Biochimie (1998) 80. 475~82
© SociEt6ffanqaisede biochimieel biologic mol6cukanv/ Elseviea. Paris
Scanning transmission electron m croscopy study of the molecular mass of
amph pol/cytochrome b6f complexes
C T r i b e t ", D Mills b**, M H a i d e r b***, J L P o p o t c*
aCNRS-UMR 7615 amt Universitd Paris-6, l~cole Supdrieure de Physique et de Chimie lndustrielles.
i0, rue 14mquelin, F-75231 Paris cedex 05, France:
bEtCml,ea~t Mole,'ular Biology Laboratot3; Meyerhofstra~e !, Heidelberg, Germany:
CCNRS-UPR 9052 ~tmt Univer,~itd Paris. 7, ln.~litut de Biol~gie Phy.~ico-Chimique.
13,row P-et~M~Cm'ie, F~75005 P, wis ,'cdc.~'05, F~m,'e
(Received 23 December 1997: accepted 23 April 1998)
Summary - - The composition and mass of complexes betv,'een Chhmavdomona.s rei~thardtii cytochrome b~)[ and low molecuku"mass
amphipathic polymers ('amphipols') have been studied using biochemical analysis and scannirig tran,,mission electron microscopy at liquid
helium temperature (cryo-STEM). Cytochrome bqfwas trapped by amphipois either under its native 14-mericstate or as a delipidated, lighter
forn'. A good consistency was observed between the masses of either form calculated from their biochemicalcompositionand those determit~ed
by cryo-STEM. These data show that association with amplfipolspreserved the original aggregation ~tate of the protein in detergent sotuti~m.
Complexation with amphipois appears to lhci!itate preparation of |he samples and mass determination by cryo-STEM as compared to
conventional solubilization with detergents (© Soci6t~ fi'anqaisede biochimie et biologie mol6culaire / Ekevier, Paris).
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 telnperature (cryo-STEM) and biochemical ineasurelucnts.
Cytochrome bqf (plastoquinol:plastocyanin oxidoreduclase), 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, HeinrichHoffmann-Stral]e7, 60528 Frankfurt-am-Main,Ger,nany.
***Present address: CEOS GmbH, hn Neuenheimer Feld 519.
69120 Heidelberg, Ger,nany.
Abbreviations: AmAc, ammonium acetate: AP, alnlnoniuni phosphate; cryo-STEM, scanning transmission electron microscopy at
liquid helium temperature; cmc. critical miccllar concentration;
DPPC, dipalmitoylphosphatidylcholine;EM, electron microscopy;
HG, 6-O-(N-heptylcarbamoyl)-methy1.0t-D-glycopyranoside (Hecameg); LM, dodecyl-ll-D- maltoside (lauryhnaltoside); M~, molecular 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 a n d 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~ 2il 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, apparently monodisperse
species 161. The surfactant/protein mass ratio in the cornis 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 association 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 temperature 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 membrane proteins has been hitherto complicated by technical difficulties 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,phosphatidylcholine ( 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+flamphipolcomplexes
Cytochrome h6fwas purifie.d from C winhardtii thylakoid membranes using a three-step protocol (specific solubilization, fl'actionation on a sucrose gradient and hydroxylapatite
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 hydroxylapatite 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 complex treated in this way had retained or not the small suhunit PetL
(3,4 kDa; ++,'/'141),
Amphil~dlhd'complexes were I~repm'edby supplementing ,,olu~
lions el' ¢ltlae¢ the !i~h! or the heavy fi~rl!~(aholll 5 ~IM cyt~c!lrolne
l) m HG will'+a COllCelllraledamphil~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 concentralion, ie while keeping the lipid concentration al~we O,I g/L during
the whole p ~ e d u ~ , 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
(< 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 concentrations: 9+10 mM for the heavy foma. 17 mM for the light tbrm).
Amphil~+l//M'complexes were separated from HG and free amphipols 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) sucrose 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 containing 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.
+
"
S
477
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 solutions 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 ~-activity of a 20-laL aliquot diluted in 5 mL Aqualuma-plus. Total lipid
concentration and specific activity in the samples layered onto sucrose 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 solutions, and assuming the presence of 20 bound iipids (egg PC) per
cytochrome f 14] in HG/PC solutions.
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~ operaled 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 magnification t l0 000 ×) and image correction. Mass anat3 sis was carried 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 generated 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 transferred to amphipols A8-35 or A8-75 161, and the bight Ibrm
to amphipoi A8-75. Both polymers are polyacrylates derivatized to - 2 5 % with octylamine, with an overall M~ of ,-8
kDa. In A8-75, the remaining 75% of carboxylates have
Pr,.'paratitm of samples./or cO'o-STEM an,dysis
o.8 7
Folh)wing fractionation on sucrose gradients (see above), the yellow-brown bands containing the purified b(Jlamphipol complexes
(about 400 I.tl,) were dialyzed in Spectra-Por tubes (MWCO 1012 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 Airfuge l Beckman) at about 210 000 g (20 PSI ). Supernatants were
kept a! ()°C before STEM analysis. 'Delipidatcd 14-1net' was i)repured 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 concentration 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.
0.7 -~
[]
Cryo.STEM analysis
Two mL of the protein sample were deposited on a 6(X) mesh carboncoated 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
0~
,6
0.5
0.4 O.3
.2 -~
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
-.--,, ~ m m ~ e ~ D a , ~
"--
. . . . . .
protein
I~s..-Cytochrome be
Rieske
-
--
~,~~
, ~
a
Flit 2, Rate zonal centrifugation analysis of the subunit
composition of the b~fheavy tbrm comp!exedwith amphipol 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 centrifuration on a 5-20% sucrose gradient in 20 mM Tricine (a)
or 2 ~ mM phosphate (b) buffer, pH 8,0, either immediately (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.PAGEin 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,
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 observations),
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 denaturation (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 illust ~ t ~ in figure 2: when centrifuged immediately after comple~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-
15 '~
~
'*-'Cytoehromef
~
....
~
Subunit W
~. . . . . . . . . . . .
.....
4 " - Pet G,L,M
M
5
10
15
~"-Cytochromef
protein
I ,~-. Cytochrome b6
Rieske
Subunit IV
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 stability, 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 ['4CIlabeled dipalmitoylphosphatidyicholine (II4CIDPPC) prior
to transfer to amphipols, and the resulting complex analyzed by ultracentrifugation on surfactant-free sucrose gradients, 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 essentially constant throughout the bu"-containing fractions of the
gradient (fig 3), consistent with the formation of complexes
with a defined composition.
479
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).
m
o
o
3
v
2
A
1
0
i
i
T
I
|
i
a
[
0,0 -
?,
."
O.e
-1
,.,
i
0.8
,
*'~
/\
'
,
CO,o-STEM analysis
1.o
0.4
o.o
1.2
,,
......... . .... ,o
tiaction
,;,
:.
o
o.o
number
Fig 3. Rate zonal centrifugation analysis of the lipid content of the
complex between the beefheavy 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 complex was fractionated by centrifngation on a 5-20% sucrose gradient in 20 mM tricine buffer, pH 8,0, Fractions were analyzed for
cytochrome f(Q)and 114CIDPPC (O). lbp. [ 14CIDPPC/cytochroreef molar ratio (A).
Estimates of the absolute number of !ipids bound per
'
'
' s e the specific aetiv°
pamele
are somewhat imprec~se,
beeau,
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
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 solution to be comparable to that measured in dilute LM solutions [4], expected particle mass under these conditions
varies between -220 and ~350 kDa. Range limits correspond 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 14mer 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 lipidfree 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.
"
3
4
E~periment
1
[A8-75]t = o (g/L)
[Pelt = 0 (g/L)
PC/14-mer
0.5
0.065
30 4- 2
0.45
0.228
100 + 4
0.37
0.206
90 5:12
0.37"
0.206
102 +_16
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
35
30
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).
25
count
20
15
10
f;
0
50
~)
~0
ooun|i
~0
8)
t¢
0
Fig 4. STEM ana!ysis of tl~e mass of tile heavy ta, c) and light (b)
tk~rnls~ffcylt~hrome bt~fadsor~d to Ihe ca"rboll film either fl'Ollla
solution in FIG (a) or as complexes with amphil~is A8-75 (b) or
A8-35 (¢).
~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 agglxgates were formed (not shown), Excess free amphipols, on
the other hand, did not induce aggregation (not shown }, The
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~ estimated 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 associated with half the amount of amphipols and to have lost
the lipids and the Rieske protein leads to a predicted molecular mass of 106~ 110 kDa, depending on whether subunit
PetL has been retained or not 141. Cryo-STEM mass determinations are in excellent agreement with these estimates
(table !1).
Discussion
The pr~ess of complexation of detergent-solubilized membrane 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 amphipols 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 ~'~+mt~|"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
Protein/cofactors a
Lipids
Detergent
Amphipol
Expected mass
STEM mass
Heal3,fimn in
HG
Light form in
HG
Light,form in
A8- 75
Heavy.form in
A8-35 ( "low I#,~id')
Heavy,form in
A8-35
Heavy form in
AS- 75
211
27 ± 16b
83-86
4 4 + 3c
127 ± 5
500 ± 100
83-86
.
23 + 2e
108 _ 5
m33 ± 21
211
22 + 2~
.
46 + 5 l
279 ± 7
260 _+58
211
75 + 8':
211
75 + 8~:
46 _+5f
332 +_.! 3
300 _++70
46 _+5~
332 ± 13
312 _.+60
87 + 7 d
325 ± 23
700 _+450
.
.
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 amphipol 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 solution contain homogeneous particles that exlfibit the molecular 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.
¢omplexation with amphipols can be used to ' heeze
~.~,' ~' mere o
brahe protein oligomers under the state they exhibit in detergent 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 complexation of the crude supernatant with amphipols, distinct complexes 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 homogeneous, 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 fragmentation, 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 perspectives 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 detergents, 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 proenvironment
often o~anicrystals (Miiller and Engel,
" ns ass~iated with the use
which originate from the
necessity of washing the sample with detergent-free solution prior to freezing it: protein aggregation and the unknown extent of detergent removal, While this particular
point was not investigated in detail, it seems that amphipoistabilized ~complexes lend themselves to electron microscopy more readily than similar preparations solubilized in
detergent or lipid/detergent micelles: under most circumstances, 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 amphipol,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 determined ~n the present study were extremely close to those
expecled on the basis of the particles' biochemical composition, 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 protein is approximately known ([71 and unpublished observations), 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 transmembrane 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, amphipoistabilized membrane proteins may also o ~ n interesting
perspectives in other electron microscopy applications
where the p~senc¢ of detergent is detrimental, either because its loss induces aggregation, or because it affects the
binding properties of the supporting f,";'dll or the surface tension 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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