Fig. 2B). A ttal f 7.5% f negatively charged C 16:0 C 18:1 PG was included t facilitate cmplete dissciatin f denatured OmpA frm the membranes (Fig. 2C). OmpA has a calculated pi f 5.6, and therefre is negatively charged at high ph. Denaturatin and dissciatin was incmplete in pure C 16:0 C 18:1 PC bilayers (data nt shwn). Full thermdynamic reversibility was bserved in the ph range , but dissciatin f the denatured state frm the membrane was favred at higher ph values (data nt shwn). The bilayer structure itself is maintained in high cncentratins f urea as has been demnstrated by x-ray diffractin and [ 31 P]NMR (21, 22). Lipid bilayers are quite permeable t urea (23). Therefre, we d nt expect the bilayers t be subject t smtic pressure r tensin under equilibrium cndititins. Previus kinetic studies indicate that OmpA reflds via several kinetic intermediates in essentially 0 M urea (17, 24). Hwever, this des nt imply anything abut whether urea-induced equilibrium unflding is a tw-state r multistate prcess, which is imprtant t knw fr further analysis f the equilibrium data. T assess whether equilibrium unflding f OmpA is tw- r cgi di pnas Hng and Tamm

3 Fig. 2. Characterizatin f denatured states f OmpA. (A) Flurescence spectra f native membrane (C 16:0 C 18:1 PC PG, 92:5:7.5)-inserted OmpA (slid line), denatured OmpA btained by treating native membrane-inserted OmpA with 8 M urea (dashed line), and denatured OmpA in the absence f lipids (dtted line). The emissin maxima f the native and denatured states were nm and nm, respectively. (B) Far-UV CD spectra f native membrane-inserted OmpA (slid line), denatured OmpA btained by treating native membrane-inserted OmpA with 6 M urea (dashed line), and denatured OmpA in the absence f lipids (dtted line). (C) Airfuge fltatin assay t measure degree f membrane-assciatin f denatured OmpA in 6 M urea. NBD-labeled vesicles in the absence (pen circles) r presence (filled circles) f denatured OmpA flat t the tp, and denatured OmpA in the absence (pen squares) r presence (filled squares) f vesicles remains at the bttm, indicating separatin f denatured OmpA frm the vesicles. multistate, unflding was analyzed at the same lipid-t-prtein rati (800:1) by Trp flurescence and SDS PAGE, i.e., tw parameters that reprt n a very early and a very late kinetic phase f the flding reactin, respectively (17, 24). As shwn in Fig. 1B, the unflded fractins btained frm SDS PAGE and flurescence cincide as expected fr a tw-state, but nt a multistate, prcess. Tw-state unflding by slvent denaturatin beys the law D D N exp m H D N denaturant G 2O D N RT H 1 exp m D N denaturant G 2O D N RT [3] where [D] and [N] are the cncentratins f the denatured and native states, respectively, G D N,H2 O G u,h2 O is the free energy f unflding in water (0 M urea), and m D N d G D N d[denaturant], which is cmmnly believed t be independent f the denaturant cncentratin. m D N (hencefrth abbreviated m) has been linked t the amunt f prtein area that becmes slvent-expsed upn unflding and is als a Fig. 3. Representative unflding curves measured by Trp flurescence with increasing ml fractins f a shrt-chain saturated PC (A), a lng-chain mnunsaturated PC (B), a lng-chain mn-unsaturated PE (C), and a dubleunsaturated PC (D). The C 16:0 C 18:1 PG fractin was kept fixed at 7.5% in A C and at 12.5% in D. The remainder f the bilayer was filled with C 16:0 C 18:1 PC. All experiments were carried ut at 37.5 C. measure f the cperativity f unflding (25, 26). When the data are analyzed with this mdel (see Methds), we btain best fits fr G u,h2 O f 3.4 kcal ml and an m value f 1.1 kcal ml M 1. Based n the reversible flding system develped, the effects f lipid cmpsitin and bilayer thickness n OmpA stability were investigated by increasing the fractin f the lipids having varius mlecular structures as guest lipids at a fixed fractin (7.5%) f C 16:0 C 18:1 PG. The remaining fractin was filled with the reference lipid C 16:0 C 18:1 PC. First, we examined the effect f shrt saturated acyl chain PCs with chain lengths ranging frm 10 t 14 carbns n the stability f OmpA. Representative transitin curves fr the example f dic 12 PC measured by SDS PAGE and flurescence are displayed in Figs. 1C and 3A. Tw-state behavir and reversibility was maintained in all cases. The resulting values f G u,h2 O and m decreased prprtinally t the amunt f the guest lipid fractin in the bilayer (Fig. 4 A and B). This effect was mst prnunced fr the shrtest acyl chain lipid dic 10 PC and least prnunced fr dic 14 PC. BIOPHYSICS SEE COMMENTARY Hng and Tamm PNAS March 23, 2004 vl. 101 n

4 Fig. 4. Dependence f G u,h2o and m-value f OmpA unflding n lipid cmpsitin. Effect f saturated and mn-unsaturated PCs and PEs n G u,h2o (A) and m (B). Effect f cis-duble-unsaturated PCs n G u,h2o (C) and m (D). All experiments were carried ut at 37.5 C. When a lipid with lnger acyl chains (C 18:0 C 18:1 PC) was tested (Fig. 3B), the trend was reversed (Fig. 4 A and B). An even mre dramatic increase f G u,h2 O and m was fund fr anther lipid (C 16:0 C 18:1 PE) with the same chain cmpsitin, but with a smaller plar headgrup than the reference lipid (Fig. 3C). Unsaturated lipids with PE headgrups are cne-shaped and therefre assume inverted hexagnal (H II ) phases at higher temperatures (6). The investigated lipid mixtures cntaining C 16:0 C 18:1 PE still frm bilayers at 37 C, but with an increased internal lateral pressure caused by intrinsic curvature stress (6, 27). These lipids als have larger area expansin and bending mduli than shrter saturated PCs (28). Including 30% C 16:0 C 18:1 PE in reference bilayers, increased G u,h2 O and m by up t 60% (Fig. 4 A and B). T further test the effect f lipid shape and internal membrane pressure n the thermdynamic stability f OmpA, a series f cis-duble-unsaturated lipids (dic 14:1 PC t dic 20:1 PC), which induce smaller elastic mduli and larger curvature stresses in lipid bilayers (29), were examined (Fig. 3D). G u,h2 O and m increased in the case f all these lipids, but cntrary t the saturated lipid series, the lipids with the shrtest acyl chains (dic 14:1 PC) induced the largest increases in G u,h2 O (Fig. 4 C and D). The values f G u,h2 O and m f all tested guest lipids exhibited a linear dependence n their ml fractin in the membrane. Therefre, each data set was linearly extraplated t the pint where the ml fractin f the guest lipid reached 100%. The extraplated values fr 100% f each examined lipid were pltted as a functin f hydrphbic thickness (30) f the lipid bilayer (Fig. 5). Straight lines were btained fr the saturated and duble-unsaturated PC series. The tw lipids with a single cis duble bnd extended the range f the saturated series. The majr differences between the tw series are that the saturated lipids yielded a psitive (337 cal ml Å) and the dubleunsaturated lipids yielded a negative ( 166 cal ml Å) slpe and larger values f G u,h2 O. The free energies f transfer f unflded OmpA int membranes cupled with flding are rather small (0 t 8 kcal ml depending n lipid species). This is cnsistent with theretical free energy estimates fr -helical (31) and -barrel (32) membrane prteins frm whle-residue hydrpathy values taken frm the thermdynamically derived Wimley White (WW) hydrpathy scale (31). A simple calculatin f the free energies f Fig. 5. Dependence f G u,h2o (A) and m-value (B) n the hydrphbic thickness f PC bilayers with saturated and mn-unsaturated acyl chains (filled circles) and cis-duble-unsaturated acyl chains (pen circles) cgi di pnas Hng and Tamm

5 Fig. 6. Cartn depicting structures and bilayer frces acting n OmpA flding unflding under equilibrium cnditins. When flding int mst bilayers (left path), the prcess is tw-state. The large black arrws indicate lateral bilayer pressure imparted n the lipid prtein interface in the hydrphbic cre (red) f bilayers cmpsed f lipids with negative intrinsic spntaneus curvature. Increasing this pressure increases the thermdynamic stability f the prtein. The small black arrws indicate lipid defrmatin frces caused by hydrphbic mismatch between the prtein and unstressed bilayers. These frces decrease the thermdynamic stability f the prtein. When flding int thin bilayers (right path), the prcess is multistate, i.e., at least ne equilibrium intermediate ccurs. Water mlecules penetrate mre easily int the hydrphbic cre (blue arrws) f mre flexible and mre dynamic thin bilayers, stabilizing equilibrium intermediates, decreasing the m- and G u,h2o values until, in very thin bilayers cmpsed f saturated lipids, cmplete unflding (secnd step) can n lnger be bserved under any f ur experimental cnditins. transfer f the apprximately 8 10 residues f OmpA that span the membrane using the augmented WW scale yields a value very clse t zer ( 0.95 kcal ml) if ne takes int accunt that tw Arg Glu pairs frm salt bridges in the interir f the barrel (15). Because the scale nly cnsiders transfer f residues frm water int the hydrphbic envirnment, we cnclude that 1 t 9 kcal ml f energy likely cmes frm flding. This is 13 t 110 cal ml per residue, if 80 residues fld int the -barrel. These estimates bviusly are extremely crude because they are based n the sums f many differences f large numbers, which may cmpund small errrs int large nes, and because cntributins frm tertiary structure and fine-tuned lipid interactins, such as thse discussed belw, are neglected. In view f many such pssible cmplicatins that are almst impssible t predict, the relatively gd agreement between experiment and predictin is remarkable. T estimate the cntributin f the hydrphbic effect t the increase f membrane prtein stability with increasing membrane thickness, we calculated the hydrphbic surface area f OmpA frm its crystal structure by rlling a prbe f 1.4 Å radius ver the hydrphbic surface (33). This surface, which shuld rughly represent the area f the prtein lipid interface, is 2158 Å 2. Because ur best estimate f the hydrphbic thickness f the OmpA transmembrane dmain is 26 Å, the average hydrphbic perimeter is 83 Å. If fully develped, the hydrphbic effect is expected t cntribute 25 cal ml fr each Å 2 f hydrphbic cntact (34). Therefre, 2,075 cal ml shuld be gained fr each Å f increased membrane thickness. This value is abut six times larger than the experimentally fund increment. Even if ne uses smaller values f cal ml Å 2 as cmmnly assumed in prtein flding (33), it is clear that the hydrphbic effect grssly verestimates the experimental value. There must be ppsing frces, such as mechanical energy stred in the membrane due t hydrphbic mismatch at the prtein lipid interface (35). Lipids need t be stretched if t shrt and cmpressed if t lng cmpared t the hydrphbic 26 Å f the prtein. Bth effects cst energy (up t 1.7 kcal ml Å accrding t ur measurements). Because there is n discntinuity at 26 Å, the energy f lipid defrmatin seems t be symmetric, i.e., the energy csts f lipid cmpressin and stretching are similar. The G u,h2 O vs. d hydrphbic lines f the saturated and unsaturated lipids meet at 30 Å, i.e., at lipids with 20 carbn acyl chains. The increase f G u,h2 O f the shrter unsaturated lipids is mst likely the result f an increased lateral pressure exerted n the prtein in the interir f the bilayer (6, 27). Bilayers f shrt-chain PCs with tw cis duble bnds exhibit increasingly larger negative spntaneus curvatures as their chains are shrtened (36). Intrinsic curvature stress is stred in these bilayers. This stress is relieved when OmpA, which because f girdles f armatic residues near bth interfaces is hurglassshaped, is incrprated int such bilayers. The stred curvature stress energy thus stabilizes the flded structure f OmpA. The largest stabilizing effect is bserved with C 16:0 C 18:1 PE, which als exhibits the largest negative spntaneus curvature. This study als reveals an interesting aspect f the m value, which is highly crrelated with the degree f hydrphbic mismatch, the acyl chain cis-unsaturatin, and G u,h2 O. Theries f the m value fr water-sluble prteins agree that the parameter is prprtinal t the expsed hydrphbic surface area upn unflding, A, and the change in surface tensin, d d[denaturant] (25, 26) G slvatin A A D A N [4] d m A d denaturant A d D A N d denaturant, [5] BIOPHYSICS SEE COMMENTARY Hng and Tamm PNAS March 23, 2004 vl. 101 n

6 where is the surface tensin f the hydrphbic surface f a prtein in water. Assuming that A is little affected by the chice f lipids, we cnclude that the bserved m value changes are the result f lateral bilayer pressure changes, which in turn will change the f membrane prteins. Fig. 5B shws that m increases as the hydrphbic thickness and chain unsaturatin f the bilayer increase. The frmer increases the lcal stress arund the prtein by lipid defrmatin (7, 35), and the latter increases intrinsic curvature stress and lateral pressure in the bilayer (6). This interpretatin is cnsistent with and supprts the interpretatin f the previusly discussed changes f G u,h2 O.Asthe saturated PC bilayers becme very thin, the m values vanish and OmpA unflds withut dissciatin frm the surface f the bilayer (data nt shwn). In these cases, the tw-state cnditin may n lnger be valid and full sigmidal curves are n lnger bserved (data nt shwn). The abslute values f G u,h2 O and m measured in SUVs may be shifted relative t thse in LUVs because f additinal curvature strain impsed by the smaller radius f SUVs cmpared t LUVs. Hwever, the lipid-specific changes f these thermdynamic parameters shuld nt be affected because they are measured relative t the reference C 16:0 C 18:1 PC PG bilayer in SUVs and because the sizes f ur vesicles measured by light scattering are independent f lipid cmpsitin (data nt shwn). Mrever, the effect f the verall macrscpic membrane curvature n even the abslute values f G u,h2 O is prbably small because the free energies f binding f hydrphbic 20- t 30-residue peptides t lipid bilayers are nt much affected by macrscpic membrane curvature, even if enthalpies and entrpies depend n curvature in a cmpensatry fashin (37). Fig. 6 schematically depicts hw elastic bilayer prperties mdulate flding and unflding f OmpA. When OmpA is under the elastic stress f bilayer frces, it unflds cmpletely and is fully excluded frm the bilayer. In additin t the hydrphbic effect, intrinsic lipid shape-induced bilayer curvature stress and lipid defrmatin due t hydrphbic mismatch cntribute significantly t G u,h2 O and m. Hwever, OmpA incrprated in very thin lipid bilayers unflds incmpletely with smaller values f G u,h2 O and m. It is likely that the prtein assumes multiple fluctuating and partially membrane-assciated cnfrmatins under these cnditins. These insights int the stability f membrane prteins are likely general and als hld true fr ther membrane prteins, including helical nes, which because f their limited slubility in urea and guanidine hydrchlride are mre difficult t study by these methds. Hwever, ther cmbinatins f denaturants that may include strng detergents such as SDS might be suitable fr similar studies n helical membrane prteins (27, 38). Our studies using the OmpA urea mdel system suggest that nnspecific physical bilayer interactins and lipid packing are mre imprtant factrs determining the stability f membrane prteins than specific chemical lipid interactins. We thank S. Gruner and S. H. White fr helpful cmments n an early versin f the manuscript. We als thank Mr. D. Rinehart fr his expert technical assistance and V. Kiessling fr prviding sftware fr data analysis. This wrk was supprted by Natinal Institutes f Health Grant GM Martinac, B., Adler, J. & Kung, C. (1990) Nature 348, Perz, E., Crtes, D. M., Smprnpisut, P., Klda, A. & Martinac, B. (2002) Nature 418, Keller, S. L., Berzrukv, S. M., Gruner, S. M., Tate, M. W., Vdyany, I. & Parsegian, V. A. (1993) Biphys. J. 65, Brwn, M. F. (1994) Chem. Phys. Lipids 73, Navarr, J., Tivi-Kinnucan, M. & Racker, E. (1984) Bichemistry 23, Gruner, S. M. (1987) Prc. Natl. Acad. Sci. USA 82, Cantr, S. C. (1999) Biphys. J. 76, Dill, K. A. (1990) Bichemistry 29, White, S. H., Ladkhin, A. S., Jayasinghe, S. & Hristva, K. (2001) J. Bil. Chem. 276, Abramsn, J., Smirnva, I., Kash, V., Verner, G., Kaback, H. R. & Iwata, S. (2003) Science 301, Zhu, Y. & Bwie, J. U. (2000) J. Bil. Chem. 275, Oxenid, K., Sönnichsen, F. D. & Sanders C. R. (2002) Bichemistry 41, Stwell, H. B. & Rees, D. C. (1995) Adv. Prtein Chem. 46, Haltia, T. & Freire, E. (1995) Bichim. Biphys. Acta 1228, Pautsch, A. & Schulz, G. E. (1998) Nat. Struct. Bil. 5, Arra, A., Ablidgaard, F., Bushweller, J. H. & Tamm, L. K. (2001) Nat. Struct. Bil. 8, Kleinschmidt, J. H. & Tamm, L. K. (1996) Bichemistry 35, Kleinschmidt, J. H. & Tamm, L. K. (2002) J. Ml. Bil. 324, Mann, C. J., Ryer, C. A. & Matthews, C. R. (1993) Prtein Sci. 2, Arra, A., Rinehart, D., Szab, G. & Tamm, L. K. (2000) J. Bil. Chem. 275, Feng, Y., Yu, Z.-W. & Quinn, P. (2002) Chem. Phys. Lipids 114, Kleinschmidt, J. H. & Tamm, L. K. (2002) Biphys. J. 83, Flinkelstein, A. (1976) J. Gen. Physil. 68, Kleinschmidt, J. H., den Blaauwen, T., Driessen, A. J. M. & Tamm, L. K. (1999) Bichemistry 38, Shrtle, D. (1995) Adv. Prtein Chem. 46, Myers, J. K., Pace, C. N. & Schltz, J. M. (1995) Prtein Sci. 4, Curran, A. R., Templer, R. H. & Bth, P. J. (1999) Bichemistry 38, Marsh, D. (1996) Bichim. Biphys. Acta 1286, Szule, J. A., Fuller, N. & Rand, R. P. (2002) Biphys. J. 83, Lewis, B. & Engelman, D. M. (1983) J. Ml. Bil. 166, Jayasinghe, S., Hristva, K. & White, S. H. (2001) J. Ml. Bil. 312, Wimley, W. C. (2002) Prtein Sci. 11, Richards, F. M. (1985) Methds Enzyml. 115, Tanfrd, C. (1980) The Hydrphbic Effect (Wiley, New Yrk). 35. Nielsen, C., Gulian, M. & Andersen, O. S. (1998) Biphys. J. 74, Tate, M. W. & Gruner, S. M. (1987) Bichemistry 26, Seelig, J. (2002) Curr. Tpics Membr. 52, Lau, F. W. & Bwie, J. U. (1997) Bichemistry 36, cgi di pnas Hng and Tamm