Ca++ induced conformational changes in the Ca++ binding component of troponin

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ca++ INDUCED CONFORMATIONAL CHANGES IN THE Ca++ BI"IJDING COMPONENT OF TROPONIN Jean-Paul van Eerd and Yukishige Kawasaki Institute of Molecular Biology and Department of Physics, Nagoya University, Nagoya, Japan 464.

Received April

17, 1972

The Ca++binding component of troponin undergoes reversible conformational changes in the presence or absence of micromolar amounts of Ca++. The presence of Ca++ causes an increase in the fluorescence intensity of the tyrosine residues as well as an The binding constant is 5.106M-1. increase of the helix content. Fluorescence polarisation shows that the motion of the tyrosine residues is much smaller in the presence than in the absence of Ca++. Thus the Ca++ binding component of troponin appears more rigid in the presence than in the absence of Ca++. Summary.

Muscle which

contraction

released

the nerve,

from the

bind

assumed that

troponin,

which

troponin. into

interest In three

(Greaser

Ca++. This usually

Phe/Tyr

component

conformation

induces

1972, by Academic

to myosin.

has been fractionated

only

substructure

et al. 3),

one of which identified

because of the

and 4 sub-

appears as it

absence

of

to bind

has an unof tryptophan

ratio5. whether

indeed

the conformation

on the presence

Ca++ induces

component. 859

Copyright 0

actin

and ulti-

troponin

3 (Ebashi

is

change in

in the

can be readily

(CBC) changes

of this

filaments

a conformational

of

of 1 . It

recently

and Mueller2),

to test

that

on excitation

on the thin

in the binding

ratio

amounts of Ca++,

a change in tropomyosin

laboratories

low E2So/E2b0

We have found

located

and Gergely4),

We set out ding

reticulum

has arisen

component

and the high

sarcoplasmic

resulting

2 (~Hartshorne

units

by small

Ca++ induces

in turn

in actin,

Accordingly

regulated

to troponin

generally

mately

is

Press, Inc.

of this

or absence

a striking

Ca++ binof Ca++.

change in the

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS. Preparation rabbit

of

skeletal

muscle

al. 6 . Troponin inability

in the

troponin

described

the

absence

and CBC. Troponin

according

was checked

to regulate

myosin from

troponin

for

to the

procedure

the absence

of

electrophoresis

in the presence

and showed very

little

in 6M ura as

CBC showed a single of sodium dodecyl

tryptophan

acto-

CBC was prepared

on DEAE Sephadex

and Gergely4.

et

by its

synthetic

of any added tropomyosin.

from

of Ebashi

of tropomyosin

superprecipitation

by chromatography

by Greaser

was prepared

fluorescence

band on gel

sulphate

(SDS)'

on excitation

at

290 nm. Ultraviolet with

absorption.

The UV spectrum

a Zeiss model PMQ 2 spectrophotometer. Fluorescence

cence

An Hitachi

spectroscopy.

spectrophotometer

was used for

was water-jacketed

Haake thermoregulator.

Tyrosine

after

always

rin's

less

and connected

fluorescence

a

at

306

than

at 276 nm. The OD value of the sample was 276 0.10. Polacoat ultraviolet polarising filters measuring

the degree

P, at

a number of temperatures

was used to change the viscosity Optical

Rotation

Dispersion

was a Jasco Autospectrometer ted from

with

was observed

of

fluorescence

The rotary molecular motion was calculated 8 equation , from observations of the degree of

polarisation cerol

measurements.

excitation

(105 UV) were used for risation.

model MPF-2A fluores-

fluorescence

The sample compartment

nm.,

of CBC was measured

the

[ml ;33 value was taken 9 .

specific of 14,700,

ca++ concentration.

at constant

measurements.

and for

at

+-I- concentrations Ca

860

Per-

fluorescence Gly-

temperature.

The apparatus content

233 nm. For

0% helix

using

and viscosities.

UV 5. The helix

rotations

pola-

was calcula-

100% helix

a [ml;33

used

value

a of

were adjusted

2200

using

BIOCHEMICAL

Vol. 47, No. 4, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

++ of Ca and 0.5 mM EGTA (ethylene

a Ca-buffer

tetraacetic

ethyl

ether)-N,N' 10 at pH 6.8 . Protein

acid)

bis

(n-amino-

in 10 mM cacodylate-HCl

concentration.

Protein 11 the method of Lowry .

mined using

glycol

concentrations

buffer

were deter-

.15-

d d

.I o-

.05-

I 260

I 260 WQVO

Figure

I 300 Length (n m )

1. Absorption spectrum of the Ca+' binding component of troponin. The protein concentration is 0.26 mg/ml in 0.3 mM NaHC03. The spectrum is identical in the presence and absence of Ca++.

RESULTS AND DISCUSSION. Ultraviolet gure

spectrum.

1. The maximum at

tyrosine

absorption.

The UV spectrum

275 nm. corresponds

The maxima at 259,

pond to maxima of the phenylalanine the

finestructure

served than

of the

because those

the molar

of tyrosine

phan is

present

structure

of

extinction

and the

tyrosine

intensity

In most proteins cannot

of phenylalanine

absorption and helix

861

is

low5,

easily

content.

be ob-

is much lower

In CBC however,

content

the

266 and 269 nm. corres-

absorption

and tryptophan.

shown in Fi-

to the maximum of

absorption.

phenylalanine

the phenylalanine

Fluorescence

of CBC is

no trypto-

so that

the

fine-

can be detected. The helix

content

Vol. 47, No. 4, 1972

BIOCHEMICAL

RESEARCH COMMUNICATIONS

content and change in fluorescence intensity 2. The helix of CBC versus Ca++ concentration at 250C. o-o ; helix content,AA; fluorescence intensity. Vertical bars represent possible errors due to line width. Protein concentrations are 0.45 and 0.12 mg/ml for the helix content and the fluorescence measurements respectively. The binding constant calculated from these curves is 5.106M-l.

Figure

and the

tyrosine

fluorescence

the Ca++ concentration distinct crease

AND BIOPHYSICAL

increase in the

The binding curves

is

5.4x106M-1 method.

intensity

are plotted

in helix

fluorescence

constant

for

5x106M-1 . This

of

CBC as a function

in Figure which

content,

2. There

is

paralleled

The increase

a very by an in-

intensity of the tyrosine residues. ++ estimated from these the Ca -CBC complex,

in good agreement with 12 by Hartshorne and Pyun using

obtained

is

of

is

in helix

content

and the

the value

of

the Chelex

increase

100

in fluores-

cence

intensity could be reversed by adding EGTA to bind any ex++ in fluorescence intensity could not have cess Ca . The increase been caused by aggregation

of CBC molecules

but

only

by an intra-

molecular

change because the UV spectrum was identical in the pre++ sence and absence of Ca . The conformational change in CBC occurs ++ concentrations in the micromolar range, i.e. at the phyat Ca siological

concentration

Fluorescence the

Perrin's

plot

where muscle

polarisation. a straight

With line

contraction changing

is regulated. temperature

in the presence

a62

shows

of Ca++ (see

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7

a6 \

+-+++-h T/q

(OK / poise

)

Figure

3. The effect of temperature and viscosity on the fluorescence polarisation of CBC. o-o ; heating curve at pCa>8 a-0 ; heating curve at pCa=5.7.A-A; isothermal curve at 30°c and pCa>8.A-A ; isothermal curve at 30°C and bars represent the standard deviation pCa=5.7. Vertical of 3 observations. T is the absolute temperature and qis the viscosity of the solvent.

Figure

3).

straight

On the other

hand there

is a large

in the absence of Ca+*. This suggests ++ the tyrosine residues occupy sites of Ca

protein

molecule

the absence thermal

which allow only ++ of Ca , the tyrosine

activation

of motion

very

limited

residues

independent

the CBC molecule.

More accurate

tyrosine

residues

in CBC can be obtained

changing

viscosities

see Figure

of motion

is

at a constant 31, because

constant.

in that

For the

However,

overall

relaxation

times

fluorescence

thermal

of

of

plot

(isothermal

case the

in

motion

from a Perrin's

temperature

the

can undergo

of the

rotational

in the

within

motion.

easily

from a

that

line

presence

plot,

deviation

the with

Perrin's

activation

lifetimes

at 30°c we

used the values of 1.8 and 2.2 nsec in the absence and presence ++ These values were derived from a value of of Ca respectively. 2.6 nsec for at 23OC

the

fluorescence

13 and corrected

for

lifetime fluorescence

863

of

free

tyrosine

intensity

in water

changes

(Fi-

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Temperature

Figure

gure of

( “C

)

4. The temperature effect on the fluorescence intensity of CBC. o-o ; at pCa>8, m-o ; at pCa=5.7, e-8 ; the relabetween the tive difference in fluorescence intensity 2 conditions. Vertical bars represent possible errors due to line width. Using

4).

14.5

these

data

we obtained

a rotational

relaxation time ++ respecand absence of Ca

and 29.2 nsec in the presence

tively

at

30°C.

ture

variation

These values that

becomes rather

rigid

The temperature in Figure

4. Strong

observed,

while

Ca++ -free

CBC is

mational siological

support

CBC is

flexible

on the

addition

effect

on the

thermal

change in CBC is

in the of

with

of

therefore

tempera-

Ca++ and

Ca++.

the

in intensity raising

of the

absence of

fluorescence

quenching

the difference increased

the results

intensity

is

shown

fluorescence

can be

of Ca++-bound

CBC and

temperature.

The confor-

much more pronounced

at phy-

temperatures.

ACKXOWLEDGEMENTS. The authors like to thank shi for stimulating discussions. of us, JPvE, held a postdoctoral for the Promotion of Science.

professor F.Oosawa and Dr. K.MihaThis work was performed while one fellowship of the Japan Society

REFERENCES. 1. 2.

S.Ebashi and M.Endo, Progr.Biophys.Mol.Biol. D.J.Hartshorne and H.Mueller, B.B.R.C. 864

31,

18, 123, (1968). 647, (1968).

Vol. 47, No. 4, 1972

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T.Wakabayashi and F.Ebashi, J.Biochem. 69, 441, (1971) S.Ebashi, M.L.Greaser and J.Gergely, J.Biol.Chem. 246, 4226, (1971). M.L.Greaser and J.Gergely, Fed.Proc. 29, 463, (1970). A.Kodama and F.Ebashi, J.BiGhem. 64, 465, (1968). S.Ebashi, K.Weber and M.Osborn, J.Biol.Chem. 244, 4406,(1969). F.Perrin, J.Phys.Radium, 7, 390, (1926). N.Greenfield, B.Davidson and G.D.Fasman, Biochemistry, 5, 1630 (1967). G.Schwartsenbach, H.Senn and G.Anderegg, Helv.Chim.Acta, -'40 1886, (1957). O.H.Lowry, N.J.Rosebrough, A.L.Farr and R.J.Randall, J.Biol. Chem. 193, 265, (1951). D.J.Hartshorne and H.Y.Pyun, Biocim.Biophys.Acta, 2, 698, (1971). G.G.Vurek and N.Alexander, Science, 156, 949, (1967). R.F.Chen,

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