BIOCHEMICAL

Vol. 86, No. 3, 1979 February 14, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 605-612

VACUUM ULTRAVIOLET

CIRCULAR

DICHROISM

SPECTRUM OF B-TURN IN SOLUTION

Samir K. BRAHMACHARIy! V.S. ANANTHANARAYANAN+* S. BRAHMS" , J. BRAHMS= , Rao S. RAPAKA' and Rajendra S. BHATNAGAR' t

II

Molecular

Institut

§

Received

Biophysics Unit, Indian Bangalore 560 012,

Institute India

of Science,

de Recherche en Biologie Moleculaire University of Paris VII, 75005 Paris,

School

of Dentistry, University San Francisco; 94143,

October

26,

(C.N.R.S.) France

of California, U.S.A.

1978

SUMMARY The vacuum ultraviolet circular dichroism spectrum of an isolated 4 + 1 hydrogen bonded B-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at - 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the B-turn conformation. This spectrum, particularly the presence of a strong'negative band around 180 nm and a large ratio , can be taken as a characteristic feature of the i-ej2Ol "!225 isolated B-turn conformation. These CD spectral features can thus be used to distinguish the B-turn conformation from the B-structure in solution. INTRODUCTION The ubiquitous conformational recognized

feature in recent

presence

of the

in globular years

proteins

(1,2).

While

has been made on the delineation E-turn

from

on the

specific

it

from

theoretical

the other

secondary

x To whom to address

of the structures

as an important

has been well

a good deal

of the various

considerations

characteristics

$-turn

of effort

forms

of the

(3,4),

experimental

eturn

that

like

will

the B-structure,

data distinguish are few.

correspondence. 0006-291X/79/030605-08$01.00/0 605

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in anyform reserved.

Vol. 86, No. 3, 1979

High

resolution

dichroism B-turn

BIOCHEMICAL

nuclear

magnetic

(CD) spectral peptides

regard

in solution

to the

wavelength bands, ations,

are

conformation in the

250-200

the CD spectral in the

of a small

linear

is not

the presence from Brahms

of the

communication,

bonded

is

the first peptide

B-turn

to arrive

S-turn

(9)

data

have reported

and the

B-structure

region,

extending

the vacuum ultra-

N-acetyl-Pro-Gly-Leu-OH conformation

report containg

of the

(Fig.1).

in To our

vacuum ultraviolet

an isolated

at

B-turn

the CD spectral et al.

(8)

two conform-

possible of the

is

of the

these

we present

of the tripeptide,

hydrogen this

the magnitudes

in the vacuum ultraviolet

CD spectrum

knowledge,

ultraviolet

It

characteristics

polypeptides

the 4 +1

in the

Recently,

down to 165 nm. In this violet

and shape

and proteins

run region.

B-turn

B-structure

to be different.

in peptides

of the

of the

200 nm and 220 nm in both

about

some of the

to that

rm, although

conclusion

for

The CD spectrum

resemblance

near

found

an unequivocal

(5-7).

190-250

(NMR) and circular

have been reported

band position

region,

appearing

resonance

studies

seen to have a general with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CD

B-turn.

EXPERIMENTAL The synthesis of the tripeptide N-acetyl-Pro-Gly-Leu-OH is reported elsewhere (11). Its purity was checked by microanalysis and it showed a single spot on precoated silica gel G plates. Trifluoroethanol (TFE) from Merck was distilled before use. The vacuum ultraviolet CD spectra reported here were recorded down to 164 nm in the apparatus constructed in the I.R.B.M. Laboratory (Paris). The details of the instrumentation and the calibration procedure are given elsewhere (9). The spectra were recorded in TFE using from 0.005 cm to 2.0 cm pathlength cells. The concentration used was from 2 mg/ml to 0.03 mg/ml. The ellipticity values are expressed in deg cm2 dmole-1 per residue. RESULTS AND DISCUSSION It crystal

is well

structure

data

known from

the

on globular

are among the most favourable

statistical proteins

residues

606

analysis (2,lO)

to occur

that in the

of the Pro a.nd Gly 2nd and 3rd

BIOCHEMICAL

Vol. 86, No. 3, 1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

C -o----"--N /aL

Rl\ H '"\1

figure

Schematic diagram of a 4 +1 hydrogen-bonded B-turn in a tetrapeptide sequence containing Pro in the 2nd position and Gly in the 3rd position.

1

position

of the

B-turn,

Fig.1).

In addition,

nature

of the residue

end) of

of the @turn

type

B-turn

respectively

as we have demonstrated in the 4th plays

conformation

Z-Pro-Y-X

of peptides

X = Gly,

Ala,

in a given

of the

best

in stabilizing

conformation.

This

is substantiated

the globular

protein

for

and Phe,

data

the characterization

features

of the

(10).

@turn

An important large

negative

reveal

region

peptide

concentration

feature

of these

band arond

199 run and a weak negative

the extent

sequence

of the (11,12)

-Pro-Gly-X-OH, that

the

Leu in the

by the statistical Our choice

on

where 4th position

the 4 + 1 hydrogen-bonded

was dictated

in TFE in the wavelength at the

N-acetyl

(lo),

in governing

Bturn

analysis

of

of N-acetyl-Pro-Gly-Leu-OH

of the vacuum ultraviolet

The vacuum ultraviolet

ures,

role

in

the C-terminal

tetrapeptide

type

Ile

the

(i.e.,

Our NMR and CD studies

Leu,

is by far

elsewhere

position

an important

or Z-Pro-Gly-X.

a series

(as shown schematically

CD spectral

by the above considerations. CD spectra

164-250

of N-acetyl-Pro-Gly-Leu-OH

run, taken

of 2 mg/ml, spectra

is the

180 nm, besides band near

607

at three

temperat-

is shown in Fig.2. presence

a positive

225 nm. All

these

of a relatively band around bands

are

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Wavelength

Figure

2

slightly positions

for

molecular

interaction,

CD spectra

The magnitude

these

the

compared type-11

B-turn

spectra

extrema

at - 4",

between

at the lowest spectrum

for

as quite

good.

our

However,

mg/m ) using

experimental

pos tions

CD spectrum

(Fig.3)

studied

the

any intra-

a larger

path-

shown in Fig.3.

band at 225 mt decreases

The exact

type-II

band

the vacuum ultraviolet

B-turn

experimental

608

in Table

I.

of The

at low concentration

and the (13)

and the

and magnitude

- 20" and - 40°C are given

temperature an isolated

to avoid

down to 174 t-m are

of the weak negative to 201.

expected

In order

recorded

(0.03

measured

theoretically

(13).

we have also

band shifts

agreement

to the

at low concentration

These

positive

nm

Vacuum ultraviolet CD spectra of N-acetyl-Pro-Gly-Leu-OH in TFE, (---) at - 4'C ; (-) at - 20°C and (-.-.-.) at - 40°C ; concentration 2 mg/ml and pathlength used 0.005 cm.

blue-shifted

length.

,

theoretical

can thus

spectrum

be considered

is displaced

by

Vol. 86, No. 3, 1979

BIOCHEMICAL

I 180

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 200

I

WAVELENGTH

Figure

I

I 240

.nm

Vacuum ultraviolet CD spectra of N-acetyl-Pro-Gly-Leu-OH in TFE, (---) at - 4°C ; (-) at - 20°C and (-.-*-a) at - 4O“C ; concentration 0.03 mg/ml and pathlength used 0.5 cm.

3

TABLE VACUUM ULTRAVIOLET

I

CD DATA OF N-acetyl-Pro-Gly-Leu-OH

Temperature "C

I 220

ce,J

9

a

ml

@,'l

a*

a

nm

IN TFE AT DIFFERENT x3

P,i

a

TEMPERATURES*

R = Ce,l $1

nm

-4

225.0

-1,650

201.0

+ 9,200

182.5

- 16,600

- 5.6

- 20

225.0

-1,300

201.0

+12,300

182.5

- 20,600

- 9.5

- 40

225.0

-1,200

201.0

+13,800

182.0

- 22,400

-11.5

peptide

OIP3

*

concentration

a expressed

of

in deg cm* dmole

mg/ml. ,

609

Vol. 86, No. 3, 1979

about

BIOCHEMICAL

5 nm to the

the transitions. is

smaller

blue,

that

NL protons

of a series

N-acetyl-Pro-Gly-X-OH, the extent R (11,12).

In the

Gly-Leu-OH

(about

Our R value

is

for

also

-G1y2)

(9).

equilibrium

possibility

very

is

from

saturation

and attains

B-turn spectrum B-turn

close

At lower shifted

to the

the Fig.3,

conformation. significantly conformation

around

Further

cooling

indicating

calculated

of Venkatachalam to be about

6 (13)). for

the coil

+

due to a lesser of

the coil

the ratio expected

complete

610

agreement spectrum

of 12 observed

to - 6OY

peptide

N-acetyl-Pro-

concentration,

- 4O"C,

theoretically

in TFE. The

in excellent

stabilization

the

of this

is

is found

right,

an increase

tripeptide of R for

value

peptide

and the

indicating

conformation

to the

of any intermolecular

As evident

correlation

the theoretically

type-1 B-turn

the

type

N-acetyl-Pro-Gly-Leu-OH,

studied

B-turn

shift

value of the ratio

11.5)

for

of the

(Fig.3),

temperature

conformation.

conformation

absolute

(about

was found

NMR chemical

tripeptide

value

12)

ratio

is a direct

of this

the type-II

for

(The R value

B-turn

the

band around

of B-turn

B-turn

of the

to distinguish This

conformation

at the lowest

of Woody (13)

(Ala2

there bonded

lowered,

observed

the value

(9)

of tripeptides

case of our

of B-turn

experimentally

poly

is

values

dependent

225 is found to increase

in the extent

(3).

of hydrogen

temperature

k! 201 /Id

with

show that

201 run band

of the negative

and temperature

on

(13).

ellipticity

in the extent

data

as the

the

COMMUNICATIONS

effect

of the

value

the increase

dependent

ratio

theoretical

the B-structure.

The solvent

solvent

magnitude

from

with

RESEARCH

from

by Brahms et al.

conformation

to increase

between

arise

205 run and that

225 nm, has been utilized

of the

could

R, between

peak around

the B-turn

BIOPHYSICAL

the observed of the

The ratio, positivie

which

Also,

than

AND

at the

state.

R shows value

for

100 %

does not change

attainment lowest

the

of the folded temperature.

Thus

Vol.

86,

No.

the

3, 1979

BIOCHEMICAL

spectrum

as representative

in solution.

Another

in an analogous

single-crystal

supporting

180 MI, as predicted

shift,

for

provides

in solution.

also

the cross

would

for

the isolated

that

presence

of

by

conformation from

band around l3-turn

a slight

characterization

/18],,5

the availability

be of use in the

negative

and particularly

B-structure

the

with

for

[812,,

the B-turn

In addition,

strong

by Woody (13)

a large

the fact

has been demonstrated

of a distinct

spectrum

conformation

(12).

new possibilities

with

distinguish

conformation

COMMUNICATIONS

temperature

B-turn

comes from

N-acetyl-Pro-Gly-Leu-OH,

This

band along

evidence

crystallography

The presence

here

at the lowest

N-acetyl-Pro-Gly-Phe-OH,

B-turn X-ray

RESEARCH

of an isolated

compound,

type-II

observed

BIOPHYSICAL

of N-acetyl-Pro-Gly-Leu-OH

can be taken

a typical

AND

from that

of B-turns

can thus

of 180 nm be used to

the B-structure,

and

B-structure.

of the antiparallel

of the CO spectrum

interpretation

blue

the presence ratio

and

of the B-turn

of the CD spectra

of globular

proteins. Towards another

the completion

tetrapeptide,

195-240

about

run wavelength

paper of the blocking

region

However,

4-7 run from the

CO bands as well obtained

by Kawai and Fasman (1.4).

the CD spectral

by Woody (13).

the CD spectrum

of

of this

resemble

those

the observed theoretical

as those

of the ratio,

from those

tetrapeptide and protecting

of Kawai

for

the

Our CD data

611

calculated by

different

from

presented

in this

and Fasman (14)

in addition

B-turn

is blue-shifted

R, are

in

(a)

authors

to presence

in

in the

and the magnitudes

- the one used by the latter groups

solutions

tetrapeptide

spectrum

spectrum

ester,

In dilute

features

by us and by Woody (13). differ

work,

Cbz-Gly-L-Ser-(0-But)-L-Ser-Gly-0-Stearyl

has been published cyclohexane,

of this

of the those

the nature has bulky of the

long

Vol.

86, No.

3, 1979

BIOCHEMICAL

hydrocarbon used and, ours

chain most

extending

AND

at the carboxyl

importantly,

(c)

the CD spectrum

BIOPHYSICAL

end (b) the

range

the

RESEARCH

nature

of wavelength

to the vacuum ultraviolet

COMMUNICATIONS

of the solvent covered, region.

ACKNOWLEDGEMENT The work reported Paris, by a grant from N.I.H. USPHS grant at California.

here was supported by DGRST grant (PL-480) at Bangalore and by a

at

REFERENCES 1. 2. 2 2 7. 8. 9. 10. 11. 12. 13. 14.

A.V., Pandya, U.V. and Chandrasekaran, R., Lakshminarayanan, Ramachandran, G.N. (1973) Biochim. Biophys. Acta 303, 14-27. Chou, P.Y. and Fasman, G.D. (1977) J. Mol. Biol. llc135-175. Venkatachalam, C.M. (1968) Biopolymers 6, 1425-14X Zimmermann, S.S. and Scheraga, H.A. (1977) Biopolymers 16, 811-843. Urry, D.W. and Ohnishi, T. (1974) Biopolymers 13, 1223-1242. Kopple, K.D., Go, A. and Pilipauskas, D.R. (19x) J. Am. Chem. Sot. 97, 6830-6838. T. and Jacobs, M. (1974) Urry, Ox., Long, M.M., Ohnishi, Biochem. Biophys. Res. Comm. 61, 1427-1433. T.F., Timasheff, S.N., Fasman, G.D. and Townend, R., Kumosinski, Davidson, 8. (1966) Biochem. Biophys. Res. Comm. 23, 163-169. Brahms, S., Brahms, J., Spach, 6. and Brack, A. (1977) Proc. Natl. Acad. Sci. USA 74, 3208-3212. Brahmachari, S.K. and Ananthanzayanan, VS. Paper presented at the Int. Symp. on Biomolecular Structure, Conformation, Function and Evolution, Madras, January 1978. V.S., Rapaka, R.S. and Brahmachari, S.K., Ananthanarayanan, Bhatnagar, R.S. Submitted for publication. Brahmachari, S.K. (1978) Ph.D. Thesis, Indian Institute of Science, Bangalore, India. Polypeptides and Proteins" (Blout, Woody, R.W. (1974) in "Peptides, E.R., Bovery, F.A., Goodman, M. and Lotan, N., Eds.) pp. 338-350, John Wiley, New York. Kawai, M. and Fasman, G.D. (1978) J. Amer. Chem. Sot. -100, 3230-3232.

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