membrane proteins I detergents / amphipols / electron microscop) ~ / cytochrome b~f
Introduction
In the present article, we report on a comparative study of cytochrome b0/'/amphipol complexes using scanning transmission electron microscopy II] at liquid helium teln- perature (cryo-STEM) and biochemical ineasurelucnts. Cytochrome bqf (plastoquinol:plastocyanin oxidoreduc- lase), one of the major complexes of oxygenic photosyno thesis, catalyses the transfer of electrons between photosystems I1 and I and tratlsduees part of the fi'ee en° thalpy drop into a transmembrane proton gradient 121. The complex l'rom Ctthtmydotttottas teinhaldtii is isolated under its native form as an enzymatically active 14-met comprised
*Correspondence and reprints. **Present address: Max-Planck-lnstitut fiir Biophysik, Heinrich- Hoffmann-Stral]e 7, 60528 Frankfurt-am-Main, Ger,nany. ***Present address: CEOS GmbH, hn Neuenheimer Feld 519. 69120 Heidelberg, Ger,nany. Abbreviations: AmAc, ammonium acetate: AP, alnlnoniuni phos- phate; cryo-STEM, scanning transmission electron microscopy at liquid helium temperature; cmc. critical miccllar concentration; DPPC, dipalmitoylphosphatidylcholine; EM, electron microscopy; HG, 6-O-(N-heptylcarbamoyl)-methy 1.0t-D-glycopyranoside (He- cameg); LM, dodecyl-ll-D- maltoside (lauryhnaltoside); M~, mole- cular mass; PC, phosphatidylcholine; SDS-PAGE, polyacrylamide gel eleetrophoresis in the presence of sodium dodecylsulfate; STEM, scanning transmissi~l electron microscopy; TMV. tobacco mosaic virus; Tricine, N-tris (hydroxymethyl)methylglycine.
of two identical heptamers 13.4]. Each heptamer comprises a copy each of four high molecular mas.~ O.4,) subunits (cytochronws l and b.. the 'Rieske' h'onosulfur protein, and subunit IV) and at least three small (3-4 kDa)hydrophobiz peptides (PetG. PetL and PetM)+ ~lb these protein~ are bound five prosthetic gloups per heptan]er: three heroes, one [2l:eo 2SI cluMcr, ~llld ~'qle chloropilyll. [3.51. The aggregate M, of the protein aad c,,ff,~;tor.~ ~;a!culatcd lot lhi~ 'hca~y' !4 ~ meric !brm i~ 2i l kDa 141. Following transfer from Hecao meg (HG)/egg phosphatidylcholine (PC) mixed micel!es to dilute laurylma!toside ([.M) solution, the heavy tbrm i~ found to retain ~36 molecules of lipids per 14omer 141. The stability of the 14°mer critically depends on the pre~ence of lipids. Upon delipidation, it loses the two copies of the Rieske protein and breaks down into a 'light'. chlorophyll° less form 14]. Depending on exact conditions, the light form retains one copy of each remaining subunit or lose.~ subunit PetL, resulting in an aggregate M, of 83~86 kDa. The preo sence of the Rieske protein, which is easily assessed by polyacrylamide gel electrophoresis in the presence of so~ dium dodecylsulfate (SDS-PAGE), is a strfligent text of the integrity of the complex.
We have previously described the complexation of cyto~ chrome bt~f by a new class of surfactants called '.'tmphipols" 16, 71. Amphipois are low M, amphipathic polymer~ that bind non-covalently but quasioirreversibly to the hydmphoo bic surface of integral membrane proteins. Following COmo plexation of the proteins with amphipols in detergent
476+
solution, amphipoi/protein complexes can be separated from detergent and free amphipols. The complexes migrate upon rate zonal centrifugation in surfactant-free sucrose gradients as water-soluble , apparent ly monodisperse species 161. The surfactant/protein mass ratio in the corn-
is typi. lassical
A s ~ i f i c pro~rty of amphipols is that. once complexed with t ~ ~lyme~. membrane proteins can be handled in aqueous buffers without being exposed to free surfactant, as is the case when using conventional detergents. This may offer an interesting road towards characterising the associa- tion state of proteins or protein complexes that are easily disrupted during purification in the presence of detergents. Such an application, however, depends on the ability of amphipols to 'trap' proteins under the aggregation state they experience in detergent solution, without promoting either dissociation or aggregation. In the present work, we have tested this point using cytochrome b~f as a model protein and mass determination by STEM at liquid helium tempe- rature as a means to directly establish the Mr of b6f/amphipol complexes, As compared with alternative approaches to Mr determination (small angle scattering, equilibrium ultracentrifugation), STEM offers the attractive features that the morphology of the objects can be directly visualised and that it can be more easily applied to heterogeneous samples ([1, 8~101: MOiler and Engel, submitted). On the other hand, its application to detergent+solubilized membra- ne proteins has been hitherto complicated by technical dif- ficulties linked to the elimination of the detergent during sample preparation (see Discussion). In the pre~ent expert° ments, the b~/" complex was trapped with amphipols from detergent solutions in which it existed under either its heavy or its light form, Protein composition and amphipollhdTli° pid mass ratios in the resulting particles were established hi,heroically, Molecuhw masses we~ established by ¢ryo° STEM and compared with masses expected for both forms on the basis of their biochemical composition, The results indicate: i) that amphi~l/protein complexes faithfully re.. fleet the aggregation state of the protein in detergent solu. tion; and it)that the combination of trapping with amphi~ls and cryo+STEM yields accurate determinations of the molecular mass of integral membrane proteins.
Materials and methods
Materials
Td¢i~ (N-lds(hydmxymethyl)methylglycine), egg yolk L-0t, phos- phatidylcholine ( e ~ ) , ~'-Iabeled dipalmitoylphosphatidyl- ¢holi~ (II~IDPPC~, pretense inhibito~ (phenyhnethylsulfonyl, fluoride, ~+aminocapt~ic acid, benzamidine) and sucrose were pur. chased from Sigma; Hecameg (6-O-tNheptylcarbamoyl).methyl. ot-D-glycopyranoside: HG) from Vegatec (Villejuif, France):
hydroxylapatite from Bio-Rad; Aqualuma-plus from Lumac LSC (Groeningen); ammonium acetate and ammonia from Prolabo (France). Amphipols were synthesized from poly(acrylic acid) with a molecular mass of 5000 (Aldrich) as previously described 161. When 25% of the acid units were grafted with octyi groups, the resulting amphipol, denoted A8-75, contained 75% charged sodium ac~late units at pH >- 8. Less charged amphipols were obtained with the same grafting ratio of octyl groups and the additional grafting of 40% mol/moi isopropyl groups. When fully charged, the polymer A8-35 contained 35% sodium acrylate units.
Purification and analysis of IJ+flamphipol complexes
Cytochrome h6fwas purifie.d from C winhardtii thylakoid membra- nes using a three-step protocol (specific solubilization, fl'ac- tionation on a sucrose gradient and hydroxylapat i te chromatography) as described previously i31. The native 14+mer ('heavy form') was obtained by elution fi'om the hydroxylapatite column with a solution of detergent (20 mm HG), lipids (0.1 g/L egg PC), and pretense inhibitors in ammonium phosphate-NaOH buffer 4(X) ms, pH 8,0 (AP buffer) [31. The bet 'light form' was generated at the last step of the purification procedure: the sample collected from the sucrose gradient was layered onto a hydroxyl- apatite column pre-equilibrated with 20 mM Tricine-NaOH, pH 8.0, 20 mM HG. and protease inhibitors; the complex, strongly ass~iated with the stationary phase, was washed with 2-3 column volumes of 100 mM AP-NaOH, pH 8,0, containing 40 mM HG and protease inhibitors. Previous experiments have shown that, upon delipidation, the complex loses the Rieske protein and dissociates into a hexameric or pentameric light Ibrm 141. The complex was then eluted with 400 mM AP-NaOH. pH 8,0, containing 20 mM HG and plotease inhihitors, It was not exmnined whether the com- plex treated in this way had retained or not the small suhunit PetL (3,4 kDa; ++,'/'141),
Amphil~dlhd'complexes were I~repm'ed by supplementing ,,olu~ lions el' ¢ltlae¢ the !i~h! or the heavy fi~rl!~ (aholll 5 ~IM cyt~c!lrolne l) m HG will'+ a COllCelllraled amphil~o! sohuion up tO a l'hta! a!uplfil}ol concenlrauon of (}, I U l wlw}, In ~wder to imptawe !he p!vservatim~ of the l~mer, complexes were prepared at 'high' lipid concentra- lion, ie while keeping the lipid concentration al~we O, I g/L during the whole p~edu~ , In these eXl~riments, the stock solution of amphipols {9 g/L) used to complex the 14-met also contained egg PC (3 g/L}. in order to reach a lipid/octyl group ratio of !/3 sol /sol in the final mixture+ Alternatively, 14-mer/amphipo! complexes were form~ without supplementation with additional lipids, using pure amphi~l st~k solutions. Complexation was achieved by briefly
+ " S (< I0 sin) incubating mixtures of amphl~l, + and b~[in HG bel'om diluting them below the cmc of HG with AP-NaOH buffer {final concentration of AP+NaOH butler (pH 8,0), 2IX} raM: final HG con- centrations: 9+10 mM for the heavy foma. 17 mM for the light tbrm). Amphil~+l//M'complexes were separated from HG and free amphi- pols by centrifugation at 250 000 g ~54 {XX~ q+m) in the TLS 55 rotor of a Beckman TLIO0 ultracentrifuge lbr 6~7 h in 5-20% (w/w) suc- rose gradients, The gradients were prepared in 20 mM tricine-NaOH, pH 8,0, or 200 mM AP-NaOH buffer, pH 8,0. for measurements of lipid binding, or in 20 mM ammonium acetate-ammonia {AmAc) buffer, pH 8,5, ibr cryo+STEM ex~riments. The brown band con- taining the complex was collected with a syringe. Analysis by urea/SDS-PAGE of the COml~sition of cytochrome b#'preparations and the distribution of b~fsubunits in sucrose gradients were carried out as described 13, 41.
Measmvment ~1" boumt !ipid,~
Aliquots of HC-labeicd dipahuitoylph~sphatidytcho~inc (I~(']DPPC) in elhanol/beneene mixed solvem were dried under nitrogen al room ~emperature. Lipids were mdispersed at 40'C in 40 mM HG ir~ water under gen|le stirring, The concentration of lipids in the final stock solution (7(>50 iaM) was determined from the [~-activity of a 20-1aL aliquot diluted in 5 mL Aqualuma-plus using a LS ! 801 liquid scintillation counter (BeckmanL Stock solu- tions of purified b~l" in 20 mM HG (90-150 ~.tL, about 5 ~M cytochmme,f) were supplemented with labeled lipids (10-30 pL} prior to overnight incubation either in the absence or presence of polymer (A8-75, about (I,1% w/w final) using a stock solution of polymer (9 g/L) with or without egg PC (3 g/L, final lipid/octyl group ntolm" ratio 1/3), if not already present, the polymer was added on the lbllowing day, 10-20 min befl-)re two-fold dilution in water below the cmc of HG. Complexes were purified by rate zonal ultracentrifugation on sucrose density grudients as described above. Gradients were collected from the top by 120-1aL fractions. Protein concentrations were determined from the absorbance at 554 and 564 nm (redox difference spectrum of cytochrome bd'. see 13]}. The concentration of [ )'~C]DPPC was determined from the ~-acti- vity of a 20-laL aliquot diluted in 5 mL Aqualuma-plus. Total lipid concentration and specific activity in the samples layered onto suc- rose gradients were calculated taking into account the presence of 0.1 g/L free egg PC in protein stock solutions, the addition of [~CIDPPC and, if applicable, of egg PC from anlphipol stock sol- utions, and assuming the presence of 20 bound iipids (egg PC) per cytochrome f 14] in HG/PC solutions.
Pr,.'paratitm of samples./or cO'o-STEM an,dysis
Folh)wing fractionation on sucrose gradients (see above), the yel- low-brown bands containing the purified b(Jlamphipol complexes (about 400 I.tl,) were dialyzed in Spectra-Por tubes (MWCO 10- 12 kDa, (').4 mM diameter) for 24--48 h against St)mL ammonium acetale/mnmonia buffer 20 hiM, ptl 8,5, with one bat!h change. Samples were centrifuged ft)r 10 rain in the A I l0 rotor of ;|11 Air- fuge l Beckman) at about 210 000 g (20 PSI ). Supernatants were kept a! ()°C before STEM analysis. 'Delipidatcd 14-1net' was i)re- pured from b(j' !4omer (stock solution in 20 mM HG, 0. i g/L egg ~ , 400 mM AF) by sedimentation lbr 6 h ut 250 000 g in the TLS 55 rotor of a Beckman TL 100 ultracentril'ttge in a sucrose gradient (5=20% w/w) cont~tining 20 mM AmAc, 20 mM HG, i)rotease inhio bitors, and no lipids 141. The yellow-brown hand corresponding to the heavy h)rm was collected and supplemented with amphipol stock solution (containing no lipids) to a final polymer con- centration of 0.1% w/w. The following steps (dilution below the cmc of HG, dialysis against AmAc, centrifugation) were the same as for the other complexes.
Cryo.STEM analysis
Two mL of the protein sample were deposited on a 6(X) mesh carbon- coated grid that had been glow-discharged. The liquid was allowed to settle for 30 s. Alter blotting, the sample was washed ~wice with a droplet of AmAc buffer 20 raM, pH 8.5, in order to eliminate non-adsorbed protein, free detergent and non-volatile salts before the freeze/drying procedure. The grid was blotted again and 5 IlL of a suspension of tobacco mosaic vires (TMV) applied to it. After washing with AmAc and blotting, the grid was phmged into liquid ethane at -160°C. Vitrified samples were stored in liquid nitrogen
477
beN)re being frec~e-dried and ~ra~s~k-rred m~dcr ~acuum ie t~c scanuing transmission ele~'tron micro.~c~pe [ ~ , ~hich ~a~ oper- aled a~ iOOkV, Images ~comprised of 1024 pixc~s, each one !, 17 mn x l, I7 nm a~ the ~eve~ ol the,sample) ~ere recorded under dark-fie~d conditions at a dose of 5 e A : . Samples xvem maintained at liquid helium temperature during preseiecti¢ms at tow mag- nification t l0 000 ×) and image correction. Mass anat3 sis was car- ried out as described by Freeman and Leonard []9], Histograms of mass distributions were obtained from the collection and analysis of at least 20 images.
Results
Biochemical analysis ~" bofTamphipol comph'xes
Two tbrms of cytochrome b(,f were purified in detergent solution: i) the native form, a 14-meric 'heavy' form: and ii) a lighter, inactive ibrm that is a breakdown product gene- rated by delipidation. Their respective M,s are 211 kDa and 83-87 kDa, including proteins and cofactors, excluding bound detergent and lipids 141. "[he heavy form was trans- ferred to amphipols A8-35 or A8-75 161, and the bight Ibrm to amphipoi A8-75. Both polymers are polyacrylates deri- vatized to -25% with octylamine, with an overall M~ of ,-8 kDa. In A8-75, the remaining 75% of carboxylates have
o.8 7
0.7 -~
,6
0.5
0.4 -
O.3
.2 -~
0.0
[] 0 ~
0 2 4 6 8 10 12 14 16 18
fraction
Fig I. Rate zonal centrifugation analysis of tile dispersity of the bof heavy Ibrm complexed with amphipols. The heavy form of the hof complex was transt~rred to amphipol A875 from a solution in 21) mM HG. No lipids were added to the amphipol stock solution (conditions I in table !, 'h)w lipids' in table il). Following dilution under the cmc of HG. Ihe complex was fractionated by centrifuga o tion on a 5-20% sucrose gradient in AP 21X) ram. pH 8.0, either iramediately CA) or after 24 h of incubation at 0°C (r=ij. Fractions were collected IYorn the top and the concentration of cytocimmlef in each fi'action determined spectro.scopically (see Materials and methods).
478
Fractions _ z
10 15 '~ -.--,, ~ m m ~ e ~ D a , ~ ~ '*-'Cytoehromef
Flit 2, Rate zonal centrifugation analysis of the subunit composition of the b~fheavy tbrm comp!exed with amphi- pol A8-75, Following addition of amphipol (9 g/L stock solution, containing 3 glL egg PC) and dilution under the cmc of HG, the complex was fractionated by centrifu- ration on a 5-20% sucrose gradient in 20 mM Tricine (a) or 2 ~ mM phosphate (b) buffer, pH 8,0, either immedia- tely (a) or after 24 h of incubation at O°C (b) (see Materials and me, thetis), Fractions were collected from the top and ,nalyted by SDS.PAGE in the presence of urea, In this gel system, cyt~hrome b6 give~ a broad band and the three small subunlts (PetG, PetL, Pc|M) comigrate 131, Silver staining was lighter tn h than in a, See text,
5 10 M
15 ~"-Cytochromef
Rieske protein I ,~-. Cytochrome b6
Subunit IV
been left underivatized, while in A8o35 a further 40% has been blocked with is~propylamine (see 161), The heavy form was transcend to amphipols in the p~sence or abe sence of extra lipids, the light form in the absence of lipids (see MatertaL~' end medloas), All forms of b~'Tamphipols complexes migrated upon sucrose gradient centrifugation in surfactant-free solutions as small particles of low dis~rsity, the light form sedimenting more slowly than the heavy one ([61~ this article, figs I and 2; and unpublished observa- tions),
Upon ultracentrifugation of the amphipol-complexed heavy ibrm, all of the ~ subunits comigrated; in some samples, however, traces of the Rieske subunit were present at the top of the gradient, indicating a small degree of dena- turation (fig 2a), Optimal preservation of the native state was found to depend on the presence of lipids in amphipol S ' ,~oluttons and on a rapid separation of the complex from excess amphipol (se¢ Materials and mettuuls), This is i llus- t ~ t ~ in figure 2: when centrifuged immediately after com- ple~ation (fig 2a), cytochmme b~f migrated as the heavy form (fractions I 1-12), while after 24 h of incubation (fig 2b) some of it had convened into the light form (fraction 9), Similarly, when the heavy form was transferred in am-
phipol~ under delipidating conditions (excess amphipols, no lipids added to the polymer), the complex sedimentcd mostly as the native, heavy form, but some light form was also detectable, whose amount increased upon incubation (fig !),
This observation suggested that, upon trapping of the bq ° complex by amphipols in the presence of lipids, some lipids remain associated with the complex and improve its stabi- lity, as previously observed in detergent solutions 14]. In order to test this hypothesis, b~f solutions in HG/egg PC mixed micelles were supplemented with traces of ['4CI- labeled dipalmitoylphosphatidyicholine (II4CIDPPC) prior to transfer to amphipols, and the resulting complex ana- lyzed by ultracentrifugation on surfactant-free sucrose gra- dients, While excess lipids (fig 3) and free amphipols 171 remained in the topmost regions of the gradient, some of the II'~CIDPPC was found to comigrate with the complex (fig 3). As found previously for radiolabeled amphipols ([7] and unpublished data), the [t4CIDPPCIbO" ratio was essen- tially constant throughout the bu"-containing fractions of the gradient (fig 3), consistent with the formation of complexes with a defined composition.
479
m o
o
v
3
2
1
0
A
i i T I | i a
0,0 - [ 1.2
?, 1.o
- 1 i O.e ." ,., 0.8 *'~
' / \ 0.4
: . o.o o.o , , , , ......... . .... ,o , ; , o
t i a c t i o n n u m b e r
Fig 3. Rate zonal centrifugation analysis of the lipid content of the complex between the beef heavy form and amphipol A8-75. The heavy form in 20 mM HG was incubated 24 h with trace amounts of l l4CIDPI~ as described in Materials and methods. Following addition of amphipol and dilution under the cmc of HG, the com- plex was fractionated by centrifngation on a 5-20% sucrose gra- dient in 20 mM tricine buffer, pH 8,0, Fractions were analyzed for cytochrome f(Q)and 114CIDPPC (O). lbp. [ 14CIDPPC/cytochro- reef molar ratio (A).
Estimates of the absolute number of !ipids bound per ' i ' ' s pamele are somewhat mprec~se, beeau, e the specific aetiv°
ity of the lipids depends on the amount of b~f-bound PC in the presence of HG/egg PC mixed mice!les, which is not accurately known. Assuming: i) that the latter amount is similar to that measured in LM solution 141; and ii) that the
affimties of egg ~K~" and tt4CJDPPC Ibr the surface of the protein are comparable, we estimate that, when amphipo~ solutions were not supplemented with tipids, purified b~flamphipol complexes retained about 30 lipids/t4-mer ~table 1). This value is close to that of 36 _+ 22 lipids/14-mer measured in dilute LM solutions [4]. When the heavy form was transferred to amphipols in the presence of excess lipids, the final complexes were found to contain up to 90-- 100 lipids/14-mer (table l).
CO, o-STEM analysis
Cytochrome b~f in detergent solution Purified preparations of the heavy form of cytochrome b~" in HG/egg PC solution were deposited on EM grids, washed with dilute ammonium acetate buffer, fi'ozen, freeze-dried, examined by STEM at liquid helium temperature, and the mass distribution of the particles analyzed (see Materials and methods). Assuming lipid binding in HG/egg PC solu- tion to be comparable to that measured in dilute LM solu- tions [4], expected particle mass under these conditions varies between -220 and ~350 kDa. Range limits corres- pond to hypotheses according to which either none or all of the detergent would remain associated with the protein and lipids following washes with detergent-free solutions. Mass analysis revealed broad mass distributions ranging from less than 200 to more than 1600 kDa ~ r particle (fig 4a and table II). In HG/egg PC solutions, the b6fcomplex is a 14- mer and migrates on sucrose gradients as a monodispersed species [3, 4]. Aggregation therefore must have occurred either upon adsorption onto the carbon film or, perhaps more likely, as a result of the washing steps with detergent° free buffer. STEM examination of the light form in lipid- free HG solution similarly revealed extensive aggregation, making determination of the M, impossible (table li). Op o timization of the protocol was not attempted.
Cytochrome b~jTamphipol complexes The same procedure, when applied to b¢flamphipol como plexes, usually yielded homogeneous fields of weiiodiso
Table 1, Numbel~of b~-bound molecules of phosphatidylcholine per 14-mer in AS.751btj'complexes, as deduced fi'om the cosedimentation of protein and [ CIDPPC. Following eomplexation of eytoehrome b6f by amphipols, protein-bound lipids were separated from free lipids by sedimentation in 5-20% sucrose gradient in 20 mM tricine-NaOH buffer, pH 8.0 (see Materials and methods). [A8o75]t == 0 and [PC]t ~ o are the initial c~ncentrations of amphipol and lipids in the sample before ultracentrifugation. Calculation of the number of b~.bound lipids assumes that [ CIDPPC and egg PC share the same affinity for the surface of the complex. Errors correspond to the largest difference between the mean value and individual values calculated for the four major bt~f-containing fractions.
aThe mixture of HG, lipids, protein and A8-75 was incubated 24 h instead of incubating 24 h without the polymer and supplementing with A8-75 just before dilution below the cmc of HG and centrifugation.
480
r .
eounU
count
ooun| i
35
30
25
20
15
10
f;
0 50
~)
~0
~0
8)
t¢
0
Fig 4. STEM ana!ysis of tl~e mass of tile heavy ta, c) and light (b) tk~rnls ~ff cylt~hrome bt~fadsor~d to Ihe ca" rboll film either fl'Olll a solution in FIG (a) or as complexes with amphil~is A8-75 (b) or A8-35 (¢).
Fig $. Dark-field STEM image of the heavy form o1' cytochrome b6fcomplexed with amphipol A8-75 (the original dark-field image has been inverted tbr better printing). TMV: tobacco mosaic virus. added as an internal standard (~ 18 nm).
presence of lipids in samples that had not been purified thoroughly enough may explain the broadening of mass distribution (-100-500kDa) observed with some 14-mer samples. This point deserves further investigation.
On dark-field images of unstained samples, b ~ 14-mers complexed with amphipols featured diameters smaller than the width (18 nm)of reference TMV particles (fig 5 }. Scans of the images indicated apparent diameters of --8 ± I nm and 10-!5 nm for the light and heavy ti,'ms, respectively. These dimensions are comparable to those measured Ibr particle~ in freeze-fractured reconstituted preparations of h~l' light and heavy fi~rnls 141. and somewllat larger than observed after negative staining of thin threeodimen.,,ional crystals of b~t' 14omer 1121.
The molecular mass expected for b~d" 14.reefs comp!exed with amphil~ls in the absence of added lipids can 1~ esti- mated t ~ m their composition to --280 kDa (table II). I~k~r 14-reefs complexed in the presence of excess lipids, which bind -. 100 iipids per particle, the calculated molecular mass increases to ...330 kDa. Assuming the light form to be asso- ciated with half the amount of amphipols and to have lost the lipids and the Rieske protein leads to a predicted mole- cular mass of 106~ 110 kDa, depending on whether subunit PetL has been retained or not 141. Cryo-STEM mass deter- minations are in excellent agreement with these estimates (table !1).
~t 'sed particles with sharp mass distributions (figs 4b, c, 5}, In the presence of excess lipids, however, ie with samples that had not ~ n purified on sucrose gradients following complexation with amphipol/lipid solutions, large agglx- gates were formed (not shown), Excess free amphipols, on the other hand, did not induce aggregation (not shown }, The
Discussion
The pr~ess of complexation of detergent-solubilized mem- brane proteins by amphipols has not been studied in any detail yet, From a comparison of the water solubility of matrix porin (OmpF) samples supplemented with amphi- pols either prior to or simultaneously with dilution of the
48[
Table 1|. A comparison of the molecula~ mass of amphipoFb{d complexes as expected from their composition a~d as determined b~ ~ cryo-STEM, Masses are given in kDa. E|ements that enter imo the caicuRation of expected masse:< are described in the text. Em>r ranges on STEM masses correspond to the half-width of mass dislributkm histograms, The Uight ~'~+m t~|" cytuchrome b~]v,'as obtai~ed in 20 mM HG, the heavy form in 20 mM HG plus 0.1 g/L egg PC before complexation with amphipols and purification. A qow lipid" heavy form ~.as prepared by sedimenfing lhe 14-met in a lipid-free sucrose gradient containing 20 mM HG before association with A8-35 I4] ¢conditions I in table !; see Materials and methodsL All samples were extensively dialyzed against 20 mM AmAc ~containing 20 mM HG in the case of preparations in HG) prior to analysis.
Composition Heal3,fimn in Light form in Light,form in Heavy.form in Heavy,form in Heavy form in HG HG A8- 75 A8-35 ( "low I#,~id') A8-35 AS- 75
aFrom 13, 41: bassumed to be Ihe same as in dilute LM sohttions [4]: ~this work; dassumed to be the same number of molecules as LM binding in dilute LM solutions 141; eassumed to be half that to the heavy form 14, 71; rassumed 1o be identical to A8+75 binding to the heavy torm 171; gfrom 171.
detergent below its cmc, it appears that amphipols associate with the protein even in the presence of detergent 171. The oligomeric state and composition of amphipol-stabilized particles are therefore likely to mirror those which preexist detergent removal, with the reservation that additional am- phipol molecules may well be recruited by the protein upon detergent depletion. The present study shows that, upon cryo+STEM observation, preparations obtained by trapping with mnphipols the 14-meric btd'complex in detergent sol- ution contain homogeneous particles that exlfibit the mole- cular mass of tile bed' 14,incr. Similarly, preparations obtained under conditions that generate the btd' light lbrm contain homogeneous particles witl~ the molecular mass expected lbr the light form. Aggregation was observed only in preparations that contained excess lipids and may wc!l have occurred during preparation of the samples for STEM observation. It seems therefore that. given due precautions.
heeze mere o ¢omplexation with amphipols can be used to ' ~.~ , ' ~' brahe protein oligomers under the state they exhibit in deter- gent solution.
This finding has a number of consequences. It tallies with our previous observations showing that, upon solubilization of thylakoid membranes with HG followed by complexa- tion of the crude supernatant with amphipols, distinct com- plexes are formed, which can be separated one l'rom another upon centrifugation in a surfactant-free sucrose gradient (CT and JLP, unpublished observations). It is also consistent with the observation that, provided enough amphipol was added prior to elimination of the detergent, all purified membrane proteins tested to date have yielded homo- geneous, monodisperse pmtein/amphipol complexes 16. 7 I. Altogether, these data bode well for the use of amphipols in identifying supramolecular complexes and in purification protocols. Observations with cytochrome bof nevertheless indicate that some precautions may be in order: lengthy
incubations with excess amphipols can lead to frag- mentation, while the iipids used for stabilization may fad+ litate artefactual aggregation upon preparation of EM samples. Purification protocols furthermore will have to take into account the high charge conferred to the corn+ plexes by the cun'ent anionic amphipols.
The fact that amphipol-stabilized membrane proteins can be handled in detergent-free solutions offers interesting per- spectives for the study of supramolecular organization in the membrane plane. Protein/protein associations that can be surmised on the basis of functional data are not aiway+~ observed in detergent solution, pre+~umably because of the looseness of the associations and their sensitivity to deter- gents, A case in point is the tbrmation of 'supercomplexes+ between reaction centel+~ and bt'~ complexe.~ in +~onlc ,+pccic+~ of photosynthetic bacteria 113!. Similarly, ,: is often diffi° cult to ascertain the oligomerization state of proteins prior to membrane disruption: artefactual disaggregation of oligomers by detergents in the course of purification is fre° quently observed, as is the case fur cytochrome b~d~ arte° factual, detergent-induced oligomerization can also be encountered 1141. Amphipols may offer a novel approach to these questions. Amphipols by themselves have never been observed to extract integral proteins fi'om membranes. However. it is conceivable to resort to mild conditions of solubilization using conventional detergents, or mixtures thereof with amphipols, followed by amphipol-trapping of the solubilized complexes. Purification and analysis of the complexes thus stabilized may yield useful intbrmation about protein/protein and protein/lipid interaction.~ that do not stand well prolonged exposure to detergents.
Finally. the data reported in the present article indicate that cryo-STEM coupled with amphipolqrapping may be an attractive and reliable route towards determining the moleo cular mass of membrane proteins and membrane protein
482
complexes. STEM is a versatile approach to the study of membrane proteins (MUller and Engel, submitted). With few exceptions (see eg [I 5, 161), data however have usually
econstituted samples, ie on pro- environment often o~ani- crystals (Miiller and Engel,
" ns ass~iated with the use which originate from the
necessity of washing the sample with detergent-free solu- tion prior to freezing it: protein aggregation and the un- known extent of detergent removal, While this particular point was not investigated in detail, it seems that amphipoi- stabilized ~complexes lend themselves to electron micro- scopy more readily than similar preparations solubilized in detergent or lipid/detergent micelles: under most circum- stances, very little aggregation was observed, while bof preparations in miceiles yielded ex';emely polydisperse fields of particles. This is probably due to the fact that am- phipol,coated be" complexes: i) e!ectrostatically repulse each other: and ii) show no tendency to shed their surfactant upon exposure to surfactant-free aqueous solutions [71. Under the same c~rcumstances, detergent-solubilized bqf complexes aggregate and precipitate [61. The masses deter- mined ~n the present study were extremely close to those expecled on the basis of the particles' biochemical com- position, It seems safe, when determining by STEM the mass of a protein complexed with amphipol, to assume that !he proteintamphipol mass ratio is identical to that deter° mined in solution using labeled amphipols. For many appli~ cations, however, such as distinguishing between monomeric and dimeric aggregation states, direct determio nation of this ratio may not even be a requirement. Current data (which bear on a limited nunlber of proteins) suggest that the mass of amphipols bound by a given protein can be anticipated within a factor o f - 2 when the number of ~-hel:ices comprising the transmembran¢ region of the pro- tein is approximately known ([71 and unpublished observa- tions), Because amphipol binding is less extensive than detergent binding [71, even relatively large uncertainties on the extent of amphipoi binding have a moderate bearing on protein mass determination: in the present case, a priori assuming amphipol binding to be -3 kDa per reel of trans- membrane a-helix ([71 and unpublished observations) would have biased our estimate of the mass of the b6fheavy form by only -20 kDa, ie by less than 10%. More generally, and beyond the mere determination of masses, amphipoi- stabilized membrane proteins may also o ~ n interesting perspectives in other electron microscopy applications where the p~senc¢ of detergent is detrimental, either be- cause its loss induces aggregation, or because it affects the binding properties of the supporting ";' the f, dll or surface ten- sion of the solution.
Acknowledgments
Particular thanks are due to C Breyton ,and Y Pierre (IBPC) lbr many useful discussions. We are also grateful to the referees and to A Engel for useful information. This work was initiated in the frame of CNRS laboratory network "Colloi'des Mixtes' (GDR 1082) and supported in part by Biotech EC grant Bio2-CT93-0076 (to JLP). Current work on amphipois is supported in part by a grant from the CNRS interdisciplinary program 'Physique et Chimie du V/rant'.
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