The time resolved emission spectra of peptide conformers measured by pulsed laser excitation

Vol.

3, 1980

94, No.

June

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 909-915

16, 1980

THE TIME RESOLVED EMISSION SPECTRA OF PEPTIDE CONFORMERS MEASURED BY PULSED LASER EXCITATION.' A.G.

Szabo

and D.M. Rayner'

Division of Biological Sciences National Research Council of Canada Ottawa Canada KlA OR6 Received

February

15,198O

SUMMARY: The fluorescence decays of a number of peptides were measured using pulsed laser excitation and time correlated single photon counting techniques. In all cases double exponential kinetics were observed with the short lifetime component (0.5- 0.85 ns) predominating over the long lifetime component (1.622.21 ns). The lifetimes varied with the peptides measured. The time resolved emission spectra of LysTrpLys shows both components have similar spectral maxima at 345 nm. It is suggested that the dual exponential kinetics originate from different conformers of the peptides. Recently

we reported

that

the

tryptophan

was described

by double

and that

the fluorescence

spectrum

into

two components

with

consistent

rationalization

originates

from

h

fluorescence exponential

of aqueous

kinetics

of tryptophan

(3.1

could

of these about

results

was that

solutions

the

of

ns and 0.5 ns)

be temporally

350 nm and 335 nm respectively

IlEiX

rotamers

decay

resolved

(1,2).

The most

fluorescence

the Cd- CS bond of the alanyl

side

chain

of

tryptophan. In order

to assess

rationalizations

for

interpretation

the

containing

measurement

fluorescence advantage

efficiencies decays

in

for

proteins

of short

using

of the special

features

of Chemistry,

of these

and particularly

difficult

as NRCC # 18269 address; Division

the bases

kinetics

of a series

of our the we have

polypeptides

residue.

quantum

fluorescence

and extend decay

decay

spectra

and applicability

systems

tryptophan

fluorescence of their

we took

'Issued 'Present

monomer

fluorescence

a single

The low

work

generality

of multi-exponential

investigated

of their

the

pulsed

peptides

makes the

the time gas spark

of a sync-pumped,

resolution

lamps.

In this

mode-locked

NRCC.

909

0006~291X/I30/110909-07$01.00/O Copyright 3 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

94, No.

3, 1980

and cavity the

BIOCHEMICAL

dumped dye laser

temporal

Materials

and spectral

AND

system

BIOPHYSICAL

RESEARCH

as an excitation

resolution

required

source

for

these

COMMUNICATIONS

and achieved

both

studies.

and Methods

All peptides were purchased from Research Plus (Bayonne, N.J.) except for AlaTrpAlaGly (a generous gift from R. Williams, NRCC). Their purity was checked by two-dimensional chromatography and electrophoresis. The peptides GlyTrpGly, GluTrpGlu, LysTrpLys and AlaTrpAlaGly required purification which was achieved by preparative thin layer chromatography on cellulose. Solutions of the peptides (5 x 10e5 M) in 0.01 M cacodylate buffer (pH 4.9) were degassed prior to the measurement of their fluorescence decays. The fluorescence decay measurements were performed using a frequency doubled Spectra-Physics synchronously pumped dye laser system as the excitation source (X 295 nm) (3). The fluorescence decay parameters were calculated by the non-l%ear least squares iterative convolution technique. The adequacy of their fit and the decay model was judged by inspection of the weighted The emission was detected at right angles through a residual plots (4). monochromator (3 nm bandpass) using photon counting electronics described previously (5) but with a Tracer Northern Model 1705 multichannel analyser. The channel width was 0.0203 ns/ch. Results

and Discussion

The laser/instrument was typical

of those

Figure

la is

decay

function

for

(RMSR) from

2.12

for

other

data.

2.32

for

The residual

a double

to 1.008

(E'WHM = 0.85

peptides

plot

exponential

and the significant

exponential

that

kinetics.

decrease

the

"best

plot

in

in

a ratio

work. exponential

lb resulted is

the root

obtained

LeuTrpLeu,

single

fig.

decay

for

in this

fit"

It

the fluorescence The lifetimes

ns)

reported

function.

+ .Ol ns and 72 = 0.70 + .015 ns with

clIIo.p

clear

after

from

mean square of LeuTrpLeu

these residual

obeys

at Aem 330 nm were

TII =

of pre-exponentials

= 0.99. For comparison

for

function

residual

this

using

plots

double

measured

the weighted

deconvolution residual

response

a single

nearly

peptides this

system. results

with

decay

are

decay

The agreement

between

good.

and a lifetime

parameters obtained

This

lc)

(fig.

obtained

after

deconvolution

in the case of N-acetyltryptophanamide

fluorescence

results

is very

plot

a RMSR of 1.16

exponential

and their

table

residual

exponential

random Double

the

with the

together

decays are our laser with

910

were

summarized nitrogen generated

of 2.83 + 0.002 observed

for

in Table

ns.

the other 1.

lamp fluorescence data

is

and the

the N-acetyltryptophanamide

small

Included

in

lifetime lamp generated results

Vol.

94, No.

3, 1980

BIOCHEMICAL

AND

... 5

BIOPHYSICAL

RESEARCH

Leu Trp Leu RMSR= 2.72

!!

Leu Trp Leu RMSR= 1.01

IL

NATA RMSR z 1.16

c

COMMUNICATIONS

I

. I .. I cI !O( Figure

1.

that

380

there

is

nm for is

probably hence

the

does not There

and

460

560

660

Weighted residual plots which are obtained from the “best fit” deconvolutions for the fluorescence decay: a) LeuTrpLeu, single exponential decay; b) LeuTrpLeu, double exponential decay; c) N-ncetyltryptophanamide, single exponential decay; 20.3 ps/ch, x 330 nm, h ex, 295 nm. em’

confirms

peptide

300

second

exponential

originate close

agreement

all

peptides

Recently measurements

error

between

except

for

difference

due to the small higher

in

Werner on several

in the fluorescence

from any instrumental

very

a significant

term

its

in

contribution

it

determination

and Forster dipeptides,

(6)

the lifetime

value

of the

artifacts. values In this

AlaTrpAlaGly. the

decay

of

the

long

makes to the

at both latter

lifetime

total

330 case

component

fluorescence

and

(4). reported some larger

911

their

fluorescence

peptides

decay

such as glucagon

(29

Vol.

94, No.

3, 1980

BIOCHEMICAL

Table

AND

8lOPHYSlCAL

RESEARCH

Fluorescence decay parametersa 330 and 380 nm.

1.

Peptide

hem x ml-1

LysTrpLys

GluTrpGlu GlyTrpGly LeuTrpMet AlaTrpAlaGly

of peptides

at

71 x ns-'

72 x ns -1

Rb

2.21 2.25 2.20 2.12 2.22 2.14 1.83 1.86 1.64 1.71 2.02 1.99 2.72 3.10 3.38

0.85 0.75 0.89 0.70 0.54 0.80 0.71 0.77 0.71 0.75 0.74 0.71 1.02 1.02 1.06

0.83 1.07 0.97 0.99 1.05 1.06 0.95 1.03 0.60 0.57 1.0 1.08 0.038 0.075 0.027

330 33oc 380 330 33oc 380 330 380 330 380 330 380 330 33oc 380

LeuTrpLeu

COMMUNICATIONS

a typical standard errors for ~1, were i.01; for ~2 were 0.015; for R were to.05 except for AlaTrpAlaGly which was R= 0.038+.005 at 330 nm, R= 0.027+.002 at 380 nm, and R= 0.075+.02'. b ratio of pre-exponentials cmeasured with

residues),

GlyTrpGly

peptides

contrast

We find while

with

correspond attribute single

report with

ours

photon

chromator

Their

(AlaTrpAlaGly

fluorescence

only

a single

the most

this

lamp excitation.

and GlyTrpGlyGly.

two exponential they

nitrogen

difference

being

decaying

exponential

populous

fluorescence

(2 nm bandpass)

rather

compared

with

components

for

Their

observed

resolution

lifetime than

on the latter

decay.

component

to the higher

counting

results

broad

two

GlyTrpGlyGly). these

peptides

reported

lifetimes

in our measurements. of the picosecond

system band

and to our

filters

We

laser/

use of a mono-

for

the

detection

of the emission. The fluorescence the fluorescence double

exponential

each wavelength, in

the proportions

components

were

spectrum

decay

of LysTrpLys

at several

decay

kinetics

emission were

~1 = 2.22 + .07 ns, of

according

resolved

with

similar

TZ = 0.86 + .05 ns with The emission

to expressions

912

by measuring

At all

wavelengths.

observed

the two components.

calculated

was time

lifetimes

at

a small

variation

spectra

outlined

wavelenghts

of both earlier

(1)

and

Vol.

94, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNlCATlbNS

Wavelengthxnm-' Figure

are

2.

Time Curve

shown

resolved a, 2.2

in figure

concentrations from

spectra of LysTrpLys at pH 4.9 Curve b, 0.9 ns component.

fluorescence ns component;

Both

2.

have a maximum at ~350 nm.

of the 0.9 ns and 2.2 ns components

these

spectra

according

to the

where

10.9/12.2 of the

maximum

found

0.9 ns and 2.2

results

the peptides than

contrast

the short

the long

similar

"Z-3

max

fluorescence

intensities

ns components.

fluorescence

aqueous the

different

at the spectral

The concentration

ratio

was

tryptophan

fluorescence conformers

component

for

is

Moreover

rationalization fluorescence

decay

aqueous

present

tryptophan.

in higher

in LysTrpLys

tryptophyl

residue

of the indolyl

of each was estimated. ground

of the dual in

behaviour

(7) nmr study

population

populous

obtained

both

In

concentration components

have

spectra.

fractional

the most

those

component.

from our

In a recent

with

lifetime

lifetime

Following

that

was estimated

to be 1.12. These

for

of the

[0.9]/[2.2]

of the

2.2

* *

the ratio

The ratio

expression:

fi= is

26'C.

state

terms

exponential

of different

rotamers,

in the tripeptides ring

in

these

of oligopeptides of three

On examination rotamer,

that

913

originates

with

observed

we suggest from

molecules. containing

rotamers

decay

about

tryptophan,

the

the C - Cg bond a

of space the amide

filling carbonyl

of the

models

of

on the

Vol.

94, No.

3, 1980

a-carbon residue

staggered

between

may readily

take

terminal

amino

group

BIOCHEMICAL

is

group

a very

suggest populous

in

of the

of this

fractional

amount

rotamer of the

of the

330 and 380 nm for

(1)

to the that

the ammonium

Therefore

as this,

we

the most

when we compare

by n.m.r.

0.9 ns component

ratio

(for

GlyTrpGly)

in LysTrpLys

and find

the

of pre-exponentials

tripeptides

components

in these

molecules

fluorescence

decay

parameters

acid

the

fractional

and of the them to be

the

for

implies

that

changed

significantly.

the

Clearly

several

spectra

and

(b)

(a)

that

that

at the

two

the

by the nature

suggest

that

in the

interactions

peptide

rather

than

for

the indole

picosecond temporal

ratio

of the

are markedly

of pre-exponentials of the

experiments to elaborate

the dual

bonds

are

local ring

interactions is

demonstrate

this

with

the peptide

has

full

observations.

an appropriate ring

kinetics

in different

peptide

particularly

the indolyl

decay

This

in proteins.

originate

from

conformers backbone

of the at the

attached. the

dye laser/single components

with

not

different ~0.03.

uf these is

ring

in

suggested,

the origiu

exponential

of the indole

being

conformers

Pi-acetyltryptophanamide

of the peptide

changes

The results

AlaTrpAlaGly

population

show that

The results

where

the

in order

interactions

to resolve

measured

1 suggests

are not much affected

additional

resoiution,

pumped,

in Table

similar

tetrapeptide

with

for

point

have

fractional

Uur resuits model

the

tripeptides

spectral

given

and lifetimes

residues.

The results from

found

component

supported

as measured

proximity

indolyl

(0.53).

Inspection

amino

is

COMMUNICATIONS

the

fluorescence.

decaying

assignment

closer

We have

of indole

fast

RESEARCH

on the b-carbon,

in even

tryptophan.

quencher

This

population

identical

up a position

efficient

rotamer.

EIIOPHYSICAL

the two hydrogens

than

the assignment

AND

improved photon

in peptide

914

capability counting studies.

of a synchronously fluorescence

decay

system

Vol.

94, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Acknowledgements The authors for

their

Northern

co-operation for

Biomathematics invaluable

thank

Dr.

Lawrence

in making

Cramer

the laser

the Loan of the multichannel section computational

his

equipment analyser;

of the Division assistance;

for

of Biological Mr.

D. Krajcarski

assistance;

Spectra

available

to us;

Mrs.

Lise

Sciences for

Bramall

Physics Tracer of

NRCC for

expert

technical

assistance. References 1. 2. 3. 4. 5. 6. 7.

Szabo, A.G., and Rayner, D.M. (1980) J. Am. Chem. Sot. (in press). Rayner, D.M., and Szabo, A.G. (1978) Can. J. Chem. 56, 743-745. Szabo, A.G., and Rayner, D.M. submitted. McKinnon, A.E., Szabo, A.G., and Miller, D. (1977) J. Phys. Chem. &, 1564-1570. Rayner, D.M., McKinnon, A-E., Szabo, A.G., and Hackett, P.A. (1976) Can. J. Chem. 54, 3246-3259. Werner, T.C., and Forster, S.L. (1979) Photochem. Photobiol. 2, 905-914. Baici, A., Rizzo, V., Skrabal, P., and Luisi, P.L. (1979) J. Am. Chem. Sot. 101, 5170-5178.

915