BIOCHEMICAL

Vol. 72, No. 1, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE y-TURN AS AN INDEPENDENT CONFORMATIONAL FEATURE IN SOLUTION Md. Abu Khaled,

Dan W. Urry

and K. Okamoto

Laboratory

of Molecular Biophysics and the Cardiovascular Research and Training Center University of Alabama Medical Center Birmingham, Alabama 35294

Received

July

6,

1976

SUMMARY: The Y-turn, an 11-membered hydrogen-bonded ring formed by a 1 + 3 type H-bond, has been observed in chloroform in the tripeptide, t-Boc-Glyl-LValp-Gly3-OMe, by proton magnetic resonance spectroscopy. This tripeptide sequence occurs in repeat pentaand hexapeptides of elastin where it also forms a y-turn. The dihedral angles of y-turns, which were proposed previously on the bases of theoretical calculations and x-ray diffraction, are compared with the results obtained by proton magnetic resonance. The results reported here demonstrate for the first time that the y-turn can exist in solution as an independent conformational feature. INTRODUCTION A "Turn" defined

in terms

atoms ing

which

H-bond

is

forming

peptide the

another turn

of H-bond

y-turn

ring

(Clo).

possible. studies

Copyright All rights

is

called

giving

tripeptide

(7)

in Figure

showed in terms

1 where basis

on peptides

0 1976 by Academic of reprodlrction in my

sequence,

of three

formed sets

Inc. reserved.

proton

162

of

bond involving (5)

have pro-

formed

by a 1 -+ 3 type diffraction

by residues

25-27.

of 4 and II, dihedral ring

and carbon-13

II.

to be a preferred

X-ray

(8-ll),

fold-

of angiotensin

(6)

bonded

in solution

a-carbon

proposed

et al.

models

(C,,).

a 7 atom hydrogen

of tropoelastin

Press. form

a "y-turn"

of extensive

Printz,

Gly-Gly-Gly, ring

the

frequently

is a hydrogen

their

has been

by a 4 -+ 1 type

calculations

to an 11-membered

also

connecting

1.

for

"y-turn"

vectors

This

shown by theoretical

rise

On the

the

in a protein

stabilized

4 and the C-O of residue

turn

folding

The most

(1).

a lo-membered

can be described

labelled

residues

which

in the

of thermolysin

for

by the

(2-4)

has been

conformation

formed

B-turn

the NH of residue posed

as a basis

of the angles

in sequential

structure

This

functions

is

also

magnetic 11-membered

studies The angles

seen to be resonance hydrogen

BIOCHEMICAL

Vol.72,No.1,1976

AND BIOPHYSICAL

‘t--H ---- 0-y ‘i I H-/c H

i+2 \

i +bc,o---H\,

JI i+2

YH

4

\H i+2

I

I

RESEARCH COMMUNICATIONS

H’;>&

y - Turn FIGURE 1:

bonded

General representation of a v-turn and third residues and an L-amino

rings

were

Gly3-Val4-Gly5

The y-turn

angles

of this

VPGVG, differ the

repeat

the turn the

somewhat peptides

secondary

dihedral

exists

the

1.

angles

the

differ

peptide

CH2 and Gly3

ring

closure

those

angles

there

also

B-turn

from those

and,

patterns

of course,

on the $2 and xi

as another

previously

would

it

protons

analysis

is

occur

angles.

163

that

as ABX spin

valyl

4 and the y-

so,

If the

expected

information

if

(6,7).

non-equivalent

of the

prominent

as to whether and,

(12), In

the NH of residue

was studied.

provides

dihedral

infra).

(vide

feature

then

make the methylene

(6,7)

arises

reported

structure systems

of elastin

involving

therefore,

The conforma-

pentapeptide

occurs,

conformational

stable

first

Vail-Pro2-

Gly3-Val4-Gly5.

earlier

t-Boc-Glyl-Va12-Gly3-OMe

ABX spin

pentapeptide,

repeat

reported

the

CH2 proton

would

glycine

information

within

as an independent

G1yl

dihedral

sequence

The question,

as a relatively

of the

sequence

feature,

C-O of residue can occur

in the

from

as the residue.

Alal-Pro*-Gly3-Val4-Gly5-Va16

occurred

of tropoelastin

structural

reasons

in the repeating

(VPGVG) and hexapeptide,

(APGVGV). tional

shown to occur

containing glycine acid as the second

whether For these Y-turn

both

patterns (13-15).

the since Analysis

on the $1, $I~, #3 and JIM a@

quartet

provides

Vol.72,No.1,1976

MATERIALS

BIOCHEMICAL

X-Gly,-Val2-Gly3-OMe

as t-butyloxycarbonyl protons.

(t-Boc)

Solutions

made using

(0.02

CDCl3 as the

were

performed

ture

of 20°C and equipped

were

obtained

PS-100

COMMUNICATIONS

AND METHODS

The tripeptides

resonance

AND BIOPHYSICALRESEARCH

and variable spectrometer

and acetyl

Proton

solvent. HR-220 with

a Varian

spectrometer

Data Machine

equipped

with

operating

spin

experiments

system.

simulation were

a JEOL JNM VT-38

with

X

the Gly,

and Ac-GVG-OMe

resonance

computer

(16)

to assign

t-Boc-GVG-OMe

magnetic

an SS-100

temperature

synthesized

(AC) in order

M) of each of the

on a Varian

using

(GVG) were

were

(PMR) measurements at a probe

tempera-

Simulated

spectra Double

program.

carried

out

temperature

on a JEOL controller.

RESULTS The 220 MHz spectrum Va12-Gly3-OMe protons

appear

two methylene

is

shown in

of the

expanded

a-proton

Figure

2A where

it

as ABX spin groups

of Gly,

patterns. and Gly3

of t-Boc-Gly,-L-

can be seen that

The assignments were

region

achieved

of the

both

the Gly CH2

signals

of these

by assigning

first

their

I

L

4.50

4.00ppm

FIGURE 2:

A. 200 MHz spectrum of t-Boc-Glyl-L-Val2-Gly3-OMe in CDC13 B. Computer simulated spectrum. Note the exact correspondence between A and B. Also included is coupling analysis diagram.

164

,

3 50

Vol. 72, No. 1,1976

corresponding

NH proton

of t-Boc-GVG-OMe for

BIOCHEMICAL

Ni occurs

acetyl ating

region

constants

of both

simulated

angles

NH proton

the

Gly, (Figure All

aClj quartet.

for

t-Boc-GVG-OMe

Karplus-like

Ac-GVG-OMe

ppm in the

of the

relationship

was done by comparison

The signal it

t-Boc

The assignments

spectrum

the

for

at 7.38

respective

in the &H

from

while

derivative. the

This

and Ac-GVG-OMe.

t-Boc-GVG-OMe

Gly3

signals.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

spectrum.

and observing

PMR data II

the

for

were

then

from

from derived

collapses and coupling a computer

readily

in Table

obtained

coefficients

made by irradi-

shifts

Va12 were

the

ppm in the

the multiplet

obtained

are given

ppm

ppm, whereas

of chemical

CH2 were

The values

in Table

were

spectra

at 5.53

and at 7.37

The values

28).

NE appears at 7.26

derivative

CH2 and Gly3

with

appeared

of CH2 signals

signals

the

of Gly,

of the

I.

obtained

The $ dihedral

3JaCH-NHby using a by Bystrov

et al.

TABLE I. PMR PARAMETERS

AMINO ACID

FOR t-BOC-GLY,-L-VAL2-GLY3-OHe

CHEMICAL

SHIFTS

PROTON(S) RESIDUES t-BOC L-Val2

(6)

CH3

IN PPM (+ .Ol)

r

IN COC13 COUPLING

3J&H-NH

CONSTANTS 3J

Gly3

OMe

IN Hz

3JBCH-yCH

aCH-BCH

1.39

‘NH3

0.92

7.0

+ 0.1

2yCH3

0.98

7.0

+ 0.1

7.0

+ 0.1

6CH

2.14

cCH

4.38

7.8

+ 0.1

NH

7.13

7.8

+ 0.1

7.0

+ 0.1

7.0

+ 0.1

GEMINAL GUY,

(J)

COUPLING

CH(A)

3.48

5.5

-16.7

CH(B)

3.75

5.5

-16.7

NH(X)

5153

5.5

CH(A')

3.89

5.5

-18.2

CHfB')

3.93

5.5

-18.2

NH(X')

7.38

5.5

CH3

3.59

165

(2J)

(17).

BIOCHEMICAL

Vol.72,No.1,1976

AND BIOPHYSICAL

TABLE COMPARISON

OF CONFORMATIONAL

AMINO ACID RESIDUES INVOLVED IN y-TURN

COMPOUNDS

II5 Building)

Tripeptide6 (Theoretical calculations)

ANGLES

(0 AND $)

I-

ANGLES*

$.1+2

@i

@i+l

qitl

-Val3-Tyr4-Va15-

175

128

67

-61

-132

-Gly,-Gly3-Gly3-

172

128

68

-61

-131

162

92

86

-57

-114

148

i+l

it2

-148

Pentapeptide

FOR A y-TURN

CONFORMATIONAL

Thermolysin7 (x-ray) Repeat

II.

@i

i Angiotensin (Model

RESEARCH COMMUNICATIONS

-Gly3-Val4-G1y5-

56

3oa

-146

JI.1+2 -162

61

120"

1

170a

;;M;jastinl'

Tripeptide (this

*

See

Figure

from

the

The JI dihedral be compared

Drieding

Model

angles

obtained

to the work

from

other

Figure

ture

dependence

(3,8-11)

which

obtained

the

sources

2) indicates

in a fixing

are

other

(13-15)

-60a

-140

lBOa

(18),

gave the

NH chemical

following

results:

most

reasonably

of the

glycine

the

of t-Boc-GVG-OMe)

conformation.

Since

(dS/dT)

ds/dT

NH is essentially

angles.

166

is

with

values

Nl=

completely hydrogen

criteria

were

NH = 0.0043 0.0066

protons

the tempera-

one of the

experiments

of Glyl

of Gly3

by an intramolecular dihedral

can

*JAB to both

and Gly3

temperature

and dG/dT Gly,

relates

values

II together

shifts

variable

that

which

These

in Table

given

a preferred

ppm/'C

model.

Y-turns.

H-bonding,

of Va12 NH = 0.0076

solvent,

a Drieding

et al.

values for

of peptide

indicates

from

of the two Gly CH2 (G1yl

to identify

delineation the

80

Gly-Val-Gly.

of Barfield

All

Non-equivalence (see

of

were

the $ and J, angles.

ds/dT

130a

1.

aObtained

formed

-140

-Gly,-Va12-Gly3 work)

per-

ppm/%;

ppm/'C.

This

shielded

bond which

from

results

BIOCHEMICAL

Vol. 72, No. 1,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION The appearance patterns

of the

indicates

dependence

that

of the from

of these

PMR constraints

between

the NE of Gly,

in Figure

the

1.

solvent

The calculated for

t-Boc-GVG-OMe

are

listed

of thermolysin

(7),

be seen that

angles

between

tripeptide

(58,11,12). between

there

the

y-turn,

values (6)

are

involved

A short

of the

range

H-bond) forming

Gly,-Va12-Gly3 temperature

indicates

manner

consistent hydrogen

Gly3-Va14-Gly6)

consistent

the with

acid

systems

the

II it

(12)

and the

if we utiliz-

In the pentapeptide

part

can

4 and Jo dihedral

pentapeptide

Gly3-Va14

there

($ and $)

can be understood

in the

sequence

where

II that

In Table

(VlP2G3V4G5)

occur

and also

between ring

calculations

coefficient

SUpra)

amino

angles for

angles

for

(17)

of elastin

constitutes

of a 14-membered

ring

part formed

(8,12).

a 7-membered

are

that

in the y-turn.

B-turn

interaction

of theoretical

values

bond

a y-turn

dihedral

torsion

Both

et al.

molecular

This difference

features

for

Bystrov

and G1yl-Va12-G1y3.

of elastin

the

H-bond

of the

be

bond.

can be seen in Table

pentapeptide

structural

part

by a 1 -t 4 type

This

the

other

in the

repeat

which

with It

the These

are differences

(Vail-Pro2-G1y3-Va14-Gly8)

basis

II along

in Table

NKto

by a hydrogen in general

using

of structure.

Gly,-G1y2-G1y3

the other

residues

of the

type

shows this hydrogen

satisfied

angles

sequence, G1yl-Va12-G1y3.

consider ing

Q dihedral

have been proposed agreement

in an intramolecular are

as ABX spin

The temperature

ppm/'C

C-O of G1y3 as shown

a y-turn

is qualitative

of 0.0043

structure

and the

of t-Boc-GVG-OMe

are constrained.

shift

as occurs on the

fit

also

Gly residues

NH- chemical

relationships

y-turns

both

Gly,

shielded

two Gly CH2 protons

(C,) for

with

the G1y3 Nlj and Glyl was also

presence

of G1y3 NH- of 0.0066 presence

of significant

a 7-membered

bond was not observed

shown to be expected

G1yl-G1y2-G1y3

its

ring

167

tripeptide.

shielding

from the (see

of

The

in Gly,-Va12-G1y3

molecule

on the

and the + angles

ppm/'C

pentapeptide

in CDC13 (9) but was observed

(6)

in this

in this

in the

C=O (a 3 + 1 type

(vide

solvent Figure

of elastin

in the same molecule

in a 1).

(Val,-Pro2in more

VoL7i,No.

1,1976

solvents

polar

a 1 + 4 type

(8,1?). H-bond

BIOCHEMICAL

AND BIOPHYSICAL

In non-polar

solvents,

forming

a 14-membered

C=O of Vail-Pro*-Gly3-Va14-Gly5 the

7-membered

the

8-turn

hydrogen

which bonded

and can be formed

with

such as chloroform,

ring

between

disrupts

the

Therefore

ring.

RESEARCH COMMUNICATIONS

or without

the

the Vail

short

the

there

NH and Va14

range

Y-turn

is

7-membered

occurs

interaction

of

not as rigid

as

hydrogen

bonded

ring. The Y-turn

of t-Boc-Glyl-Va12-Gly3-OMe,

possible

B-turn

the Gly3

NH and would

be constrained

this

sequence

allow

as the

qualitative values

for

data

+-I# map with reasonable

for

$1,

geminal

feature

which

actions

for

does not its

nature

of chain

to the

initiation

between IQ,

require

in

of folding

ACKNOWLEDGMENT: This Grant No. HL-11310.

It

work

the

the

may also

Y-turn

basis

more stable t-Boc

to rotate be noted

0 and IJ angles

rather that

and

than

there

II and the

(ZJAB) contours.

Accordingly

it

is

conformational

of

has implications

the elastin

molecule

in proteins

associated

cooperative

specifically and generally

to

is

et al.

an independent

the

carbonyl

of Table

of the

than

Barfield

the presence This

stability. mobility

the

utilize

CH2 to be free

constant

that

is

would

$3 and 93 on the

coupling

to conclude

Gly3

indicates.

correspondence obtained

the

which

however,

(18) is

inter-

as to the with

respect

such as thermolysin.

was supported

by the National

Institutes

of Health

REFERENCES: Kuntz, I.D. (1972) J. Am. Chem. Sot. 94-, 4009-4012. Venkatachalam, C.M. (1968) Biopolymers 5, 1425-1436. 3. Urry, D.W. and Ohnishi, M. (1969) Spectroscopic Approaches to Biomolecular Conformation (edited by Urry, D.W.) 263-299 American Medical Association Press, Chicago, Ill. 1970. 4. Boussard, G., Marraud, M. and Neel, J. (1974) J. Chim. Phys. 2, 1081-1091. Printz, M.P., Nemathy, G. and Bleich, E. (1972) Nature 237, 135-140. 65: Nemathy, G. and Printz, M.P. (1972) Macromolecules 5, 7K758. Mathews, B.W. (1972) Macromolecules 2, 818-819. ii: Urry, D.W. and Long, M.M. (1976) CRC Crit. Rev. Biochem. (in press). 9. Urry, D.W., Mitchell, L.W. and Ohnishi, T. (1975) Biochim. Biophys. Acta 393, 296-306. 10. Urry, D.W., Ohnishi, T., Long, M.M. and Mitchell, L.W. (1975) Int. J. Pept. Protein Res. 7, 367-378.

::

168

Vol.72,No.1,1976

11. 12. ;:: 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Urry, D.W., Mitchell, L.W., Ohnishi, T. and Long, M.M. (1975) J. Mol. Biol. 96, 101-117. Renugopalakrishnan, V., Khaled, M.A. and Urry, D.W. (1976) J. Am. Chem. Sot. (submitted). Alberty, R.A. and Bender, P. (1959) J. Am. Chem. Sot. 81, 542-549. Pease, L.G., Deber, C.M. and Blout, E.R. (1973) J. Am. Chem. Sot. 95, 260-262. Khaled, M.A., Renugopalakrishnan, V. and Urry, D.W., J. Am. Chem. Sot. (submitted). Urry, D.W., Cunningham, W.D. and Ohnishi, T. (1974) Biochemistry 13, 609-615. Bystrov, V.F., Portnova, S.L., Balashova, T.A., Kozmin, S.A., Gravilov, Yu D. and Afanasev, V.A. (1973) Pure and Appl. Chem. 2, 19-34. Barfield, M., Hruby, V.J. and Meraldi, J.P. (1976) J. Am. Chem. Sot. 98, 1308-1314.

169