Vol. 183, No. 1, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 258-264

February 28, 1992

TOTAL SYNTBESIS OF HORSE

Carlo

*Institute

Received

Di Belle*,

Claudio

HEART

Vita+,

CYTOCHROME

and

Luigia

C

Gozzini-

of Industrial Chemistry and +Dept. of Organic University of Padua, Padua, Italy

January

8,

Chemistry,

1992

Summary: A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the aprpiotein and may represent an useful alternative to site-directed mutagenesis ’ , particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis ofanentireprotein. o 1992 ~cademlc press, 1"~.

Cytochrome face

of

rest

the

in the

structure

and properties

normally

of

all

of this

found

aerobic

the

between Studies

on several

allowed

correspond

evolution.

involving

the

the

Some

heme

between overall

to two of these

moiety,

residues

a unique

fold regions

which

complexes,

are

more

of horse

stable

and

heart

are

than

the

cytochrome because

similar

well characterized proteins such as ribonuclease A7 or 8 clease and are therefore particularly suited to understand involved

in protein

^Present

address:

Biochemistry

0006-291X/92 Copyright All rights

$1.50

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

258

Bracco

Spa,

Milan,

compleof the

39-56 (Fig. 6 c . Intere-

to be inserted of ones

du-

interactions given

by ot-

staphylococcal

nu-

cooperative

folding6. Department,

to study relation-

cleavage

residues

her

ractions

tool

for

surinte-

key role

linked

believed

presumably

its

intricate

regions 23-25

outer The

due to

non-covalently

of two permissible

namely

disrupting

they

and three-fragment

definition

chain,

without

stingly,

the

two-

on the

organisms3.

and to the fact that it represents 4 , immunogenicity 5 and, in general, 6. structure and function in proteins

have

ring

protein

membrane

evolution

polypeptide la),

studied

is

in electron-transfer

xes

a well

mitochondrial

molecule

molecular ships

c is

inner

Italy.

inte-

Vol.

183,

No.

1, 1992

Several

years

a native-like fragment

This

between of

the

by the

fragment

complex have

et

the

the

the

perate

protrude in

from

the

should

amino

complex the

ordered

formation

any modification tural

residues

structure

1-22

insertion

sequence, or

e.g.

deletion

interfere

Fiq. Fig. wing

la. Primary structure of horse lb. A schematic representaz;on refers to tuna cytochrome c c of

the

of

not

crystallographic cytochromes

with

investigations different

1 to (Fig.

(Fig.

form lb).

ferrous

either

of the

that

65-66

the

amino bond

finentire

observed

in

the

apofragment do not

complex. with

acids

(lflecoo-

Therefore,

natural

or unnaor

and it

(66-

a threeThese

and therefore

substitution of

restoration

the

and

was also

22 of the

limited

fragments,

to

bond

lb)

molecu(66-104)

native

substitutions peptide

state.

was

segments

confirming

and stabilization

in the acids,

of the

fun-

ferrous

apofragment

heme-fragment

re-

o-amino

synthetic

using

non-heme

selective

[Hse>65](23-65),

25)H:[Hse>65](1-65):(66-104) xibly

of

the

substitutions

shown,

heme

cytochrome and

of

spontaneous

reduced

[Hse> 65](1-65)

(l-25)H

Restoration

apofragment

have

CNEr

and

semisynthetic

when these

reduced

on the

in the heme

formation

[Hse> 65](1-65)H

c and

introducing

al. 13

possibility

c molecule.

obtain of

restored

the

fragments

is

COMMUNICATIONS

on the

Hse 65 lactone

heme iron

way to

between

with

extended

CNBr

RESEARCH

reported

cytochrome

possibility 10-12 .

be selectively

apocytochrome with

bond

have

[Hse>65 ](l-65)H

Gozzini

peptide

can

the

native

region

recently,

two

heart

up the

the

C-terminal

104), dings

opened

BIOPHYSICAL

C-terminal

GAUGE, provided

although

the

the

horse

reactive

of

More that

the

condensation

fragments, to

of

AND

& Harbury'

between

(66-104)

observation

les

ago Corradin

complex and

synthesis ction

BIOCHEMICAL

segments

seems like-

heart cytochrome c. of the three-fragment complex. The draand is taken from Gozzini et al. 13 . X-ray

have species23-25

259

shown

a

close

structural

homology

among

Vol.

183,

No.

1, 1992

1. Chemical

BIOCHEMICAL

synthesis

of the fragments

solid

phase

synthesis

Fully protected

1 (l-66)

on resin

Stepwise

I

BIOPHYSICAL

and activation

of (1-66)

HF

solid

1Cys

synthesis

1 (66.104)

on resin

HPLC

purification

I

I 66

of (66-104)

HF

purification 1 66

COMMUNICATIONS

lactone

phase

I

HPLC

I 104

deprotection

I

CNBr

I 1

RESEARCH

lo [Hse 651 (l-65) Stepwise

Fully protected

I 1

reaction 0

65

II. Complexation

assisted

14s L 1

synthetic

1&

25

25

joining

between

c-p 65

activated

I 66

insertion

[Hse 651 (l-65)

synthetic

lactone

and (66-104)

I 104

I Hse 65 Fully synthetic apocytochrome

I 1

Ill. Heme

AND

I 104 c

by mitochondrla

I 104 Synthetic

[Hse 651 holocytochrome

c

Fiq. 2. Strategy followed for the total synthesis of [Hse65]holocytochrome c. I. Peptides corresponding to (l-66) and (6$;;$84) sequences were synthesized by stepwise solid-phase methods on Pam resin . After HF cleavage, purification was achieved by semipreparative HPLC. Homogeneity was determined by analytical HPLC, amino-acid composition, Edman degradation, electrophoresis, 26,28 tryptic and chymotryptic peptide2tapping. Details are reported elsewhere . Cys Acm-protection was removed then CNBr treatment resulted in by IQ'+, cleavage of the 65-66 peptide bond with simultaneous formation of activated [Hse65](l-65)-lactone. The native (l-25)H heme-fragment was obtained by tryptic6cleavage of reduced (1-38)H:apocythocrome complex according to Taniuchi et al. II. "Optimized" rejoining conditions involve incubation of synthetic [Hse;65](l-65) and (66-104) with native (l-25)H in HEPES buffer, PH 7, in the absence of oxygen. (l-25)H and (66-104) were in equimolar amounts, while 50% molar excess [ Hse>65 ] ( l-65) was used to take into account for lactone hydrolysis. Dithionite and radical scavengers were added to keep the heme-iron reduced and to prevent damages to the heme. The reaction was monitored by SDS-PAGE (Fig. 3). Rejoining yields up to 80% were okgained after 48 h, as determined by HPLC (data not shown). The synthetic [Hse lapocytochrome c was purified by characterised by amino-acid composition and, ion-exchange and RP-HPLC and by W-visible and UV-CD spectroafter non-covalent binding to f2;ri (l-25]H, and biological activity . III. Mitochondria incubation was performed scopy, described in ref. 14. Briefly, 0.5 mg synthetic essentially as [Hse65]apocr;octzome c was radioactively labelBed at Lys residues by reductive ( c specific activity = 15 x 10 dpmfmg). The radioactive matemethylation a fresh beef heart mitochondria rial was incubated for 40 min at 25 OC with suspension (100 mg protein in 2.5 mL total volume) and hemin. The reaction was (Fig. 4). Then, the newly formed radioactive monitored by RP-HPLC [Hse65]holocytochrome c was extracted with 0.5 M NaCl, submitted to ionredissolved in 6 M guanidiexchange chromatography, precipitated by 10% TCA, The purified synthetic material gave nium chloride and purified by RP-HPLC.

260

Vol.

183,

No.

1, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1234567

KDa 16.9

_

14.4 t:;

=

2.5

-

Fiq. 3. SDS-PAGE time course formation by complexation-assisted bated with synthetic (66-104) tions described in the monitored by electrophoresis ty Phast Gel and silver nes 2 to 7) reveal that

ly after

6 h, while

ly that

this

se results, system

specific

sted

a possible

chrome

(66-104) is

under

2 (point

II).

The

Pharmacia-LKB

Phast

Samples

at

taken

joining

0,

reaction

using

System

1,

6,

11,

was

High Densi32,

48 h (la-

lapocytochrome c (12 KDa) becomes detectable on(4 KDa) has reacted almost quantitatively after

not detectable MW standards.

low

[Hse>

could

be extended

combined

with

the

in

also

known

the

present

to the

experimental

39-56

condi-

region.

Moreover

availability

the-

of a mitochondrial 14-17 for heme attachment to the apoprotein I have strategy for the total synthesis in vitro of horse

enzymatic also

sugge-

heart

cyto-

to

assembly

c13.

a functional of

the

procedure

holoprotein the

heme to

[Hse65]apocytochrome ments

spanning

As shown

in

the

entire

fragment. the

synthetic with

culated tially

to

synthesized which

has

ligation

of two

chemically

sequence of

beef (Fig.

band

as 80% were

determined

heart

protein no effect holoprotein

is

to the

the

on the

structure

was purified

heart to

the

frag-

(66-104)

labelled

at Lys re-

[Hse 65]holocytochrome [Hse 65]apocytochrome the

and properties

of of

yield

native

Met 65 by Hse65,

by a combination

c by was cal-

and the

ininewly

a

modification cytochrome c 18 . The

semipreparative

the expected amino-acid composition and resulted indistinguishable from authentic horse heart holocytochrome c by different criteria, including intensity and osition of the maxima of the prinsipal absorption bands, biological activity 51 and monoclonal antibody binding .

261

by

48 h by HPLC analysis.

synthetic of

by

apoprotein.

The overall

between

yield

followed

and hemin.

difference

replacement

into

inser-

good

c was

corresponding

of

synthetic

synthesized

was radioactively

mitochondria The only

with

horse

after

and converted

respect

4).

of the

electrophoretic

methylation

total

[Hse65]apocytochrome

[Hse65]apocytochrome

be > 8% with

treated

synthetic

as high

by reductive

incubation

obtained

formation

of the

Yields

sidues

has been

104-residues

3, the

2, the

by mitochondria-mediated and Cys 17 of fully

Cys 14

of

Fig.

in turn,

covalent

Fig.

in

achieved

side-chains

c which,

disappearance

Then,

illustrated

has now been the

complexation-assisted

the

stai;;ng. [Hse

(l-25)H

Fig.

concept

According tion

to

[Hse[z]apocytochrome c ](l-65) was incuthe experimental condi-

Synthetic

and native on a

Fragment (l-25) Lane 1 contains

tions.

joining.

legend

h.

32-48

of synthetic

analysis

ion-

Vol.

183,

No.

1, 1992

BIOCHEMICAL

AND

0

BIOPHYSICAL

20

10 TIME

RESEARCH

COMMUNICATIONS

30

(min)

Fig. 4. RP-HPLC analysis of mitochondria-mediated stereo-specific hemeinsertion on synthetic (Hse6']apocytochrome c. The stereospecific attachment of the heme to Cyclic l7 residues by beef mitochondria (see legend to Fig. 2, point III) was determined by RP-HPLC analysis on a Vydac C4 column (0.46 x 15 cm) eluted with an acetonitrile gradient in 0.1% TFA (5 to 28% in 5 min, 28 to 38% in 20 min, 39 to 90% in 3 min). The eluate was analysed at 220 nm far total protein detection, at 400 nm for heme-containing proteins and by C scintillation counting for [Hse65]apocytochrome c conversion. Synthetic [Hse6'Japocytochrome c elutes at 19 min, while the newly produced 14C radioactive heme-containing [Hse 65]cytochrome c is eluted at 21 min, as expected for native cytochrome c. The total radioactivity recovery for both peaks was 40%, due to losses during work up of the reaction mixture. Accordingly, the final yield of the newly formed [Hse6' lholocytochrome c corresponds to about 8% with respect to the (Hse6']apocytochrome c initially treated.

exchange heart

and

RP-HPLC

and

holocytochrome

rization.

c on

Proofs

of

ascorbate-reduced ticity by

a yeast

tro

system,

Our

molecule mediated

at

222

results the by

a

procedures.

be of

spectra

with by

in the

synthesis

combination The

the

the

synthetic,

method,

which

antibodies of

to

a functional

of

in

characteristic

presence

native

biological

spectra

monoclonal

possibility of

from and

W-visible

recognition

demonstrate

indistinguishable

spectroscopic

included:

dehydrogenase

total

to

basis

W-CD

nml';

lactate

found

the

identity

state;

band

was

the

oxidized

262

principle

and

negative 20 and

reduction

ellip-

a complete

in

lactate21.

achieve, horse

in heart

can

and be

vi-

holocytochrome

conformation-assisted in

horse characte-

used

to

c enzyme-

obtain

cy-

Vol.

183,

No.

1, 1992

tochromes

carrying

C-terminal

parts

to

the

either of

of

automated

advantages

of the

ction direct

relatively

synthesizers present

in

short

C-terminal acids

by

fragments;

extended

the

(i)

in a matter (ii)

(iii)

COMMUNICATIONS

of days

in the

can

by current

variants

synthetic

amounts

containing

easily

of

the

of

desi-

that

sequence

non naturally

The

and fun-

and purity

sequences

and

effort

structure

of a given

different

N-

HPLC techniques.

large in yield

the

be produced

of protein

relatively

of

variants

that

study

multiple

combination

reduces

peptides

and purified for

RESEARCH

modifications

sequence,

as follows:

analysis; time

BIOPHYSICAL

short

approach

can be produced

functional

made

or

apocytochrome

may be summarized peptide

AND

minute

the

preparation

by modern

red

BIOCHEMICAL

allow can be

the

N-

occurring

and amino

can be synthesized. Although

method

the

general

may be limited

permissibility means

restricted

systems

containing

of

applicability

by the the

Hse +

to the potential

case

of the

requirement Met

conformation-assisted

of tight

substitution,

of cytochrome resynthesis

complex the

c and

present it

formation

coupling and by the

strategy

may be extended

is

by no

to

other

sites.

ACKNOWLEDGMENTS Work supported in part by Consiglio Nazionale delle Italy. We thank Dr. Hiroshi Taniuchi, Laboratory of NIH, Bethesda, MD 20892, USA, for helpful discussions

Ricerche (C.N.R.), Chemical Biology, and encouragement.

Rome, NIDDKD,

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14.

Hampsey, D.M., Das, G. & Sherman, F. (1986) J. Biol. Chem. 261, 32593271 Liang, N., Pielak, G.J., Mauk, A. G., Smith, M. & Hoffman, B.M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1249-1252 Dickerson, R.E. & Timkovich, R. (1975), in The Enzymes, Boyer, P.D., Ed. (Academic Press, New York) vol. XI, Part A, pp. 397-547 Margoliash, E. & Schejter, A. (1966) Advan. Protein Chem. 21, 113-286 Cooper, H.M., Jemmerson, R., Hunt, D.F., Griffin, P.R. Yates, J.R., Shabanowitz, J., Zhu, N.Z. & Paterson, Y. (1987) J. Biol. Chem. 262, 11591-11597 Taniuchi, H., Parr, G.R. & Juillerat, M. (1986) in Methods in Ensymol. 131, 185-217 Richards, F.M. Ei Wyckoff, H.W. (1971) in The Enzymes, Boyer, P.D., Ed. (Academic Press, New York) vol. IV, pp. 647-806 Anfinsen, C.B., Cuatrecasas, P. & Taniuchi, H. (1971) in The Enzymes, Boyer, P.D., Ed. (Academic Press, New York) vol. IV, pp. 177-204 Corradin, G.P. & Harbury, H.A. (1974) Biochim. Biophys. Res. Commun. 61, 1400-1406 ten Kortenaar, P.B.W., Adams, P.J.H.M. 6 Tesser, G.I. (1985) Proc. Natl. Acad. Sci. USA 82, 8279-8283 Wallace, C.J.A., Mascagni, P., Chaitt, B.T., Collawn, J.F., Paterson, Y ., Proudfoot, A.E.I. & Kent, S.B.H. (1989) J. Biol. Chem. 264, 1519915209 Offord, R.E. (1987) Protein Eng. 1, 151-157 Gozzini, L., Taniuchi, H. & Di Bello, C. (1991) Int. J. Peptide Protein Res. 37, 293-298 Basile, G., Di Bello, C. & Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191

263

Vol.

15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

183,

No.

1, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Visco, C., Taniuchi, H. & Berlett, B.S. (1985) J. Biol. Chem. 260, 6133-6138 Nicholson, D-W., Kohler, H. & Neupert, W. (1987) Eur. J. Biochem. 164, 147-157 Dumont, M.E., Ernst, J.F. 6i Sherman, F. (1988) J. Biol. Chem. 263, 15928-15937 Harbury, H. (1978) in Semisynthetic Peptides and Proteins, offord, R.E. & Di Bello, C., Eds., (Academic Press, London) pp. 73-89 Corradin, G.P. & Harbury, H.A. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 3036-3039 Burnens, A., Demotz, S., Corradin, G.P. & Bosshard, H.R. (1987) Science 235, 780-783 Hantgan, R.R. & Taniuchi, H. (1977) J. Biol. Chem. 252, 1367-1374 Almassy, R.J. Ei Dickerson, R.E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2674-2678 T. & Dickerson, R.E. (1981). J. Mol. Biol. 153, 79-94 Takano, Ochi, H., Hata, Y., Tanaka, N., Kakudo, M., Sakurai, T., Aihara, S. & Morita Y. (1983) J. Mol. Biol. 166, 407-418 Louie, G.V., Hutcheon, W.L.B. & Brayer, G.D. (1988) J. Mol. Biol. 199,295-314 Di Belle, C., Tonellato, M., Lucchiari, A., Buso, O., Gozzini, L. 6 Vita, C. (1990) Int. J. Peptide & Protein Res. 35, 336-345 Di Belle, C., Vita, C., Gozzini, L. & Hong, A. (1990) in Peptides: CheEds., (Emistry, Structure and Biology, Rivier, J.E. & Marshall, G.R., scorn, Leiden) pp. 1049-1050 in preparation Vita, C., Gozzini, L. & Di Bello, C. manuscript Veber, D.F., Milkowski, J.D., Varga, S., Denkewalter, R.G. & Hirschmann, R. (1972) J. Am. Chem. Sot. 94, 5456-5461 Demel, R.A., Jordi, W., Lambrechts, H., van Damme, H., Havius, R. 6 de Kruiff, B. (1989) J. Biol. Chem. 264, 3968-3997

264