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Structure

Ways & Means

A Fluorescence-Detection Size-ExclusionChromatography-Based Thermostability Assayfor Membrane Protein Precrystallization ScreeningMotoyuki Hattori,1,3 Ryan E. Hibbs,1,3 and Eric Gouaux1,2,*1Vollum Institute2Howard Hughes Medical Institute

Oregon Health and Science University, Portland, OR 97239, USA3These authors contributed equally to this work

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.str.2012.06.009

SUMMARY

Optimization of membrane protein stability underdifferent solution conditions is essential for obtainingcrystals that diffract to high resolution. Traditionalmethods that evaluate protein stability require largeamounts of material and are, therefore, ill suited formedium- to high-throughput screening of membraneproteins. Here we present a rapid and efficientfluorescence-detection size-exclusion chromatog-raphy-based thermostability assay (FSEC-TS). Inthis method, the target protein is fused to GFP.Heated protein samples, treated with a panel of addi-tives, are then analyzed by FSEC. FSEC-TS allowsone to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under avariety of conditions without purification. We appliedthis method to the Danio rerio P2X4 receptor andCaenorhabditis elegans GluCl to screen ligands,ions, and lipids, including newly designed choles-terol derivatives. In the case of GluCl, the screeningresults were used to obtain crystals of the receptorin the presence of lipids.

INTRODUCTION

Growth of membrane protein crystals for the purpose of deter-

mining the underlying atomic structure traditionally requires

a labor-intensive screening approach to identify conditions

that stabilize the protein of interest. This ‘‘precrystallization’’

screening is usually performed with purified protein, which pla-

ces a requirement for large amounts of purematerial and, neces-

sarily, the ability to purify the protein of interest. Membrane

proteins, particularly those from eukaryotes, are typically ex-

pressed at low levels and are often unstable during and after

purification. The challenge of eukaryotic membrane protein crys-

tallography bears itself out in the Protein Data Bank, where <1%

of the deposited structures are from this elusive category. The

degree to which one can identify, in advance of protein purifica-

tion, conditions and compounds that stabilize a given protein

candidate, the higher the likelihood that protein purification will

Structure 20, 1293

proceed smoothly and that well-diffracting crystals will be

obtained. Atomic structures of membrane proteins are of

tremendous interest from both basic science and drug develop-

ment perspectives; relatively little structural information exists

for this class of proteins, molecular mechanisms of signal trans-

duction across cell membranes are not well understood, and

drug design for targets of many currently prescribed therapeu-

tics (Zambrowicz and Sands, 2003) is rendered relatively blind.

One useful technique for precrystallization screening of mem-

brane proteins is fluorescence-detection size-exclusion chro-

matography (FSEC) (Kawate and Gouaux, 2006). In this method,

the target protein is expressed as a GFP fusion, and the SEC

profile of the resulting fusion protein is monitored by fluores-

cence spectroscopy. This simple method allows for rapid evalu-

ation of protein expression level andmonodispersity of the target

protein using nanograms to micrograms of unpurified material

fromwhole-cell extracts. This method has proved to be a power-

ful tool for screening panels of membrane protein orthologs,

detergents, and conformationally sensitive antibodies, and

thus has led to the recent determination of a number of mem-

brane protein structures from this laboratory, including LeuT,

ApcT, ASIC, P2X, GluA2, and GluCl (Hattori and Gouaux, 2012;

Hibbs and Gouaux, 2011; Jasti et al., 2007; Kawate et al.,

2009; Krishnamurthy and Gouaux, 2012; Shaffer et al., 2009;

Sobolevsky et al., 2009; Yamashita et al., 2005).

There are several methods to evaluate protein stability that

have already been described, but these techniques often require

large amounts of pure protein and, therefore, are unsuitable for

high-throughput screening of eukaryotic membrane proteins.

Fluorescent dye-based thermal stability assays using a thiol-

reactive fluorophore N-[4-(7-diethylamino-4-methyl-3-coumar-

inyl)phenyl]maleimide (CPM) (Alexandrov et al., 2008) have

been developed to evaluate the thermostability of membrane

proteins in a high-throughput manner. Although the method

requires only microgram samples of protein, because of the

high sensitivity of the CPM dye, it requires free cysteine residues

embedded in the protein core. A thermostability assay that relies

upon analytical size-exclusion chromatography was used effec-

tively to screen compounds and conditions for stabilizing

membrane proteins for crystallization (Czyzewski and Wang,

2012; Mancusso et al., 2011), but this latter method requires

a relatively large amount of purified sample because of the low

sensitivity of UV absorbance.

–1299, August 8, 2012 ª2012 Elsevier Ltd All rights reserved 1293

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Fluorometer

SECcolumn

Proteinsample

Thermalcycler

Fractioncollector

Fluorescence

Void

Oligomer

Protomer

Figure 1. Flow Chart of FSEC-TS

The protein sample, after heat treatment and centrifugation, is loaded onto

a SEC column, connected to a fluorescence detector to monitor GFP or

tryptophan fluorescence. The panel labeled ‘‘Fluorescence’’ shows a hypo-

thetical elution profile from GFP or tryptophan fluorescence.

Structure

FSEC-Based Thermostability Assay

In the current study, we present a FSEC-based thermosta-

bility assay (FSEC-TS). In this method, nanogram-to-microgram

quantities of purified or unpurified proteins are incubated over

a range of temperatures and then are applied to a SEC column

in line with a fluorescence detector to monitor GFP or tryptophan

fluorescence. The results from FSEC-TS provide a denaturation

or ‘‘melting’’ temperature (Tm), which is used as a reference point

to test the degree of protein thermostabilization by a panel of

small molecules. We applied this simple and rapid method to

the D. rerio P2X4 receptor and the C. elegans GluCl channel to

screen ligands, divalent cations, and lipids, including new

synthetic cholesterol derivatives. We find that thermostabiliza-

tion of both proteins by different compounds is not qualitatively

altered by the presence of GFP and that, in proof-of-principle

experiments, Tm by FSEC-TS agrees well with Tm determined

by a radioligand binding assay. The results are useful in identi-

fying compounds for stabilizing the proteins during purification

and in crystallization and, in the case of GluCl, have been used

to obtain a distinct, lipid-dependent crystal form.

RESULTS

Validation of FSEC-TSTo validate our assay, we used three proteins: EGFP, D. rerio

P2X4 (zfP2X4) receptor, and C. elegans glutamate-gated

chloride channel a (GluCl). Protein samples were incubated

over a range of temperatures for 10 min using a thermal cycler

(Figure 1), followed by ultracentrifugation to remove precipitated

material. The supernatant was then applied to a SEC column

attached to a fluorescence detector to monitor GFP or trypto-

1294 Structure 20, 1293–1299, August 8, 2012 ª2012 Elsevier Ltd Al

phan fluorescence (Figure 1). In these experiments, we used

detergents that we knew from previous experience would stabi-

lize the native oligomeric state of the receptors.

As shown in Figure 2A, peak profiles from EGFP samples

incubated between 4�C and 65�C were sharp and symmetrical,

indicating that the protein remained monodisperse. In contrast,

a clear thermal shift of the peak profiles was observed from the

samples incubated at temperatures near to and above 70�C(Figure 2A). Aggregation and precipitation after heating resulted

in a significant decrease in the peak height, and a melting curve,

from the fluorescence signal intensity at the respective temper-

atures, was observed (Figure 2B). The calculated Tm is 76�C,which is consistent with the reported melting temperature for

GFP (76�C), the temperature where one half of the intrinsic

fluorescence is lost (Bokman and Ward, 1981; Ward, 1981).

P2X receptors are nonselective cation channels gated by

extracellular ATP and are implicated in diverse physiological

processes, such as synaptic transmission, inflammation, and

taste and pain sensing (Jarvis and Khakh, 2009; North, 2002;

Surprenant and North, 2009). Seven P2X receptor subtypes

assembled into either homomeric or heteromeric trimers (Jarvis

and Khakh, 2009; North, 2002). Recently, our group reported the

crystal structure of zfP2X4 in an apo, closed-channel conforma-

tion (Kawate et al., 2009). Precrystallization screening of P2X

orthologs by FSEC had identified zfP2X4 as a promising target

for the crystallization trials.

The well-behaved, crystallized construct of an N-terminal

EGFP fusion to zebrafish P2X4 (DP2X4-A) (Kawate et al., 2009)

was expressed with an octa-histidine affinity tag and purified

for the assay (see Figure S1A available online). FSEC peak

profiles from the EGFP-tagged DP2X4-A samples incubated

between 4�C and 40�C were sharp and symmetrical (Figure 2C);

these peaks correspond to the functional P2X receptor trimer. A

clear thermal shift of the peak profiles was observed from the

samples incubated around 50�C (Figure 2C). Protein denatur-

ation led to a significant decrease in the trimer peak height and

concomitant increase in the putative monomer peak height. A

clear melting curve from the trimer peak height at the respective

temperatures was observed (Figure 2D). The calculated Tm is

49�C, on the basis of the trimer peak height.

To verify that the result from FSEC-TS is relevant to binding-

competent receptor, we measured the ATP binding activity of

the heat-treated samples (Figure S1B). The experiment was

performed at 100 nM ATP concentration, approximately 5-fold

above the Kd value of 19.6 ± 3.8 nM (Figure S1C). The calculated

Tm is 47.8�C ± 0.3�C, consistent with FSEC-TS (49�C). To assess

the effect of theGFP-tag on Tm, we also obtained amelting curve

from EGFP-free DP2X4-A samples by monitoring tryptophan

fluorescence from the endogenous Trp residues in DP2X4-A

(Figure 2D); the estimated Tm is 50�C, not significantly different

from the Tm for EGFP-tagged DP2X4-A (49�C).GluCl is a pentameric chloride channel gated by glutamate

and ivermectin (Cully et al., 1994) and is a member of the Cys-

loop receptor superfamily (Thompson et al., 2010). This receptor

family also includes the ion channels activated by acetylcholine,

serotonin, g-aminobutyric acid, and glycine. GluCl was identified

as a promising target for crystallization by FSEC screening of

Cys-loop receptor orthologs, which subsequently led to the

determination of the structure of a GluCl-Fab complex with the

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Figure 2. Thermostability Profiles of zfP2X4 and GluCl

(A, C, and E) Representative FSEC profiles from EGFP (A), EGFP-DP2X4-A (C), and GluClcryst (E), heated at the respective temperatures, detected by GFP

fluorescence (A and C) or by Trp fluorescence (E).

(B, D, and F) Representativemelting curves of EGFP (B), EGFP-tagged and EGFP-freeDP2X4-A (D), andGluClcryst andGluCl-EGFP (F). Melting temperatures (Tm)

were determined by fitting the curves to a sigmoidal dose-response equation. The fitted curves are shown as a black line.

See also Figure S1.

Structure


allosteric agonist ivermectin bound and the channel in an open

conformation (Hibbs and Gouaux, 2011).

We carried out FSEC-TS with GluCl to test the validity of the

assay and to identify compounds for stabilizing the receptor in

a resting, closed channel state. The crystallized construct of

EGFP-free GluCl (GluClcryst) was purified (Figure S1D) and incu-

bated at 4–80�C and applied to a SEC column, with the eluted

material monitored by tryptophan fluorescence (Figure 2E). We

used the TSKgel SuperSW2000 column for GluCl, which requires

only 15 min per sample, to test many different lipid combinations

in a high-throughput manner, as described later. A melting curve

based on the peak height of the pentameric species was

observed (Figure 2F), with a calculated Tm of a remarkably high

Structure 20, 1293

59�C. Determination of Tm based on a radioligand binding exper-

iment using 3H-ivermectin yields a similar melting temperature

of 58�C (Figure S1E).

The FSEC-TS results from EGFP, zfP2X4, and GluCl demon-

strate that a combination of heat treatment and FSEC can be

applied to monitor the temperature-induced unfolding of soluble

and membrane proteins by detecting GFP fluorescence from

GFP-tagged proteins or by measuring tryptophan fluorescence

from endogenous Trp residues.

Evaluation of Ligands for Cocrystallization by FSEC-TSTo test whether FSEC-TS can be used to estimate the effect of

ligands on membrane proteins for crystallization, we evaluated


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Figure 3. Effects of Additives on the Thermostability of zfP2X4

(A) The normalized peak heights from EGFP-fusion DP2X4-A, heat-treated at

50�C for 10 min, in the presence of the respective additives. The peak heights

were normalized to that from the sample at 4�C without any additive.

(B–D) Chemical structures of novel cholesterol derivatives, hexaethyleneglycol

hydroxyethyl cholesterol (B) maltoside hydroxyethyl cholesterol (C), and

phosphocholine hydroxyethyl cholesterol (D), employed in the FSEC-TS

studies are shown. Structures were created using the ChemDraw (Cambridge

Scientific Computing).

See also Figure S2 and Table S1.

Structure


the thermostabilizing effects of ATP on zfP2X4 and of ivermectin

on GluClcryst. Both ligands bind to their cognate receptors with

high affinity, and, thus, we can use them to examine whether

we can pick up stabilizing effects of tightly bound ligands. In

FSEC-TS, both ATP and ivermectin had dramatic stabilizing

effects, increasing the calculated Tm by 21�C for DP2X4-A and

by 22�C for GluCl (Figures 2D and 2F, respectively). In accor-

dance with these results, crystallization of zfP2X4 with ATP

yields crystals that diffract to 2.8 A resolution (Hattori and

Gouaux, 2012), and crystals of the GluCl-ivermectin complex

diffract to 3.3 A resolution (Hibbs and Gouaux, 2011).

FSEC-TS with Unpurified ProteinOne of the advantages of FSEC using GFP-tagged protein is that

unpurified protein can be analyzed because of the wide spectral

separation between GFP fluorescence and the fluorescence

intrinsic to most proteins. To test whether unpurified protein

can be analyzed using FSEC-TS, EGFP-tagged DP2X4-A and

GluCl obtained from whole-cell lysates were heat-treated in the

presence and absence of ATP and ivermectin, respectively,

and were applied to a SEC column after centrifugation.


For EGFP-DP2X4-A, the calculated Tm values in the absence

and presence of ATP are 52�C and 69�C, respectively (Fig-

ure 2D), consistent with the Tm values from the purified EGFP-

fusion DP2X4-A, which are 49�C in the absence of ATP and

70�C in the presence of ATP.

For GluCl-EGFP, the apparent Tm values in the absence and

presence of ivermectin are 41�C and 53�C (Figure 2F), respec-

tively. These values are significantly different from the apparent

Tm values from the purified GluClcryst (58�C in the absence of

ivermectin and 81�C in the presence of ivermectin). There are

two likely sources of the difference in Tm between GluCl-EGFP

andGluClcryst. First, unlike in zfP2X4, wherein EGFP is positioned

at the receptor terminus, in GluCl, the EGFP is inserted into an

internal loop, between the third and fourth transmembrane

helices. We suggest that insertion of GFP into this loop destabi-

lizes the protein. Second, GluCl-EGFP contains the full-length,

wild-type sequence of the GluCl gene, whereas GluClcryst is

a truncated construct used for crystallization. Regardless of

the differences in Tm, however, the qualitative effect of iver-

mectin on GluCl thermostability remains unchanged. Taken

together, we conclude that FSEC-TS can be used to analyze

unpurified GFP-tagged protein.

Effect of Divalent Cations on zfP2X4The effects of three different divalent cations (Ca2+, Mg2+, and

Zn2+) on zfP2X4 thermostability were tested by FSEC-TS (Fig-

ure 3A). EGFP-tagged DP2X4-A samples in a series of buffers

containing the respective additives were heat-treated at 50�Cfor 10 min, which is close to the calculated Tm of apo protein.

As in the case for other P2X receptors, ATP-responses at P2X4

receptors can be modulated by divalent cations, such as Zn2+

(Garcia-Guzman et al., 1997; Wildman et al., 1999) and Mg2+

ions (Negulyaev and Markwardt, 2000). The significant divalent

cation-induced stabilization of the trimer peak is observed in

the presence of all tested divalent cation (Figure 3A). Consis-

tently, best crystals of zfP2X4 in an apo, closed state (Kawate

et al., 2009), as well as crystals in the presence of ATP (Hattori

and Gouaux, 2012) were obtained in presence of Mg2+.

Effect of Ligands on zfP2X4The effects of different P2X ligands, 20,30-O-(2,4,6-trinitrophenyl)

adenosine50-triphosphate (TNP-ATP) (antagonist) andpyridoxal-

phosphate-6-azophenyl-20,4’-disulphonic acid (PPADS) (antag-

onist) on the thermostability of GFP-fusion DP2X4-A were tested

by FSEC-TS (Figure 3A). TNP-ATP stabilized the zfP2X4 trimer to

a similarly strong extent as was observed with ATP (Figure 3A).

PPADS, by contrast, destabilized theprotein (Figure 3A). In corre-

lation to the effects of ligands on thermostability, we have

successfully crystallized zfP2X4 in the presence of TNP-ATP

but have not been able to produce crystals with PPADS.

Effect of Lipids on zfP2X4Purification of membrane proteins from their native environment

requires the use of detergents. However, detergent solubilization

of membrane proteins often leads to aggregation, because

membrane proteins tend to be less stable in detergent micelles

than in the lipid bilayer. To remedy this behavior, the addition

of lipids during purification or crystallization can be used to

improve membrane protein behavior for the purposes of

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Figure 4. Effects of Lipids on the Thermostability of GluCl

The normalized peak heights fromGluClcryst heat-treated at 57�C for 10min, in

the presence of the respective lipids. The peak heights were normalized to that

from the sample at 4�C without any additive. See also Figure S3 and Table S2.

Structure


biophysical characterization, crystallization, and structure deter-

mination (Guan et al., 2006; Hattori et al., 2007; Hibbs and

Gouaux, 2011; Krishnamurthy and Gouaux, 2012; Lemieux

et al., 2003; Sobolevsky et al., 2009; Zhang et al., 2003).

The effects of ten different lipids on the thermostability of

GFP-fusion DP2X4-A were examined by FSEC-TS (Figure 3A)

and also by ATP binding assay (Figure S2). Among the tested

lipids, phosphocholine hydroxyethyl cholesterol (CH-PC), malto-

side hydroxyethyl cholesterol (CH-maltoside), and a hexaethyle-

neglycol hydroxyethyl cholesterol (CH-HEG) are newly designed

cholesterol derivatives (Figures 3B–3D). Hydroxyl groups of

cholesterol are connected to phosphocholine (CH-PC), maltose

(CH-maltoside), or hexaethyleneglycol (CH-HEG) via a hydrox-

yethyl group. Cholesteryl hemisuccinate (CHS), as well as

cholesterol, have been successfully used for purification and

crystallization of GPCRs (Cherezov et al., 2007; Rasmussen

et al., 2011; Rosenbaum et al., 2011; Soriano et al., 2009).

However, the ester group of CHS can be hydrolysis sensitive,

and cholesterol itself is sparingly soluble. Our cholesterol deriv-

atives are more stable because their head groups are connected

to hydroxyl groups of cholesterol via a hydroxyethyl group. Addi-

tionally, the new head groups of the derivativesmight have stabi-

lizing effects on membrane proteins. Therefore, we tested these

cholesterol derivatives as well as other lipids by FSEC-TS.

Surprisingly, most of the lipids except for the phosphoinosi-

tide, PI(4)P, significantly stabilized the trimer peak of zfP2X4 in

FSEC (Figure 3A), which is largely consistent with thermostabili-

zation observed by the same compounds in a radioligand

binding assay (Figure S2). Among phosphocholine-containing

lipids, the lipids with longer alkyl chains more effectively stabi-

lized the trimer peak (Figure 3A). We also tested one of the phos-

phoglycerol-containing lipids, POPE (Figure 3A). Although the

chain length of POPE is the same as that of POPC, POPC stabi-

lized the trimer peak of zfP2X4 more effectively than POPE did.

Among cholesterol derivatives, CH-HEG was the poorest

additive in terms of both FSEC-TS and the radioligand binding

assay (Figure 3A and Figure S2). On the basis of FSEC-TS,

CHS was slightly better than CH-PC and CH-maltoside, but on

the basis of the binding assay, CH-PC had the strongest stabi-

lizing effect among the tested lipids (Figure 3A and Figure S2).

We also tested monoolein (MO), which has been used for

membrane protein crystallization in the lipidic cubic phase

(Johansson et al., 2009). MO also stabilized the trimer peak of

zfP2X4 by FSEC-TS but was not better than other ‘‘good’’ lipids

such as POPC (Figure 3A).

Among all tested lipids, only PI(4)P totally destabilized the

trimer peak of zfP2X4 by FSEC-TS (Figure 3A), and consistently

in the presence of this lipid the receptor showed almost no ATP

binding activity (Figure S2). Interestingly, phosphoinositides are

known to regulate the activity of P2X4 receptors through direct

interactions (Bernier et al., 2008).

Effect of Lipids on GluClWe previously found that either POPC or DPPC was required for

growth of well-diffracting crystals of the GluCl-Fab-ivermectin

complex (Hibbs and Gouaux, 2011). To screen lipids for

a more stabilizing effect on GluCl, we tested 24 different

lipid combinations using FSEC-TS with pure protein (Figure 4).

GluClcryst samples in a series of buffers containing the respective

Structure 20, 1293

lipids were heat-treated for 10 min at 57�C, which is close to the

calculated Tm of apo protein. Most lipids had some significant

stabilizing effect on GluCl, whereas total soy lipid, brain ceram-

ide, and DMPE did not stabilize GluCl (Figure 4). Among tested

lipids, sphingomyelin and PO-containing lipids consistently had

strong stabilizing effects, and the addition of CHS often further

increased thermostability. The lipids identified in this experiment

have been fed into crystallization trials of GluClcryst with the goal

of obtaining well-diffracting crystals in an ivermectin free condi-

tion, and a new, to our knowledge lipid-dependent crystal form

of GluClcryst has been obtained (Figure S3A). These crystals

are from theC2 space group, currently diffract X-rays to between

3 and 4 A (Figure S3B), and are a promising lead in obtaining the

structure of a closed-channel conformation of a eukaryotic

Cys-loop receptor.

ConclusionsIn this study, we applied FSEC-TS to zfP2X4 and GluCl. This

method carries over the advantages of conventional FSEC: it

requires only nanogram-to-microgram quantities of protein and

allows for screening of different stabilizing conditions in an

efficient manner with both purified and unpurified protein. Using

this method, we screened various conditions, such as ligands

and lipids, to stabilize zfP2X4 and GluCl. Ligand screening

demonstrated that ATP, a biological agonist of P2X, and its

synthetic analog, TNP-ATP, dramatically stabilized zfP2X4,

suggesting that these are suitable ligands for cocrystallization.

Lipid screening showed that our designed cholesterol deriva-

tives and many other lipids significantly stabilized zfP2X4 or

GluCl (Figures 3 and 4). Employing the ligands and lipids identi-

fied in this study has led to a distinct well-diffracting crystal

form of GluCl.

EXPERIMENTAL PROCEDURES

Expression and Purification

The crystallized construct of zfP2X4 (DP2X4-A) was expressed as an

N-terminal EGFP fusion and was purified as described previously (Kawate


Structure


et al., 2009). The EGFP-tagged and EGFP-free purified proteins were concen-

trated and dialyzed overnight at 4�C against three changes of buffer I (20 mM

HEPES-NaOH [pH 7.0], 80mMNaCl, 20mMKCl, and 2mMcymal-6), and then

were stored at �80�C before use. The crystallized construct of C. elegans

GluCl (GluClcryst)) with a C-terminal 8x-His tag was expressed in Sf9 cells

and purified as described previously (Hibbs and Gouaux, 2011), omitting the

addition of Fab before the SEC purification step.

For GluCl experiments in crude cell extracts, a fusion of GluCl with EGFP

was made with GFP inserted into the full-length, wild-type C. elegans GluCla

receptor between Asn392 and Ser393 in the mature protein sequence. Sf9

cells at a density of 3–4 3 106 cells per ml were infected with baculovirus en-

coding GluCl-EGFP and were harvested 72 hr later by centrifugation; cell

pellets were stored at �80�C.

Radioligand Saturation Binding Assay for zfP2X4

Measurements of total ATP binding was obtained by adding GFP-fusion

DP2X4-A to a final concentration of 30 nM in 250 ml buffer I containing

0–1000 nM radiolabeled ATP where the hot ATP was diluted with cold ATP

in a ratio of 1:4, yielding a final specific activity of 6 Ci/mmol. Reactions

were incubated at 4�C overnight and then were terminated by filtering through

GSWP 02500 nitrocellulose membranes pre-equilibrated with buffer I contain-

ing 100 mM cold ATP. Filters were washed three times with 2 ml of buffer I,

placed in 6 ml of Ultima Gold scintillation cocktail, and counted after 1 hr. Esti-

mates of nonspecific binding were obtained by reactions in the presence of

100 mM cold ATP. The experiment was performed in triplicate, and the data

were fit to a rectangular hyperbola using the GraphPad Prism4 program.

FSEC-TS with Purified Proteins

For EGFP, 500 ng of EGFP in 50 ml of buffer II (20 mM Tris-HCl [pH 8.0]

and 150 mM NaCl) were incubated at 4�–90� for 10 min, using a thermal

cycler and then were centrifuged at 87,000 3 g for 20 min. A fraction of the

supernatant (25 ml) was loaded onto a SEC column (Superose 6 10/300 GL,

GE Healthcare) pre-equilibrated with buffer II and run at a flow rate of

0.5 ml/min.

For zfP2X4, 1 mg of GFP-fusion andGFP-freeDP2X4-A in 50 ml of buffer I was

incubated at 4–80�C and 4–70�C for 10min and was centrifuged at 87,0003 g

for 20 min. Twenty-five microliters of the supernatant were loaded onto a SEC

column (Superose 6 10/300 GL) pre-equilibratedwith buffer III (20mMHEPES-

NaOH [pH 7.0], 80 mMNaCl, 20 mM KCl, and 1 mM n-dodecyl b-D-maltoside,

C12M) and run at a flow rate of 0.5 ml/min.

For GluCl, 2 mg of GFP-free GluClcryst in 100 ml of buffer IV (20 mM Tris-

HCl [pH 7.4], 150 mM NaCl, and 2 mM C12M) were incubated at 4–80�Cfor 10 min, and centrifuged as described above. Twenty-five microliters

of the supernatant were loaded onto a TSKgel SuperSW2000 column

(TOSOH Bioscience), pre-equilibrated with buffer IV, and run at a flow rate of

0.35 ml/min.

The eluent from the SEC columnwas passed through a fluorometer (Kawate

and Gouaux, 2006) (lexc of 480 nm and lem of 510 nm for GFP fluorescence;

lexc of 280 nm and lem of 325 nm for tryptophan fluorescence). The peak

heights from EGFP (Figure 2B) or oligomers (Figure 2D and 2F) were normal-

ized to that from samples incubated at 4�C, and were fit to the Hill equation

using the GraphPad Prism 4 program. Melting temperatures (Tm) were deter-

mined by fitting the curves to a sigmoidal dose-response equation.

Gaussian fitting was performed using PeakFit software (SeaSolve Software,

Inc) as described previously (Kawate and Gouaux, 2006). Initial peak detection

and fitting were performed with the AutoFit peaks I Residuals option. The

obtained Gaussian functions were further refined and fitted to the original

FSEC profile using a least-squares minimization procedure (Figure S1F). The

obtained melting curves based on the peak height are consistent with the

melting curves based on the peak area of EGFP (Figure S1G), zfP2X4 trimer

(Figure S1H) or GluCl pentamer (Figure S1I) estimated by Gaussian fitting.

To test ATP and ivermectin effects, GFP-fusion DP2X4-A in the presence of

1 mM ATP and EGFP-free GluClcryst in the presence of 10 mM ivermectin were

incubated at 4�C for 1 hr and 30 min before heat treatment, respectively.

FSEC-TS with Whole-Cell Lysates

The GFP-fusion of DP2X4-A was expressed in Sf9 insect cells as described

elsewhere (Kawate et al., 2009), and the harvested cells were stored


at �80�C before use. Sf9 cells expressing GFP-fusion DP2X4-A from 7.5 ml

culture were resuspended in 1 ml of buffer V (50 mM Tris-HCl [pH 8.0] and

150 mM NaCl) containing 40 mM C12M in the presence and absence of

1 mM ATP, rotated at 4�C for 1 hr, and then centrifuged at 87,000 3 g for

40 min. One hundred microliters of the supernatant was dispensed into thin-

walled PCR tubes and was incubated at the respective temperatures for

10 min, using a thermal cycler. After centrifuge at 87,000 3 g for 20 min,

50 ml of the supernatant was applied to a Superose 6 column, pre-equilibrated

with buffer V containing 1 mM C12M.

Sf9 cell pellets from 30ml culture containing GluCl-EGFP were resuspended

in 3ml of buffer IV containing 40mMC12M, rotated at 4�C for 40min, and then

centrifuged at 87,000 3 g for 40 min. The supernatant was divided into two

tubes, and ivermectin (100 mM final concentration from a 10 mM stock in

DMSO) was added to one of the tubes, and was rotated at 4�C for 10 min.

One hundred-microliter aliquots of the supernatant were dispensed into thin-

walled PCR tubes and were incubated at the respective temperatures for

10 min using a thermal cycler. After centrifugation at 87,000 3 g for 20 min,

20 ml of the supernatant was applied to a TSKgel SuperSW2000 column,

pre-equilibrated with buffer IV.

The eluent from the SEC column was passed through a fluorometer (Kawate

and Gouaux, 2006) (excitation, 480 nm; emission, 510 nm for GFP fluores-

cence). Data were normalized and fit to the Hill equation, as described above.

FSEC-TS Screening of Additives

To determine the effect of lipids on the stability of zfP2X4, 5 mg of GFP-fusion

DP2X4-A in 250 ml of buffer I were rotated at 4�C for 1 hr after addition of 2.5 ml

of a 1003 lipid suspension (20% DMSO and 80% buffer I). The mixture was

next incubated at 50�C for 10min and then centrifuged. Twenty-fivemicroliters

of the sample was used for FSEC as described above. The rest of the sample

was stored at 4�C for use in binding assays; 1003 additive solutions were

added to GFP-fusion DP2X4-A at the respective final concentration shown

in Table S1.

For GluCl, 850 ng of GluClcryst in 10 ml of buffer IV were rotated at 4�C for

40 min after the addition of 90 ml of lipid stock solution in buffer IV containing

40 mM C12M. The mixture was next incubated at 59�C for 10 min and then

centrifuged. A 25 ml aliquot of the sample was used for FSEC as described

above. Lipids were added to GluClcryst at the respective final concentrations

as shown in Table S2.

Binding Assay of Heat-Treated DP2X4-A

These experiments were performed as described above except that the

binding was initiated by adding heat-treated GFP-fusion DP2X4-A to a

final concentration of 30 nM in 250 ml of buffer I containing 100 nM ATP

(1:4 3H:1H; 6 Ci/mmol final specific activity). The entire experiment was

performed in triplicate.

Binding Assay of Heat-Treated GluClcrystBinding of 3H-ivermectin to GluClcryst after heat treatment was performed

using a scintillation proximity assay (SPA). A solution of pure GluClcryst was

made in buffer IV at a subunit concentration of 200 nM, and 70 ml were aliquot-

ted into thin-walled PCR tubes and incubated for 10 min at temperatures

ranging from 4�C to 80�C. A suspension was made containing 2 mg/ml

SPA beads (YSi copper his tag, GE Healthcare) and 100 nM ivermectin

(1:9 3H:1H; 5 Ci/mmol final specific activity; 3H-ivermectin purchased from

American Radiolabeled Chemicals and 1H-ivermectin purchased from Sigma)

in buffer IV. The SPA suspension was mixed 1:1 (50 ml to 50 ml) with the protein

solution by pipetting in a 96-well microtiter plate, and the cpm data were

collected after 18 hr of incubation at room temperature.

SUPPLEMENTAL INFORMATION

Supplemental Information includes three figures and two tables and can be

found with this article online at http://dx.doi.org/10.1016/j.str.2012.06.009.

ACKNOWLEDGMENTS

We thank A. Penmatsa for providing the lipid screening kit for GluCl, J. Michel

for technical help, and L. Vaskalis for assistance with figures. This work was

l rights reserved


Structure


supported by the Uehara Memorial Foundation (M.H.), the American Asth

Foundation (E.G.), and the National Institutes of Health (R.H. and E.G.). E.G.

is an investigator with the Howard Hughes Medical Institute.

Received: April 6, 2012

Revised: June 7, 2012

Accepted: June 13, 2012

Published: August 7, 2012

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