AFM S rand Nand T
Olga I. K IrVadim Y*Faculty oInstitute
The aption of mdimensiomicrosomtochromeductaseportshite (HOPhemo- anbe adsortively chdissociatgomers dimages ochrome Pnm high,forms ofrespectivplexes coP450 2B4 and its reductase and to measure the heightsof these complexes (7 nm). The method is applicablefor visuaalso thei
Key Wchrome Pforce mic
The cyygenase sdrugs, ca
wnngedsso4a d5).r dimtiomhna
bic (graphite) and hydrophilic (mica) supports. The 2B4
1 To whoBiomedicalRussia.
0003-9861/99Copyright All rights of
Archives of Biochemistry and BiophysicsVol. 371, No. 1, November 1, pp. 17, 1999Article ID abbi.1999.1412, available online at http://www.idealibrary.com onlization of not only individual proteins butr complexes. 1999 Academic Pressords: cytochrome P450 2B4; NADPHcyto-450 reductase; oligomers; monomers; atomicroscopy (AFM); complex formation.
tochrome P450-containing microsomal monoox-ystem plays the key role in the metabolism of
rcinogens, mutagens, and other xenobiotics (1).
and Fp monomers and oligomers can be visualized ongraphite support. It was shown that electrostatic pro-teinsupport interactions play an essential role in thedissociation of 2B4 and Fp oligomers on mica. The binarycomplexes formed from 2B4 and Fp monomers were onlyobserved on the mica support.
MATERIALS AND METHODS
Chemicals. Emulgen 913 was purchased from Kao Atlas (Osaka,Japan). Other chemicals were purchased from Reakhim (Moscow,Russia).tudy of Membrane Proteins, CytochADPHCytochrome P450 Reductaseheir Complex Formation
iselyova,* Igor V. Yaminsky,* Yuri D. Ivanov,,1
u. Kuznetsov, and Alexander I. Archakovf Physics, Moscow State University, Moscow, Russia; andof Biomedical Chemistry RAMS, Moscow, Russia
arch 8, 1999, and in revised form July 26, 1999
plication of the AFM technique for visualiza-embrane proteins and for measuring their
ns was demonstrated. The AFM images of theal monooxygenase system componentscy-P450 2B4 and NADPHcytochrome P450 re-were obtained by using two types of sup-
ydrophobic, highly oriented pyrolytic graph-G) and hydrophilic mica. It was shown thatd flavoprotein monomers and oligomers can
bed to and visualized on HOPG. On the nega-arged mica matrix, flavoprotein oligomersed to monomers while hemoprotein oli-issociated into less aggregated particles. Thef cytochrome P450 2B4 and NADPHcyto-450 reductase monomers were about 3 and 5respectively, while the images of oligomeric
these proteins were about 10 and 8 nm high,ely. We were able to observe the binary com-mposed of monomeric proteins, cytochrome
As knoteractiP450 rproteinWhenand 2Bmeric,oped (used foas sedcorrelathe atoing tecsuringjectsproteinclathrichromAFM sas 2B4m correspondence should be addressed at Institute ofChemistry RAMS, Pogodinskaya St. 10, Moscow, 119832,
2 Abbrevcytochromeite; AFM, a
$30.001999 by Academic Pressreproduction in any form reserved.ome P450 2B4,
ina P. Kanaeva,
, cytochrome P450 2B4 (2B4)2 functions by in-with its redox partner, NADPHcytochrome
uctase (Fp). Both 2B4 and Fp are membranewhich hampers the study of their functions.lubilized from the microsomal membrane, Fpbecome oligomeric (24). To make them mono-etergent monomerization technique was devel-Various experimental methods are currentlyetermination of protein sizes and weights such
entation, gel chromatography, electrophoresis,n laser scattering, and others (2, 3, 5). Recently,ic force microscopy (AFM) and scanning tunnel-iques (STM) have been put to use for size mea-nd visualization of individual nanomeric ob-ch as nucleic acids, biological membranes,ystals (6), photosynthetic reaction centrum (7),(8), concanavalin A (9), and NADPHcyto-450 reductase (10). In the present work, the
dy of oligo- and monomeric 2B4 and FP, as wellcomplexes, was performed by using hydropho-iations used: 2B4, cytochrome P450 2B4; Fp, NADPHP450 reductase; HOPG, highly oriented pyrolytic graph-tomic force microscopy.
Preparation of proteins. Oligomers of membrane proteins 2B4and Fp were isolated from liver microsomes of male New Zealandrabbits treated with 0.1% (w/v) sodium phenobarbital in drinkingwater for 1 week as described earlier (11, 12). Specific content andspecific actand 4043tively. SpecA276/A418 5
Monomelows: to 5 n913 solutioture, the prmM and 0.buffer, pH24 h (5).
Analyticamined by tcoefficientoxidized mtration of pof A456 5 2
AFM expcarried ousupports, Hmonomers913 was dilwere deposeach sampThe 2B4/Fpmeric protemM in 100the mixturbuffer, andreported easurface isimaged inbeing 607molecules u
All AFMmultimodements, Santilevers forlength of th308340 k
AFM imsoftware FRussia) (17
AFM ismeter-sc(6). An imeasurelateral deffect (18guishingtein formimages oter of anbell-shapexperimeproteinsdomly diments w
Visualization of oligomeric and monomeric 2B4 andFp images and of their complexes was carried out ontwo types of surfaceshydrophobic HOPG and hydro-
lowdimB (ehcouhihodre. Cialonithheveighermerthad
2 KISELYOVA ET AL.ivity of Fp at 30C were 1313.5 nmol Fp w mg21 proteinmmol of cytochrome c w min21 w mg21 protein, respec-
ific content of 2B4 was 1718 nmol w mg21 protein and its1. Both proteins showed a single band on SDSPAGE.rization of Fp and 2B4 oligomers was carried out as fol-mol (2030 ml) of either protein 13 ml of 2% (w/v) Emulgenn was added. After 10 min incubation at room tempera-otein and Emulgen 913 concentrations were brought to 525 g/liter, respectively, with 100 mM K-phosphate (KP)7.4, and the mixtures were incubated at 4C for another
l measurements. The concentration of 2B4 was deter-he method of Omura and Sato (13) using the extinctionof A450490 5 91 mM21 cm21 for the CO complex of theinus reduced 2B4 in the difference spectrum. The concen-urified Fp was determined using the extinction coefficient1.4 mM21 cm21 (14).eriments and samples preparation. Experiments were
t using the direct surface adsorption method (15). AsOPG and mica were used. The solution of 2B4 or Fp
(5 mM) in 100 mM KP buffer with 0.25 g/liter Emulgenuted with the same buffer up to 0.2 mM. Protein samplesited on the HOPG or mica surface and left for 2 min. Thenle was rinsed with distilled water and dried in air flow.
complexes were obtained by mixing appropriate mono-ins in solution at the concentration for each protein of 5mM KP buffer, pH 7.4, with 0.25 g/liter Emulgen. Thene was incubated for 10 min, diluted 50 times in the sameimmediately placed onto support. According to the datarlier (16), with the air humidity 45% and higher, the micacovered with water layer. In this study, samples wereair under ambient conditions, with relative humidity0%. There is reason enough to suppose that the proteinnder study remain hydrated.experiments were carried out in the tapping mode on aNanoscope IIIa atomic force microscope (Digital Instru-ta Barbara, CA). Commercially available Nanoprobe can-the tapping mode (Digital Instruments) were used. Thee cantilevers was 125 mm and the resonant frequency was
Hz.ages were processed with the aid of the user-friendlyemtoscan 001 (Advanced Technologies Center, Moscow,).
a novel high-resolution instrument for nano-ale objects size measuring and visualizationmportant property of AFM is its ability tothe exact height of observed rigid objects. Theimensions are broadened due to a tip-related). Therefore, the main criterion for distin-between the monomeric and oligomeric pro-s is the height of the observed object. The
f objects have bell-shape profiles. The diame-object was determined as the width of its
ed image measured at half height. For controlnts the appropriate buffer mixture withoutwas applied to a support and imaged. Ran-stributed contaminations in control measure-ere less than 1 nm high.
philicdictateconcenmanyby muview olower
As woligomg/litermers (presenAFMlightersuredmatelythe mabuffernm inclusionand 1Bsize owhoseare, reThe ennmieffect (more cing 0.2KP bu1A). Itter atthree-Fig. 1compr
FiguHOPGessentand mules wsuredtheir athe heoligomthat ofoligomThus,with rca. The choice of protein concentration wasby the demands of the AFM technique: hightions (about 5 mM) lead to the adhesion of toolecules to the surface; they cover the surfaceayers which do not ensure good resolution. Inthis, the protein concentrations used weren 5 mM.shown earlier, the incubation of Fp or 2B4
s in 100 mM KP buffer, pH 7.4, with 0.25ulgen 913 led to their dissociation to mono-The images of 2B4 monomers on HOPG are
d in Figs. 1A and 1B. Here and further in theages darker regions correspond to pits andegions correspond to protrusions. The mea-
ensions of these protrusions were approxi-.53 nm high and 1518 nm in diameter. Asmum height of imaged contaminations in KPes not exceed 1 nm, the protrusions of 2.53ight may be considered as proteins. The con-bout the monomeric state of 2B4 (Figs. 1 As based on the assumption that the molecularB4 is close to that of cytochrome P450camight and width as measured by X-ray analysisectively, 3 and 5 nm (index PDB 2CPP) (19).rged lateral diameter of 2B4 images15 to 18pparently due to the tip-induced broadening). The monomers are better visualized and aretrasting in 20 mM KP buffer, pH 7.4, contain-g/liter Emulgen 913 (Fig. 1B) than in 100 mMr at the same detergent concentration (Fig.
ppears that monomers adsorption occurs bet-rather than high buffer ionic strength. The
ensional image of monomers (light color) inbottom) is shown for illustration and better
ension of the image perspective.ld not obtain the images of 2B4 monomers onlic mica supportin view of slight binding ofbic protein molecules to the negatively
hydrophilic surface.1C represents the images of 2B4 oligomers onomparison of Figs. 1A, 1B, and 1C revealsdifferences between the images of oligomers
omers. In Fig. 1C oligomers show up as glob-unresolved structure (white spots). The mea-
ight of these oligomers is about 10 nm andrage diameter is about 50 nm. By comparingts of oligomers and monomers, the volume of
s was calculated. It was 48 times larger thanonomers. It was supposed therefore that eachconsists of 4060 molecules of monomers.
e volume (Vm) occupied by a 2B4 monomerius rm is
Vm 5 (4/3)prm3 
FIG. 1. AexperimentpH 7.4, wit1.0 3 1.0 m
3PROTEIN COMPLEX FORMATION IN THE MONOOXYGENASE SYSTEMFM images of 2B4 monomers (A, B) and oligomers (C) on HOPG. In B, their three-dimensional image is also presented. Theal conditions were 0.2 mM 2B4 in 100 mM KP buffer, pH 7.4, with 0.25 g/liter Emulgen 913 (A); 0.2 mM 2B4 in 20 mM KP buffer,h 0.25 g/liter Emulgen 913 (B); and 0.5 mM 2B4 in 100 mM KP buffer, pH 7.4 (C). The image areas were 0.8 3 0.8 mm (A, B) andm (C).
while thegomer (intween its
Two-ditom) viewbuffer, pHwhich cademonstr2B4 oligo
on HOPG. On mica, apart from large 50-nm protru-sions, a number of small-sized images are seen. Accord-ing to the sizes, all protrusions can be divided into four
FIG. 2. TAFM imagewere 1.7 m0.48 3 0.48in Table I.
4 KISELYOVA ET AL.volume occupied by n monomers in the oli-the approximation neglecting the space be-monomeric units) may be expressed as
Vo 5 nVm. 
g that the height of the monomer (hm) or oli-o) is 2rm,o, we obtain
n 5 Vo /Vm 5 (ho /hm)3. 
mensional (top) and three-dimensional (bot-s of 2B4 oligomers on mica in 100 mM KP7.4, are shown in Fig. 2. Unlike monomers,
nnot be adsorbed on mica support, oligomersate high adhesion to mica. Apparently, themer images on mica differ greatly from those
groupsto refesincematelysecond8 was2.75 nwere cizationthat apromogregatbe expbetweechargetermoltheir obic intphobicciation
Thein Figseen. T2022withouwide 3the pasize fowere uof hydof oligOligommensiin Fig(Fig. 3was abBasedmonomgomermonom
wo-dimensional (top) and three-dimensional (bottom)s of 2B4 oligomers on mica. The experimental conditionsM 2B4 in 100 KP buffer, pH 7.4. The image area wasmm. Arrows 14 indicate groups of protrusions presentedrepresented in Table I. It seems to us naturalo the particles of the first group as monomers,
height of these 2B4 molecules is approxi-.53 nm and their diameter is 15 nm. Theoup of particles was defined as octamers (n 5
lculated from Eq.  using the average hm 5and ho 5 5.5 nm). The rest of the particlessidered as those being at a higher oligomer-vel. Comparing Figs. 1C and 2, one can seeydrophilic, negatively charged mica surfacedissociation of 2B4 oligomers to the less ag-particles. The dissociation of oligomers mayned by the fact that electrostatic interactionsthe charged groups of 2B4 molecules and
mica support are more intensive than the in-ular interactions between 2B4 molecules inomeric complexes. On HOPG, the hydropho-ctions between 2B4 molecules and the hydro-rface of the matrix are insufficient for disso-
f the oligomeric complex to occur.ages of Fp monomers on HOPG are presentedA. The space-separated objects are clearly
e imaged Fp molecules are 45 nm high andm in diameter. It is known that sizes of Fpits hydrophobic tail are 5 nm deep 3 7 nmnm high (20). Indeed, the 4- to 5-nm height ofcles observed agrees well with the expected
onomeric Fp. The images of Fp monomersbtainable on mica because of poor adsorptionhobic molecules on the mica surface. Imageseric Fp on HOPG are represented in Fig. 3B.s cohere with each other forming a two-di-l network. Clearly, oligomeric images shownB differ greatly from monomeric Fp images. The average height of Fp oligomer imagest 78 nm, and their diameter was 4060 nm.Eq. , where the average hm 5 4.5 nm for Fps and the average ho 5 7.5 nm for Fp oli-e suggest that each oligomer consists of five
s. In contrast to monomers, high affinity of Fp
and Surface Concentrations of 2B4 Monomersand Oligomers on Mica Surface
53 1518 100 Monomer56 2225 35 8-mer89 2237 10 12- to 30-mer
215 5060 0.5Aggregates of higher
oligomertion betwFp monomonomerand 202of Fp olFpmicaFp molec2B4 oligoexplained
FIG. 3. AmM Fp in 1were 0.8 3
5PROTEIN COMPLEX FORMATION IN THE MONOOXYGENASE SYSTEMs to mica support was registered. The interac-een the negatively charged mica support andmers in oligomer was so strong that only Fps were seen, most of them being 45 nm high2 nm in diameter (Fig. 3C). The dissociation
igomers on mica suggests that electrostaticinteractions surpass the attraction betweenules in oligomers. The dissociation of Fp andmers to less aggregated particles is probablyby mica-induced stripping of monomers from
the agmica s
FM images of Fp monomers (A) and oligomers (B) on HOPG and Fp00 mM KP buffer, pH 7.4, with 0.25 g/liter Emulgen 913 (A) and 0.20.8 mm (A), 1.1 3 1.1 mm (B), and 0.5 3 0.5 mm (C).gates with their subsequent spreading on theface.ieve that impossibility for 2B4 and Fp mono-ds...