Supported phospholipid bilayers for two-dimensional protein crystallization

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 81 9-826

January 29, 1986

SUPPORTED PHOSPHOLIPID BILAYERS FOR TWO-DIMENSIONAL PROTEIN CRYSTALLIZATION E. E. U z g i r i s G e n e r a l E l e c t r i c R e s e a r c h and D e v e l o p m e n t C e n t e r S c h e n e c t a d y , N.Y. 12301

Received December 6, 1985

Phospholipid bilayers, s u p p o r t e d on UV i r r a d i a t e d carbon shadowed n i t r o - cellulose e l e c t r o n m i c r o s c o p e g r i d s , have been used to induce t w o - d i m e n s i o n a l c r y s t a l g r o w t h o f IgE and IgG a n t i - D N P m o n o c l o n a l a n t i b o d i e s . The UV i r r a d i a tion renders the grids hydrophilic i n a v e r y u n i f o r m f a s h i o n and a l l o w s f o r the transfer o f p h o s p h o l i p i d m o n o l a y e r s from an a i r / w a t e r interface in a sequential dipping procedure. The s u r f a c e c o v e r a g e a c h i e v e d was n e a r l y 100~ a s m e a s u r e d by a n t i b o d y b i n d i n g and by t h e f o r m a t i o n o f p r o t e i n a r r a y s on t h e bilayer covered grids. The s u p p o r t e d b i l a y e r s a p p e a r t o be s t a b l y h e l d and a r e a p p r o p r i a t e f o r slow b i n d i n g c o n d i t i o n s a n d l o n g i n c u b a t i o n t i m e s w i t h low concentrations of binding protein. © 1986 ~ademic Press, Inc.

Under a p p r o p r i a t e dimensional (1).

arrays

Heretofore,

allow

for

either

their

when bound

rearrangement

Blodgett

monolayer

realized

by d i p p i n g

the

a free

"X" t y p e

with just

to

on an (2).

films

that

air/water

interface,

In the

latter

the air/water

formation used.

(3),

films

i.e.

or

prior body

a

bapten

to the withdrawal directed

against

ordered antibody was n o t w i t h o u t to control,

phospolipid

hapten

in

difficulties.

and an e x p o s u r e

and

have been monolayers, as

a Langmuir--

s u p p o r t e d m o n o l a y e r was microscope

The c o n d i t i o n s layer

on

grid

were f o r

insertion

only

into air,

T h i s was v e r i f i e d

by i n t e r -

an

unconjugated

that

occurred

fashion

phospholipid

t h e b i n d i n g o f an a n t i -

(4).

The h y d r o p h o b i c c h a r a c t e r of

fashion

through the interface

with

question

arrays were formed in this

oriented

electron

pickup of

o f t h e g r i d and o b s e r v i n g the

the

interface.

Upon w i t h d r a w a l

conjugated

an

i n t o o r d e r e d two

supported

shadowed,

a s e c o n d m o n o l a y e r was n o t p i c k e d up i n g e n e r a l . changing

in

arrays

instance,

carbon

organize

serve to anchor the proteins

crystalline

monolayer at

one i n s e r t i o n

will

into

a hydrophobic,

of l a y e r

proteins

phospholipid

the phospholipid

unsupported,

through

circumstances,

unimpeded. However,

Highly-

this method

o f t h e g r i d s was h a r d

e v e n one day t o room a t m o s p h e r e was enough

819

to

0006-291X/86 $1.50 Copyright © 1986 by Academic- Press, Inc. All rights of reproduction in any ,form reserved.

Vol. 134, No. 2, 1986

render (1)

the

procedure

were n o t v e r y

stalline large area

arrays

crystal

through

uniform

I

Modifications

a

in the

insertions)

reliable

did not

seemed t o

cry-

preclude

the

surface

binding conditions, surface

area

by t h i s

improved coverage.

(e.8.,

Most

likely

hydrcphobic interactions

used

step.

and much

improved

The r e c o v e r y

of p r o t e i n

arrays

method

hydrophilic

c a n be

close

to

for

achieving

surfaces 100~

of

supported

by UV i r r a d i a t i o n .

the

surface

area

by

approach

is

method. A further

important

that

protein

just

miniscule

array

feature

of a s u p p o r t e d p h c s p h o l i p i d

f o r m a t i o n c a n be i n d u c e d from q u i t e

amounts of protein.

The a l t e r n a t i v e air/water

approach,

interface

used

and

desired.

small

areas

the protein observed

are

arrays in

interface

The

procedure

v o l u m e s and s m a l l arrays,

formation

(6),

but

is

lectin

of

just

arrays

coated grids

can

0.5~g of protein. on f r e e

monolayers at points

the

of view,

and

a n awkward a p p r o a c h when s m a l l v o l u m e s is

supports

of

small volumes requiring

a p p r o a c h from s e v e r a l

There

onto solid

studies

air/water

say e . g . ,

i s an a t t r a c t i v e

i n d e e d was t h e f i r s t

film

For e x a m p l e , p h o s p h o l i p i d

be i n c u b a t e d on 10X d r o p s c o n t a i n i n g ,

tein

yield

hard to control,

f i l m s b a s e d on g e n e r a t i n g

as

total

of

procedure to achieve multilayers,

phospholipid

this

film

patches

was o n l y 20% o f

c o u l d be p u s h e d t o ~50~ o f

monolayer transfer

report

small

in the

arrays

in these experiments

With c a r e and r a p i d p r o t e i n

was due t o t h e u n r e l i a b l e ,

in the first

films

only relatively

R e c o v e r y of p r o t e i n

of a r r a y s

sequential

-

Discontinuities

experiments.

(5).

The p h o s p h o l i p i d

in extent

were formed.

in typical

technique

unreliable.

growth.

the recovery

this

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also

after

binding

the the to

problem of poor arrays

form a t

gsnglioside

recovery

the

of

interface

monolsyers

at

the

(7). described quantities

here of

is

facile,

proteins,

and s h o u l d be a p p l i c a b l e

suitable

for

has a high recovery

t o most p h o s p h o l i p i d

use

with

of s u r f a c e

small pro-

systems.

MATERIALS AND METHODS The p h o s p h o l i p i d s w e r e p u r c h a s e d from A v a n t i P o l a r L i p i d s ampules. The h a p t e n d e r i v i t i z e d phespholipid, DNP-Cap-PE, 820

i n 1 ml s e a l e d dinitrophenyl-

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

caproyl-phosphatidylethanolamine i s d e r i v e d from egg l e c i t h i n , PC, a~ i s PE, phosphatidylethanolamine. The p h o s p h o l i p i d s w e r e s p r e a d on a 20cm teflon t r o u g h by a d d i n g 1 0 k - 2 0 k d r o p s o f t h e l i p i d (~400~g/ml i n c h l o r o f o r m ) on t h e water surface. The p h o s p h o l i p i d f i l m s , upon s a t u r a t i o n o f t h e w a t e r s u r f a c e , r e a c h e d a n e q u i l i b r i u m p r e s s u r e o f 3 5 - 4 0 d y n e / c m f o r DNP-Calr-PE, PE, and PC a s m e a s u r e d on a L a n g m u i r f i l m b a l a n c e ( 8 ) . T h i s was c o n s i d e r e d a d e s i r a b l e p r e s s u r e f o r t r a n s f e r o f f i l m s and f o r p r o t e i n b i n d i n g and f u r t h e r c o m p r e s s i o n was n o t u s e d , nor w e r e more d i l u t e f i l m s i n v e s t i g a t e d . IgE mouse monoclonal anti-DNP antibody was purchased from Miles Laboratories. The IgE was prepared by an affinity purification procedure, which resulted in some contamination by Ig6 and by low molecular weight components. However, no further purification was performed for these experiments. Mouse monoclonal IgG1, anti-DNP was a gift from R. Kornberg and L. Herzenberg and came in pure ~orm. Polyclonal goat snti-DNP IgG was purchased from Gateway Immunosera Inc. This was also purified on an affinity column and contained various DNP binding contaminants. Protein binding and self-organization was assayed by electron microscopy. The g r i d s w e r e w a s h e d o n c e w i t h a d r o p o f w a t e r a f t e r b e i n g l i f t e d from t h e p r o t e i n s o l u t i o n s and t h e n s t a i n e d w i t h 1% u r a n y l a c e t a t e . P r o c e d u r e A: n i t r o c e l l u l o s e coated electron microscope grids were carbon s h a d o w e d i n a staandard way and t h e n i r r a d i a t e d w i t h a s h o r t w a v e l e n g t h UV l i g h t a t ~ 20mW/cm i n a i r ( a n d t h e r e f o r e a l s o o z o n e a t t h e s e i n t e n s i t i e s ) for 2 to 3 minutes. Longer exposures resulted in o v e r h e a t i n g and some f i l m damage. Monolayer transfer from the water surface to the irradiated grids was done in the following way: I) Prior to spreading the phospholipid the grids were immersed, the monolayer was then spread, and the grids were withdrawn s l o w l y a t 900 t o t h e s u r f a c e w i t h a p a i r o f f i n e e l e c t r o n m i c r o s c o p e t w e e z e r s ; 2) t h e g r i d s w e r e a i r d r i e d f o r any d r o p s o f w a t e r t h a t may h a v e b e e n c a r r i e d o u t ( a l t h o u g h t h e y u s u a l l y were q u i t e d r y ) a n d t h e n s l o w l y immersed t h r o u g h t h e m o n o l a y e r a t 900 t o t h e s u r f a c e ; 3) t h e g r i d was t h e n w i t h d r a w n i n t o a i r and a l l o w e d t o d r y b e f o r e f l o a t i n g t h e g r i d w i t h t h e f i l m down, o n t o an a n t i body solut ion. P r o c e d u r e B: t h e c a r b o n c o a t e d g r i d s w e r e n o t i r r a d i a t e d , u s e d on t h e same day a s when t h e c a r b o n s h a d o w i n g was d o n e , and u s e d i n t h e "X" i n s e r t i o n t y p e l a y e r p i c k u p mode d e s c r i b e d a b o v e . , i . e . a s i n g l e i n s e r t i o n f o l l o w e d by a withdrawal into air. Phospholipid transfer was a c h i e v e d a l s o on i n d i u m c o a t e d p l a s t i c and glass slides after UV i r r a d i a t i o n and by p r o c e d u r e A. The f o r m a t i o n o f m o n o m o l e c u l a r l a y e r s c o u l d be v i s u a l l y d e t e c t e d b o t h as t h e p h o s p h o l i p i d monol a y e r s w e r e p i c k e d up a n d s u b s e q u e n t l y a f t e r a n t i b o d i e s bound t o t h e e x p o s e d h a p t e n groups ( 9 ) . RESULTS AND DISCUSSION Phospholipid are after

compared f o r

clustering,

various

solution)

scored

for

f o r m e d by p r o c e d u r e

stability

exposure for

(~250pg/ml then

films

of

against times

fractional

It

is

were

then

IgE or p o l y c l o n a l area

a characteristic

phospholipids.

exposure

evident

showing

A and p r o c e d u r e to b u f f e r floated

in Fig.

IgG f o r 30 m i n u t e s . high

821

1 that

1.

on a h i g h

density

The g r i d s

concentration The g r i d s w e r e

antibody

of antibody binding to mobile, from F i g .

B f o r DNP-Cap-PE

binding

and

hapten derivatized

by p r o c e d u r e B,

even a

short

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t00,

\ \ \ \ co

0

otxl

~_ea

0;

\

,

I ~,

Io,

,

I

TIME, hrs

I

2

Fin. 1. Binding of IgE or p e l y c l o n a l IgG anti-DNP a n t i b o d y to p h o s p h o l i p i d covered g r i d s a f t e r f l o a t i n g on 150 mM NaC1, 50 mbi T r i s s pH 7 . 4 b u f f e r ( a l s o c o n t a i n i n g 50~g/ml human IgG) for v a r i o u s p e r i o d s of time. Grids were l i f t e d o u t and f l o a t e d on same b u f f e r w i t h 250gg/m1 IgE or IgG anti-DNP f o r 3 0 ' t h e n s t a i n e d w i t h 1~ u r a n y l a c e t a t e , o p h o s p h o l i p i d , DNP-CaF-PE, t r a n s f e r r e d by p r o c e d u r e B; p h o s p h o l i p i d , DNP-Catr-PE, t r a n s f e r r e d by p r o c e d u r e A.

exposure

to buffer

can lead

f a c e w h e r e a s by p r o c e d u r e at

least

3

hours

consequence arrays

of

of

the

decreased patchy

when p r o t e i n

binding

however,

the

g r o w t h of l a r g e

is

3

nature slow,

preferred

used in the

arrays

The c o m p o s i t i o n phospholipid haptenated or

the

type

monolayer after

lack

of

any it

Table

I

of

would

of p h o s p h o l i p i d

tion.

of

A.

the

cedure

A on h i g h l y

a

low

as

it

is

a

can

lead

to

grids

of

most

for the

protein

frequently

solution. the

sur-

practice,

recovery

concentration

Relatively

outer

This,

nucleation

and

hydrophilic

the

in

that

procedure

results.

and bilayers surfaces. 822

is The

The

c a n be a s s a y e d from

or

A.

to

supported

c a n be e a s i l y

Fig.

surface close

2

were cover-

t o 100%.

by c h a n g i n g

hap L e n a t e d Binding

absence

exposed

shown i n

concentration

was r o u t i n e l y

interface

presence

layer

are

low p r o t e i n

film

steps the

films

o f many h o u r s .

air/water

three

summerizes

t o be i n d e e d b i l a y e r s

course would occur

phospholipid

indicate

appear

of

from

the

In normal

film

the long incubations

the

as the

to

the

over a period

the at

held

of

g r o w t h on p h o s p h o l i p i d

steps after

stably

from the g r i d

(5).

procedure

incubation

age by p r o t e i n

which as

are

span examined),

condition

2-D c r y s t a l s

following

time

of phospholipid

films

stability

E x a m p l e s o f 2-D c r y s t a l and F i g .

losses

A, t h e l i p i d

(the maxima

a very

is

to large

of

to

the non-

antibody

of one or t h e o t h e r the

antibody

phospholipid

constructed

solufilms

using pro-

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fia. 2. E l e c t r o n m i c r o g r a p h of c r y s t a l s o f mouse m o n o c l o n a l ISG1, grown on DNP-Cap-PE s u p p o r t e d b i l a ~ e r ( p h o s p h o l i p i d t r a n s f e r by p r o c e d u r e ~ ) . Incubat i o n was f o r 18 h o u r s , 23 C, and $0~8/ml ISG.

FiB, 3 . E l e c t r o n m i c r o g r a p h of m u l t i d o m a i n c r y s t a l g r o w t h of IgE on DNP-CapPE b i l a y e r d e p o s i t e d by p r o c e d u r e A. I n c u b a t i o n f o r 12 h o u r s , 23°C, and 50~g/ml IgE.

823

Vol. 134, No. 2, 1 9 8 6

B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

TABLE

I

Binding of Anti-DNP Antibody to Asymmetric Phospbolipid Films

P h o s p h o l i p i d a t Air/Water I n t a r f a c e •

Anti-DNP Antibody

Step 1 DNP--Ca It-PE

Bindinge*

"

Step 2 DNP--Ca F'-~

"

"

Step 3 DNIL CaF=PE

"

" "

PC

+

DNP"Cap - l ~

DNP- Ca p- PE

PC

PC

" "

" "

DNP-Calr-PE

n

H

PC

+

I~

~

##

PC

" "

+

w#

Step 1 i s t h e w i t h d r a w a l of an immersed g r i d t h r o u g h the s p r e a d monolayer into air. Step 2 i s t h e i n s e r t i o n

t h e g r i d t h r o u g h the s p r e a d monolayer i n t o

of

water. Step 3 is the withdrawal

÷

again through the spread monolayer.

S i g n i f i e s h i g h d e n s i t y b i n d i n g of a n t i b o d y as o b s e r v e d i n t h e electronmicrographso

S i g n i f i e s an abaence of any s i g n i f i c a n t p r o t e i n b i n d i n g . The g r i d s i n case are featureless and empty w i t h v e r y l i t t l e u r a n y l acetate staining.

that

In this which

procedure,

entails

the

in the final readily

drives

interface. last

film

some if

picking

right type

the

is

the

a

grid

spread

supported

through

layer,

a withdrawal

off

of

a withdrawal up a n o t h e r

However,

2nd l a y e r

step

step

of

composition.

Therefore,

films

the monolayer,

not at

through

a free

and back onto

monolayer

are

to

be

is

all

desired interface

the water/air

necessary

taken

out

into

on

the

air

for

manipulation.

In the results

last

step

in a film

expected

that

indeed, step

last

possibility

the

withdrawal

further

the

a

a

loss

(see

below).

water:

It

c a n be

dipped

into

assays

of

the

surface

procedure

of w a t e r

third of

of

This

film last

observed

trough, hapten

the

being carried

monolayer

spread

A,

to

out with

would reside area

at

driven

and t h e r e availability

is

no

is

not

off

at

through grid.

of

pressure stable

the

Therefore,

the water is

interface

film,

detected

against

it

I).

of

it

in

exposure

However,

antibody if

is

and,

in this

a low m o n o l a y e r p r e s s u r e

evidence

(see Table

824

the

on t o p

constant

monolayer be

withdrawal

to when

binding

the phospho-

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lipid

coated grids

are not allowed to air

d r y and a r e

tions

while

quite

as well

still

observed to destabilize cedure

A,

it

saline

solutions.

Other coated the

to

plastic

inner

to air

tested

slides,

slides,

the

and f o r m m u l t i l a m e l l e r

important

surfaces

indium

ally

is

wet,

and

indium

prepared glass

Further

insertions

deposition alized

slides,

(9).

phospholipid

a further

of

of

the

layers

in

slides

Subsequent antibody

darkening

slide

before

solu-

films

Therefore,

were

in p r o -

e x p o s i n g them t o

irradiation

Incramental

the

outermost

structures.

UV

on a q u e o u s

include

and n i t r o c e l l u l o s e

monolayers

and w i t h d r a w a l s

of monolayers.

as

by

monomolecular phospholipid

transferred

as t h e

dry the coated grids

by a d a r k e n i n g o f t h e s l i d e s the

placed

indium

films.

With

c o u l d be d e t e c t e d

vlsu-

d a r k e n i n g was d e t e c t e d the

dipping

did not

binding

due

procedure

lead

A.

t o any f u r t h e r

was a l s o

clearly

and c o n f i r m e d t h e e l e c t r o n

visumicro-

scope observations. The t r a n s f e r the at

steps

of

constant

o f m o n o l a y e r s o n t o UV i r r a d i a t e d

procedure pressure,

30

insertion

through

observed

on

ciency.

After a 3'

dyn/cm),

but

slide

upon w i t h d r a w a l

area

at

with the

slide

surface area

quent

s t e p s 2 and 3,

observed within

a

suited

In the

for

insertion

experimental

proven to

after

a deposition

transfer

surface

a

be r e l i a b l e .

and w i t h d r a w a l , error

small

a high routinely.

brief

each

of

film

transfer

was

upon i n s e r t i o n

effi( a t 30

of the monolayer onto the

coefficient,

pressure

in

but with variable

t h e r e was no t r a n s f e r

here for

working with

systems examined,

t o use

spread monolayer,

air,

slides

change of a s p r e a d monolayer

UV i n a d i a t i o n ,

t h a t was p a s s e d t h r o u g h t h e

a p p r o a c h i n g 100% r a t h e r ready

the

unit

as an a r e a

Witout

into

fixed

~he m e t h o d d e s c r i b e d well

dyn/cm.

UV i n a d i a t i o n ,

was o b s e r v e d

monolayer

A was d e t e c t e d

indium

i.e.

the

decrease

o f 30 d y n / c m was e q u a l interface. a unit

to the

Also i n t h e

transfer

in

subse-

coefficient

was

is facile

and

a t 30 d y n / c m . generating

supported bilayers

v o l m n e s and m i n u t e

surface

coverage

The g r i d s

UV i r r a d i a t i o n .

When f a i l u r e s

is

achieved

of protein.

and m a i n t a i n e d ,

c a n be p r e p a r e d any t i m e and a r e These

have occurred, 825

quantities

transfer

procedures

have

they were always a s s o c i -

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a t e d w i t h p h o s p h o l i p i d d e g r a d a t i o n caused by the e f f e c t s of s t o r a g e and o x i d a tion.

In t h e s e i n s t a n c e s ,

drastically

unstable,

the s u r f a c e e q u i l i b r i n m p r e s s u r e was measured to be

decaying

to

below 10

dyne/cm w i t h i n

5 m i n u t es

after

s p r e a d i n g f o r example.

REFERE~ClgS 1.

U z g i r i s , E.E. and Kornberg, R.D. (1983).

Nature 301, 134-136.

2.

Gaines, G.L. (1966). I n s o l u b l e Monolayers I n t e r s c i e n c e P u b l i s h e r s , New York.

3.

Ibid,

4.

R e i d l e r , J . , U z g i r i s , E.E. and Kornberg, R.D. E x p e r i m e n t a l Immunology, L. Herzenberg, ed.

at

Liquid-Gas

Interfaces,

p. 338.

5.

U z g i r i s , E.E.

6.

Fromherz, P. (1971).

(1985).

7.

Uzgiris, E.E. (1983).

8.

Ref. 2, p. 66.

9.

Giaever, I. (1976).

I.

(in press)

C e l l u l a r Biochem. ( i n p r e s s ) .

Nature 231, 267-268. Biophys. 3. 41, 389a.

J. Y,mmun. I16, 766-771.

826

in Handbook of