Conformational structure of the central nervous system proteolipid apoprotein. A Raman and infrared spectroscopic study

43

JoumalofA4olecular Structure,175 (1988) 43-48 Elsevier

Science

Publishers

CONFORMATIONAL

B.V., Amsterdam

STRUCTURE

APOPROTEIN.

A RAMAN

P. CARMONA' 1 Instituto

, J.M.

OF

AND

-Printed

THE

CENTRAL

INFRARED

RAMOS,

M.

de

Optica

(CSIC),

Instituto de 28006 Madrid

Neurob (Spain

ologia

in The Netherlands

NERVOUS

SYSTEM

SPECTROSCOPIC

COZAR

and

Serrano S.R.

STUDY.

J.

MONREAL

121,

28006

Cajal

PROTEOLIPID

Madrid

(CSIC),

(Spain).

Velazquez

144,

ABSTRACT Raman and infrared spectroscopy have been applied to investigate the structure of proteolipid apoprotein, PLA, in the solid state. These techniques reveal the presence of a-helical, B-sheet and unordered structures through the amide A, I, II, III and V bands. A fitting program for resolution of infrared amide I band provided an estimate of the protein secondary structure including 39% a-helix, 36% B-sheet and 25% coil and turns. PLA displays higher content of B and unordered structures than non-delipidated proteolipid PLP which, however, is more rich in a-helical segments. The Raman spectra also reveal the hydrogen-bonding environments of tyrosine residues in this protein. These residues are known from these Raman spectra to be exposed on the protein surface.

INTRODUCTION The of

conformational

prime

importance

architecture

and

membrane

that

proteins

on

cidated.

The

study the

the of

main

intrinsic

proteolipid

core

of

the

cant

interest,

functional

only

reconstituted

membranes

and

of

get

is

(refs. (refs. PLP

0022-2860/88/$03.50 0 1988 Elsevier

3-4)

of has

is

be

60% a

in

of

the

This

protein

into

the

but

also

interactions

is

membrane to

the

model PLP,

is

the in

be

total for that

of

reported

Publishers

on

some

B.V.

the

the is,

study

natural

signifiand of and

l-2). we

elu-

hydrophobic

structural

to

the a

so-called

of

good

form

embedded

insight

the

is

on

Myelin

myelin

about

to

l-2).

emphasizing

Science

influence

influence

myelin up

proteins

of

delipidated

membrane,

protein-protein

papers

myelin

to

myelin

their

The

makes

(PLA)

of

of

function

The

membrane

membranes.

revealed

(refs.

and

of

been

bilayer

lipid-protein

spectra

which

has

apoprotein

not

role

In earlier

fibres. and

proteins.

lipid

point

component

(PLP)

and

intrinsic

biological

nerve

protein

protein

of view

architecture

fraction of

from function

surrounds the

proteolipid protein

structure

vibrational

environment-depend-

44

ent

conformational

information

on

the

high

hydrophobicity

cult

to

ondary are

break

and

in

(refs.

most

of 4-5),

have

not

work

these

is

two

previously

have of

compare

used

which

scarce

to

are

its

diffi-

protein

sec-

spectroscopy vibrational

the

PLA

in

the

solid

state.

the

secondary

characterize

either the

of

is

due

vibrational

infrared

for

PLA

studied

to

techniques,

been

forms

pure

procedure to

There

of

estimates

using we

state

reliable

proteins.

aggregation

protein

this

proteins

and

structure

quantitative

this

In

lipids

strong on

conformational

the

structure

secondary

Data

structure

spectra

of

protein and

up.

non-existent.

Although

aspects

and

Raman

separately

structural

spectra or

study

together, of

PLA.

METHODS Proteolipid matter Lees

protein

according method

to

(ref.

(PLP)

was

a modification

6).

The

concentration

of

n-butanol.

This

was

apoprotein

(PLA)

solution by

Aguilar

ratio

found

was

Infrared

et to

lyophilized

PLA

samples

and

was

drying

9)

for

mine

in

arrangements. shape

for

Raman

and

algorithm

were

using as

the

and

the

are

reported

ferred

to

RESULTS The shows

AND

5145

to

on

i

cm-l.

internal

turns)

a Jobin-Yvon

with

to

(ref. deterof

a Lorentzian

the

amide

Ramanor

spectral

Lyophilized

diameter

used

164

slit

plasma samples

I band.

U-1000

a Spectra-Physics

The

water

polypeptide

of

calibrated

tl

heavy program

percentages

assumes

of

Deuteration

with

was

of

5998

of

fitting

program

line

molar

model

components

source.

frequencies,

mm

the

terms

as

respectively.

Samples

them

and

proteolipid

PLP,

I band

in

at

width

lines were

argon was

from

the

trans-

capillaries.

DISCUSSION

infrared some

recorded

excitation

laser,

amide

(coil of

the

techniques.

A

and

ultrafiltration

and

station.

bands.

PLA

unresolved

the

2 cm-l

0.9-l

of

Folch-Pi

lipid/protein

PLA

white

water-saturated

by

pellet

amide

brain

solubilized

a Perkin-Elmer

KBr

infrared

the

obtain

equilibrating

unordered

original

spectra

laser

by

assign

the

on

bovine

was of

The

for

a data by

structure

The the

spectrometer ion

of

B-sheet

a-helix,

studied out

to

7-8).

by

to

protein/ml

25.0

scanned

carried

secondary

and

were

order

of

a delipidation

(refs.

0.5

5)

protein

employed

assisted were

resolution

the

1 mg

of

al.

be

spectra

spectrophotometer

of

means

from

(ref.

lyophilized

a constant

described

extracted

spectrum

spectral

of

features

proteolipid indicative

apoprotein of

(see

a predominant

Fig.

1)

helical

45

t4000

3500

3000

2500

2000

BOO

1400

1600

1200

1000

600

600

400

200

cm-'

Fig.

1.

Infrared but

structure, tures

are

with

an

cm-l)

dered

and

of

the

a splitting (ref.

be

of

Raman

romporent

range

amide

at

structures,

It

and

PLA. unordered

infrared amide -1 , which cm

either

In

known

and

the and

the

wavenum,bers

and

for

and/or of

the

infrared

a-helix

with -1

be

the

con-

Raman

that

uncr-

distingbished

Raman hand

as

this

) and

spectra.

other

is

consistent

10)

can

Rarran

infrared on

cm

(ref.

arrangements

infrared

five

(1654

struc-

A band is

a-helical

agreement

infrared

is well

backbone

1668

usually

in

appear

PLA at

Other are

1625

and

cated

in

modes

caused

the

can

be

is

these

!n

the

amide there

u-helical

1685

B-structure side

due

2)

case

! fremust

be

ccnfcrmation

of

for

the 1520

usually (ref. the

also

the

of

v(~,o)

11).

and

695

assigned

cm to

Moreover,

infrared

and

amide

another unordered

Raman

polypeptide

infrared

and

shows

B-sheet

in

indicaticns

the

cf

to

located

Typical

cm-I

range

Fig.

types

following.

spectral by

tc

whereas

high-frequency

(see

qualitative

the

respectively,

PLA

frequency

assigned

modes

the

I band of I cmwhich

as this

ccnformations.

on

the

coincident,

about

of

10).

The

ture

of

their

protein

must

3290

arrangement.

polypeptide

account

unordered

near

I modes.

a-helical

b-sheet

the

fact,

powder

the position and intensity -1 cm) suppcrt the existence of

splitting

amide

into

quencies

1654

of

structure,

polypeptide

is

:1659

However, at

predocir,ant clusion

In

located

secondary

forms.

1 band

taking

and

lyophilized

amounts

present.

sharp ordered

B-sheet amide

of

significant

also

relatively

spectrum

spectral chain

secor,dary bands

of

V(O,IT)

-1

amide

I

are

lo-

bands

the

strucB-sheet

amide

II

and

a weak

shoulder

I band

near

1665

V

6

600

800

1000

I

I

1200

I

1400

1600

2800

3000

cm-’ 2.

Y
-1

Raman

indicates

agreement

Raman

to

band

with

the

I band

the

random appears

assigned

resolution

36%

b-sheet

obtained

from

amide

bands

12),

Raman

areas area

protein.

The

the

chains.

larly

infcrmative

concerning

involving

the

gation

Siamwiza

of

is

due

out-of-plane zenes.

If

donor;

the

phenolic et

to

al.

tyrosine

ratio

of

(ref.

13)

850/830

buried, cm-'

III

Raman

of it bands

known

on

is

the of

sum

of

the

f-ccnient

the

spectra

interac-

are

particu-

interactions

and

to

the

doublet the

a strong

about

C.5,

investi-

at

850

overtone

parasubstituted as

unknown

helices.

tyrosine

acts

of

that

total

According

the

were

proteins

that

the

bonding

the

a-helix,

different

a fraction

to the

program

the

the

eleven

basis

vibration

vibration is

of

as

tyrosine.

ring-breathing

ring-bending the

of

that

is well

information The

39%

of

study

with

hydrogen

group

it

fitting

components

question

as

occurs

side

protein

band

amide

percentages

resolved

related

some

of

the

B-strands

provides

in

II!

the

provided

empirical

closely

same

the

These

The

an

also

amide of

absorptivities)

spectroscopy

tions

cm-'

of

equal. on

of is

of

I band

a problem,

suggests

the

is

to a-helix and is consistent -1 component of the amide

amide

area

not

of

result

cm

therefore,

(the

This

application

area

PLA.

component

structures.

could,

I band

due

1654

infrared

are

integrated

a given

-1

The

integrated

however,

amide

cm

of

shoulder

The

unordered

represent

(ref.

830

the

One

absorptivities

of

1269

a-helix.

the

these

the

main

intensities

component

total

forms.

25%

I band.

integrated

all

1241 coil

infrared

of

powder

structure.

at

to

and

lyophilized

secondary -1 cm Raman

predominant

for

of

unordered

with

assigned

the

sprctrum

and

of

an

ben-

hydrogen-bond resulting

from

41 the

higher

most

intensity

tyrosine

The can

S-S

residues

to

that

the

configurations (ref.

infrared

the

the

chain

present

consists

of

unordered

510

to

Fig.

2,

surface.

the

and

disulfide bonds in PLA -1 (see Fig. 2). This 542 cm

and

moiety

must

trans-gauche-trans coexist

from

the

unordered

sitions

from

a-helix

Besides

the

Raman

spectra

the

of

to

in

the

protein

The

the

reported obtained revealed residues

rest

to

3-4). cause

expense

other

about some

the

in

polypeptide

in

increase

coat

about

of

a-helix.

of

Tran-

protein

(refs.

secondary

this

(36%).

an

of

of

content

loss

authors

details

which

the

B-sheet

conformations by

structure

of

and

seems

Raman

u-helical

(refs.

the

application

of

15-16).

structure, the

case

molecular

were

found

to

surface.

This The

secondary

(25%)

at

first

classical

the

reference

B-sheet

been

have

ACKNOWLEDGEMENTS. (CAICYT).

regions as

the

with

state.

and

environment

tyrosine

protein

solid

structures

information

environments on

also

determine

percent,

protein

and

have

39

represents

combination

the

protein

B-sheet

virus

to in

be

lipids

sora

According

protein of

study in

to

proteolipid

fully

at

C-S-S-C

apoprotein

Taking

be

the

band.

gauche-gauche-gauche

the

determined

the

bands

spectroscopy

proteolipid

pf3

on

cm-l

frequencies

spectroscopy

scattering

was

830

14). summary,

In of

of

the are

stretching

correspond

suggests

of

work

technical

has

been

assistance

supported of

Mrs.

by E.

the

Comision

Rubio

is

Ase-

grate-

acknowledged.

REFERENCES 1 A. Watts and J.H.M. De Pont, Progress in Protein-Lipid interactions, vol. 1, Elsevier, Amsterdam, 1985, pp. l-56. 2 L.M. Garcia-Segura, M. de CGzar, M.C. Moreno and J. Monreal, Brain Res., 380 (1986) 261-266. 3 G. Ayala, P. Carmona, M. de C6zar and J. Monreal, Eur. Biophys. J 14 (1987) 219-225. 4 P:'Carmona, J.M. Ramos, M. de C6zar and J. Monreal, J. Raman Spectrosc., in press. 5 J. Monreal J. Neurochem., 25 (1975) 539-541. 6 J. Folch-Pi and M. Lees J. Biol. Chem., 191 (1951) 807-817. 7 J.S. Aguilar, M. de C6zir, M. Criado and J. Monreal, J. Neurochem., 39 (1982) 1733-1736. 8 J.S. Aguilar, M. de C6zar, M. Criado and J. Monreal, J. Neurochem., 40 (1983) 585-588. 9 M.A. Raso, J. Tortajada, D. Escolar and F..Accidn, Comput. Chem., 11 (1987) 125-135. 10 C. .de Lizi, M.H. Baron and F. Fillaux, J..Chim. Phys., 75 (1978) 631-649.

48 11

12 13

14 15 16

G. Zundel, U. Bb;hner, J. Fritsch, H. Merz and B. Vogt, in D.W Gruenwedel and J.R. Whitaker (Editors), Food Analysis, Marcel Dekker, In., New York, 1984, pp. 435-509. M. Levitt and J. Greer, J. Mol. Biol., 114 (1977) 181-239. R.C. Lord, M.C. Chen, T. Takamatsu, I. Harada, M.N. Siamwiza, H. Matsuura and T. Shimanouchi, Biochemistry, 14 (1975) 48704876. H.E. van Wart, A. Lewis, H.A. Scheraga and F.D. Saeva, Proc. Natl. Acad., Sci. USA., 70 (1973) 2619-2623. G.J. Thomas, Jr., and L. A. Day, Proc. Natl. Acad. Sci. USA., 78 (1981) 2962-2966. G.J. Thomas, Jr., Biophys. J., 46 (1984) 763-768.