1H-1H and 13C-13C vicinal coupling constants and amino acid side chain conformation in peptides

BIOCHEMKAL

Vol. 97, No. 2, 1980 November

AND BIOPHYSICAL RESEARCH COMMUNKATIONS Pages 751-758

28, 1980

AND 13C-13C VICIHAL COUPLING CONSTANTS AND AMINO ACID SIDE CHAIN CONFORMATION IN PEPTIDES

'H-lH

F. Piriou, F. Toma, FT. Monnot, Service de Biochimie, CEN-Saclay, 1 Institut Received

October

B.P.

Pasteur,

J. Savrda' Departement

no 2, 91190 and

and S. Fermandjian+ de Biologie

Gif-sur-Yvette,

25 rue du Docteur

Roux,

France

75015

Paris,

France

7,198O 13

constants Sl!!l!?ARY : The vicinal couplin C'-13Cy were measured in aspartic acidand phenylalanine (85 % ? 3C enrichment) as free amino acids and in the peptides Asp-Pro and Gly-Pro-Phe. These coupling constants used in connection with those between the c1- and the B-protons provide the unambiguous assignment of rotamers I and II in the Asp and Phe side chains. The method is generally applicable to other amino acids and residues even in large peptides. A possible set of Jcsc and J$sC values is proposed for the use of carbon 13-carbon 13 vicinal 2oupling constants in the side chain conformational studies of amino acid residues with a free carboxyl group.

INTRODUCTION : The determination

of peptides

the

understanding

of their

was predominated

by X-rays

by far

those

to a better in solution,

of

studies

which

Nowadays, allows

especially

constitues

mode of action.

solution.

chemistry

conformation

for

an essential

The early yielded

magnetic

an improved

approach

labelled

which

excelled

resonance

of peptide

amino

towards

of such an analysis

parameters

multinuclear

when isotopically

stage

step

acids

associated conformation

are

inserted

in

peptides. This amino

report, acids

of 13C-13C

following

our

and peptides

[l-91,

and 'H-'l-I

vicinal

arrangement

of amino

acids.

By choosing

the

{85% 13C-Asp}-Pro

compounds

previous

constitutes coupling

should

on several

13C-Phe)

85 % lsC enriched

an application

constants

85 % 13C-aspartic

and Gly-Pro-185%

+ to whom correspondence

studies

for

acid,

evaluating

of the joint

use

the

chain

side

85 % 13C-phenylalanine

our major

objects

free

were

: (i)

and to

be addressed. 0006-291X/80/220751-08$01.00/0 751

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 97, No. 2, 1980

establish three

a set bonds

for

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

'H-lH

of parameters

in side

coupling attached

of rotamers

in side

residue,

residues

being

constants

two protons

proline

chains

Jg and Jt

; (ii)

to the B-carbon

either

in the sequence

the conformational

; (iii)

cis

coupling

to ascertain

and then

chains

the

effects

before

factors

by varying

of the

the assignment

steric

imnediately

about

of the side

unambiguous

specific

over

by Pachler

the assignment

yield

to show the

to learn

constants

as the one proposed

or trans , on the

; (iv)

changes

13C-13C

as reliable

Cl01

chains

for

and next

responsible the

of the

for

ionization

state

of the molecules. MATERIALS AND METHODS .: The uniformly 13C labelled amino acids Asp and Phe were obtained in our laboratory as previously reported C21. The preparation of the13C enriched peptides (85% 13C-Asp}-Pro and Gly-Pro-{85 % 1sC-Phel was tested and scaled down with natural abundance 13C amino acids by liquid phase synthesis. The 13C NMR spectra were recorded at 22.63 MHz in the Fourier transform mode under broad band proton decoupling on a Bruker WH90 spectrometer equipped with a Aspect 2000 computer (16K data points) and a lsC homodecoupler. The concentration of the samples in 2H20 (99.8% 2H, C.E.A.) was about 0.1 M. The 13C-13C coupling measurements were performed with a digital resolution of 0.25 Hz or 0.125 Hz. 13C-{1sCl decoupling was used to supress additional geminal couplings obscuring the vicinal coupling constants 3Jc,-cv (related to the X1 dihedral angle) on the carbonyl carbon signals. The 1H NMR spectra were recorded at 250 MHz in the Fourier transform mode on a Cameca TSN 250 spectrometer equipped with a Nicolet 1080 computer (16K data points). Samples (0.05 M in 2H20) were studied at a digital resolution of 0.37 Hz. In the two peptides, Asp-Pro and Gly-Pro-Phe, the ratios of the cis-trans isomerism of proline were estimated from the lsC and IH signal intensities. RESULTS AF!D DISCUSSION A - Spectral

analysis

a) ----~Proton At all

the C'H,

in a typical

coupling

Iteration

1080 package).

fractions

NMR spectra of the unlabelled ---e---w-----

resolved

two vicinal

to their

:

the pH examined,

are well

angle.

:

of these However,

respective of rotamers

resonances ABX system HaX-HBA

constants values

(Fig.

in the

752

acids

of which

related

by the

and residues

provides

the

to the X1 dihedral

ITRCAL programme

assignment

the unambiguous 1).

two amino

the analysis

was performed

prohibits

I and II

of the

and HaX-HBB

the uncertainty

protons

corn~o~n$

of the

attribution

(Nicolet

two e-resonances of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 97, No. 2, 1980

R II

RI Figure

1. Staggered

rotamers

(x1:60").

RIII

C'-cy

of the 13C spectrum

signals

shifts

the center cis

Cl-C"

; (ii)

intensities (one

bonds)

residue

labelled

13C-PheI with

of the

of the

bond)

coupling

coupling

(SC, = 175.49

fractions

of cis

in Phe for

(Fig.

spectrum

gives

and trans

proline

('L 60 Hz)

in Phe ; (iv)

trans

(2.9

us (i)

; 6c,

isomers

: 80 % ; cis

tripeptide the

conformers

form

(trans

the

2) shows the C'

13C at 85 % in the

ppm trans

and trans

two triplets constant

constant

to RI and RII.

compounds

enriched

analysis

two triplets the

related

are

of the C' atom of Phe in the cis

of the

form)

of the

of the

pH. A straightforward

chemical

RI1 (x1:180"),

to RI.

of Gly-Pro-I85%

of the phenylalanine

at acidic

and 3JHu-HBB

3JHu-HB,,

b) -_-~__~-_---_----------Carbon 13 NMR spectra Analysis

around CCL-C5 : RI (x1:-60"),

is related

35

R III

from

= 175.26 from

: 20 X)

ppm

the ratio ; (iii)

the Cl-Cy

Hz) and the cis

the

(three (3.8

Hz)

acids

Asp

conformers. B-

'H-lH

Coupling

constants

and fraction

and Phe in the

three

and 13C-13C

coupling

of rotamers

ionization

states

constants

in the free

amino

:

as well

as fraction

of rotamers

10 HZ

Figure

2.

High

resolution

in the C' region

l3C

NMR spectrum

showing

conformers.

753

of

the signals

Gly-Pro-{85%

13C-Phe)

of the cis and trans

for

Asp

Vol.

97, No.

BIOCHEMICAL

2, 1980

Table

1.

;;,";;

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Vicinal coupling constants and rotamers of aspartic acid and phenylalanine side chains in.the free amino acids at different ionization states 3J (Hz) "qp

H‘Q@* 6.4

Rotamer C' -cY

4.2

2 ::

t

,u L bc d 2

+ -

8.7

3.8

-

9.9

4.0

2

t

7.6

5.7

z 5 5

-t

7.9

5.2

zz F a

-

7.3

5.6

Populations

I 0.35

II

III

0.15

0.50

9

2.5

m

+0.07

3.2

m

0.56

0.11

0.33

+fl.Ol

3.9

0.67 -l-Km-

0.13

0.20

to.06

2.4

0.46 -ijXJ-

0.2%

0.26

2.7

0.4% ar;l-r

0.24

0.2%

2.4

0.43 7l-xl--

0.27

0.20

ARI :

difference of rotamer I populations carbon 13 and proton-proton vicinal respectively. Rotamer populations were calculated using Jg = 2.6 Hz, Jt = 13.6 Hz (proton-proton);

-0.06

-0.01

-0.03

as obtained by carbon-13 coupling constants, the Jg

sets : = 0.6 Hz,

Jt

= 5.1

Hz

(carbon-carbon).

and Phe are rotamers

listed

are

similar

Hansen

et al.

carbon

and the

Kainosho C'H,

Clll,

established

estimating and the

s-protons Cl21

and other effects

set

these

of values

amino

magnitudes partially

acids

constants coupling

acids

C3,7,81

group.

However,

of Jg and Jt reposes

for

on the

by

the carbonyl and by

deuteration

in the

9 values

to take

respectively

constants,

Jt = 5.1 Hz and J and calculated

of

between

of stereoselective

experimental

set

two amino

coupling

basis

of the carboxylic

of the

BA and HBB and fraction of H

to the H"-HBAaB

on the

free

the absolute

for

the vicinal

jointly

by considering

validity

found

who used

The 13C-13C

C13,141

substituent

1. Assignment

to those

and Ajisaka

fragment.

acids

in Table

= 0.6

into

no serious

Hz has been

on carboxylic account

the

attempt

of

has been done Cl-cy comparison between

3J

values as well as on the limits pF"H~A and 3Jc, -c~ by Pachler of confidence of the values Jt = 13.6 Hz and J = 2.6 Hz proposed 9 for l-?-H' coupling constants ClOl. Yet, Table 1 shows that the two coupling numerous

experimental

3J

Vol.

97, No.

constants I,

vary

BIOCHEMICAL

2, 1980

3J

f_HBA

in the

ionization

states

from

and 13C-13C

'H-lH

aromatic It

fair

the set

directly

RESEARCH

related

between

coupling

and that

the fraction

constants

is

COMMUNICATIONS

to the fraction

when the pH is changed

the agreement

to mention

Ji"

fractions

that

London

= 1.2 Hz and Jk"

et al.

the

of rotamer

at the

of rotamer

good for

studied

values.

However,

in this

C - Coupling

three

I determined

two amino

acids,

conformers

was then

carboxylic

group

carboxylic

group).

in the

are

of the C'-CY

coupling

for

However,

the

these

two conformers

study

is

constants

are

values

very

grounds

of rotamer

in rather

good agreement

well

to the

peptides

First

the cis

In addition,

I in Asp and Phe does the rotamer

I is

the

we find

concerning

not occur

that

the

tripeptide

the variations the agreement is good.

two types

in the

in almost chain

specific the

in the

the most populated

755

and the

of

the conformational

that

side

that

(free

in such peptides.

we note

of the

position

two techniques

jointly

conformer

underlines

again

(free

of Ha-HBA and that

of using

of all,

the amino

position

Asp-Pro

by the

I and II

the arrangement

same : this

in peptides.

those

comments

and trans

for

Phe at the C-end

I determined

several

over

estimated

2 and 3. lie find

rotamer

the cis

:

in the dipeptide

on the validity

allow

for

at the N-terminal

parallel

of rotamer

prevails

not the

Asp (dipeptide)

set

the 13C set

and for

in Tables

peptides.

conformer

of rotamer

chain)

discriminating

tranS

proline

on theoretical

Several

seem to fit

whether

respectively

conclude

the results

properties.of

side

reported

the fraction

couplings

this

peptides

the Asp residue

Asp and Phe,

Ile can already

containing

to verify

for

Gly-Pro-Phe,

between

does not

Asp.

in the Asp and Phe residues

of proline

was suitable

Data for

this

from

proposed

work.

constants

The problem

Cl51

= 4.5 Hz for

in Asp and Phe determined

our

acids

BIOPHYSICAL

and carboxylic.

is

with

both

and 3JC,-C~y

same direction

AND

two peptides

all

the cases.

of the effects

strongest

residues of cis

In under

and trans

stabilization

same conformer in the trans

the

since

for

conformer

Vol.

97, No.

BIOCHEMICAL

2, 1980

Table

AND

2 - Vicinal coupling constants side chain in the dipeptide

Form

ln z =x

83

t

58

t

50

-

17

t

42

+

50

-

and rotamers Asp-Pro.

3J (Hz)

Ionic state

%

Ha-HBA 9.0

4.0

11.2

2.9

10.6

3.4

8.0

5.6

8.8

5.2

6.4

and for

populations

Phe (tripeptide)

conformer.

Such effects

Table

* z < cf

3.

Ionic

80

t

75

t

20

z25

calculated

the

rotamer

0.59 7n7-

0.13

0.28

to.08

0.79 --rim--

0.03

0.18

4.0

0.73 3-77

0.08

0.19

3.0

0.50 --7F3--

0.28

0.22

0.57 --is-XT

0.24

0.19

0.35 -ET

0.50

0.15

1.9

state

using

I is

HU-HBB

8.6

6.0

7.2

5.5

-

7.4

5.5

t

11.2

5.0

+

10.0

4.6

-

10.2

4.6

now the most

and rotamers Gly-Pro-Phe.

3J (Hz) Ha-HBA

AR1:

difference of carbon 13 and Rotamer populations

to.02

to.03

-0.04

-0.06

I

in the cis

by the difference

of

Rotamer

C’-Cy

populated

the

phenylalanine

Populations

II

III

ARI

2.9

0.55 --fFT

0.31

0.14

2.3

m

0.42

0.27

0.31

2.3

0.44 a;57-

0.27

0.29

-0.07

3.8

0.79 a;TT

0.21

0.00

-0.08

3.3

TX0.67

0.19

0.14

-0.07

0.70 -TX-r

0.19

0.11

3.3

-0.10

u 35

to.08

as obtained by carbon 13coupling constants, respectively. the sets of Table 1.

explained

may be reasonably

Vicinal coupling constants side chain in the tripeptide

Form %

+65

were

acid

ARI

of rotamer I populations ARI : difference carbon 13 and proton-proton vicinal Rotamer

aspartic

III

3.7

8.0

the

populations

I

3.0

"

of

COMMUNICATIONS

II

C'-Cy

4.5

l-

RESEARCH

Rotamer

HCL-HBB

CL

w-l

BIOPHYSICAL

rotamer I populations proton-proton vicinal were calculated using

756

-0.04

-0.05

as obtained by carbon-13 coupling constants, respectively. the sets of Table 1.

of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 97, No. 2, 1980

steric

effects

adjacent

exerted

residues.

as the size are

since

changed.

and side very

rotamers.

for

The trends

states.

impossibility

in this

nitrogen conformer

indicate

the

other

this

that

and the

interaction

amino

group.

additional

support

?I- and C- terminal

groups

are

those

free

II in the three

amino ionization

by the geometrical the

side

chain

and the

II should

groups

the carboxylate of cis highest

in the cis

II becomes preponderant

rotamer

of rotamer

idea

in the

as the

35hamHBA and 3Jc,-cy

both

at their

groups.

as far

found

rotamer

proportions

in the cis

important

in the side

Asp residue,

the two terminal

to the

conformers

the most group

between

state

between

(both

are

II is explained

In contrast,

concomitantly

gives

over

of interactions

As the

and trans

of the N- and C- terminal

however

effects when the

proximity

The increase

the

of cis

in the

of the

charge

carboxylic

prevailing

between

respect

ionizable

in the anionic

occuring

C41 preventing

increase

group.

chains

in the molecules

the effects

well

of rotamer

two rotamers.

by interactions

chain

mimic

rotamer

of the amino

occur

of rotamers

I and III

The low fraction

over

the close

side

of Asp and Phe as well

In this

the proportions

of the

the

chains

be considered.

both

is concerned,

rotamers

side

In the dipeptide

for

towards

modifications

in the repartitions

with

proline

of the

must

the presence

conformer

acids,

nature

concern

of Asp and also

trans

the

and trans

noticeable

These

chain

likely

chain

Yet,

of the peptides

significant

pH is

by cis

be explained

in the cis

conformer

group

of the Asp side

conformer

and rotamer

level

in the anionic

of interactive

II

state)

forces

between

bonds

coupling

the

conformer.

CONCLUSION : In conclusion, constants amino

the resultsindicate

are

acids

helpful

for

the

the conformational

13C-13C

three

analysis

of side

chains

in free

and residues.

When used jointly, rotamer

that

'Ha-lHB

I and rotamer

and 13C' -13Cy

coupling

II even in difficult

mixtures

exhibited

by the proline

coupling

constants

offer

cases

containing

an alternative

constants

such as the conformational

peptides.

way for

discriminate

studies

Undoubtely, of side

1aC-laC chain

between

Vol.

97, No.

2, 1980

conformations and Ji"

BIOCHEMICAL

in peptides

values

proposed

a free

carboxylic

group.

position

another

set must

having in this

AND

BIOPHYSICAL

RESEARCH

overcrowded

lH NMR spectra.

study

exclusively

For other

apply

residues

be considered

and especially

COMMUNICATIONS

However,

to residues those

the J c,c 9 having

in non terminal

C8,91.

REFERENCES : 1. Piriou,F.,Lintner,K.,Fermandjian,S.,Fromageot,P.,Khosla,Fl.C.,Smeby,R.R.,and Bumpus,F.H. (1980) Proc.Nat.Acad.Sci.USA 77,82-86. 2. Tran-Dinh,S.,Fermandjian,S.,Sala,E.,Mermet-Bouvier,R.,Cohen,n. and Fromageot, P. (1974) J.Amer.Chem.Soc. 96,1484-1493. 3. Tran-Dinh,S.,Fermandjian,S.,Sala,E.,Mermet-Bouvier,R. and Fromageot,P. (1975) J.Amer.Chem.Soc. 97,1267-1269. 4. Fermandjian,S.,Tran-Dinh,S.,Savrda,J.,Sala,E.,Mermet-Bouvier,R.,Bricas,E., and Fromageot,P. (1975) Biochim.Biophys.Acta 399,313-338. 5. Haar,!*!.,Fermandjian,S.,Vicar,J.,Blaha,K.,and Fromageot,P. (1975) Proc.Nat. Acad.Sci.USA 72,4948-4952. 6. Vicar,J.,Abillon,E.,Toma,F.,Piriou,F.,Lintner,K.,Blaha,K.,Fromageot,P.,and Fermandjian,S. (1979) FEBS Lett. 97,275-278. 7. Piriou,F.,Toma,F.,Savrda,J.,and Fermandjian,S. (1979) Tetrahedron 35,441-446. 8. Fermandjian,S.,Piriou,F.,Toma,F.,Lam-Thanh,H.,Lintner,K.,Vicar,J., and Conference on NMR of Macromolecules", Fromageot,P. (1978) in : "Proc.European (Conti,F.,ed.) Lerici,Sassari-Sardinia,pp.229-242. 9. Fermandjian,S.,Piriou,F.,Lintner,K.,Toma,F.,Lam-Thanh,H.,and Fromageot,P. and Biological Function", Proc. Sixth (1979) in :"Peptides : Structure and Meienhofer,J.,eds.), Pierce Chem.Co., American Peptide Symp.,(Gross,E., Rockford,Illinois,pp.205-208. 10. Pachler,K.n.R. (1964) Soectrochim.Acta 20,581-587. 11. Hansen,P.E.,Feenev,J.,and Roberts,G.C.K. (1975) J.Magn.Resonance 17,

249-261.

-

12. Kainosho,i:.,and Ajisaka,K. 13. Marshall,J.L.,and Miller,D.E. 14, Barfield,M.,Burfitt,I.,and 2631-2634. 15. London,R.E.,Halker,T.E.,Kollman,V.H Chem.Soc. 100,3723-3729.

97,5630-5631. J .Amer.Chem.Soc. (1973) J.Amer.Chem.Soc. 95,8305-8308. Doddrell ,D.(1975) J. Amer.Chem.Soc. 97, (1975)

.,and

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Matwiyoff,N.A.

(1978)

J.Amer.