The effect of Mg++ on the conformation of the Ca++-binding component of troponin

Vol. 49, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFFECT OF Mg++ ON THE CONFORMATION OF ++ COMPONENT OF TROPONIN. THE Ca -BINDING Yukishige Kawasaki and Jean-Paul van Eerd Department of Physics and Institute of Molecular Biology, Faculty of Science, Nagoya University, Nagoya, Japan 464. Received

September

22,1972

SUMMARY Mg++ like Ca++ induces a conformational change in -+-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 90 % of the change in optical rotation induced by Ca++. The apparent bindin constant of Mg++ to the Ca++-binding component is than that of Ca++. Circular dichroism 5 x 10 3 M-l , much smaller measurements show that these changes are simple helix-coil transitions. Unlike the Ca++-induced conformational change, the Mg++-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning. INTRODUCTION Muscle Ca ++ '.

Troponin

of muscle,

is

subunits: that

contraction (TN), the

inhibits

the

thin

binds

amounts

of

a component

Ca ++ (TN-C),

between

actin

and myosin

to tropomyosin,

(TN-T) 3 . Recently, a large

concentration

binds

interaction

by micromolar

located on the thin filaments of three Ca ++ 2 . TN consists

for

that

that

filaments

undergoes

TN-C

regulated

a protein

receptor

a component

and a component the

is

it

conformational

another

protein

has been

reported

change

of that ++

when the Ca range 4,5 . However,

changed in the micromolar ++ the effect of Mg , whose intracellular concentration exceeds Therefore, we studied that of Ca ++ 6 , might be considerable. the

effect

is

(TN-I),

and in

of Mg ++ on TN-C,

present

evidence

that

change

in TN-C,

though

MATERIALS

AND METHODS

Preparation

of Muscle

(TM) were prepared

Mg++ can also not

communication,

induce

Troponin

rabbit

skeletal 898

we

a conformational

a physiologically

Proteins.

from

Copyright 0 1972 by Academic Press, Inc. AN rights of reproduction in any form reserved.

this

important

(TN) muscle

one.

and tropomyosin by the method

BIOCHEMICAL

Vol. 49, No, 4, 1972

of Ebashi

et a17.

of Greaser checked

electrophoresis

fluorescence prepared

in

0.3

Labelling

of TM.

NaHC03 and 0.1

M XC1 at O°C.

and was dialysed

ratio

and was 3:l

Fluorescence

after

was used

276 nm. Tryptophan excitation

fluorescence

for

for

the

respectively.

340 nm in

and Circular

at

10 mM

standing

for

two days.

The

solution

was

the

assuming

a value

of

110 for

Percentages calculated

fluorescence

306 nm after

complex

346 nm

J-20

A Jasco

spectra-polarimeter dichroism

rotation

measurements

at 233 nm,

[e],

the mean residue

were obtained

molecular

p-structure by Greenfield

and and

Fasmanl'. ca++

and Mg

++ Concentrations.

The Ca++ concentration

899

The

and TMDNS.

Measurements.

ellipticity,

as described

at

at

at 500 nm after

TN-T

and circular

e-helix,

Tyrosine

of TN-C and TN-T.

of TN-C,

Dichroism

of

spectro-

excitation

was observed

complex

mean residue

mean residue

were

fluorescent

measurements.

of TN-T

rotation

and the

random-coil

MPF-2A

and a Jasco

[m’l 233,

TN-C.

mixing

to TM was observed

the

The reduced

for

M KC1 for

fluorescence

fluorescence

optical

was left

initial

A Hitachi

ORD/UV 5 spectrophotometer

weight

0.1

the

dialysis.

of DNS labelled

Rotation

were used

was

and was dialysed

(DNS) , in

the

at 290 nm in

at

Optical

6 M urea

5-sulfonate

of TN-C was observed

excitation

tryptophan

with

against

Measurements.

fluorescence

after

in

dodecyl

of TN-C and TN-T

The mixture

of DNS to TM in

photometer

of sodium

TM was mixed

1-dimethylaminonaphtalene

molar

and TM was

showed negligible

components

method

mM NaHC03.

Fluorescence

12 hours

the

TN-T

the presence

measurements

these

using

of TN-C,

in TN-C and TM. The complex by mixing

against

4:l

The purity

Fluorescence

sulfate'.

dye,

TN-C and TN-T were isolated

and Gergely8.

by gel

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

Vol.49,No.4,

1972

regulated

using

glycol

bis

addition

neglected

than

of CaC12.

the

complete

the

constant

a Ca ++-buffer

for

that

At least

1 mM EGTA was necessary

-Ca ++ state

We

binding Mg++ is

only

Ca++

(4 x lo7

for

was regulated

biuret

the

binding

is much smaller The Mg++ concentration

at pH 7.3)12.

MgC12 in the

buffer,

presence

of

4 mM EGTA and

pH 6.8. Protein

concentrations

were determined

method13.

RESULTS AND DISCUSSION Mg ++ -induced Conformational effect

to

40 at pH 7.3 which

Concentration.

by the

in TN-C at pH 7.3. ++ Mg and EGTA because

between

by adding

10 mM Na-cacodylate Protein

of CaC12 and 1.6 mM EGTA (ethylene

ether)-N,N' -tetraacetic acid) in 60 mM buffer, pH 7.3 11 . A correction was made in the ++ of the Ca concentration for a slight change in pH

calculation

achieve

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

@-aminoethyl

Na-cacodylate

upon

BIOCHEMICAL

of MgC12 on the

Change tyrosine

in TN-C.

Fig.

fluorescence

1 shows the

intensity

(I)

and

the

optical rotation at 233 nm ([m'l,,,) of TN-C in the absence ++ of Ca . Addition of 1 mM EDTA (ethylenediamine tetraacetic acid) did is

to TN-C in the not

induce

necessary Both

means

that

any change to achieve

I and not

in TN-C.

of the

change

optical

constant

observed

in Fig. Binding the

to remove

increase but the

also

induced

by Ca++.

on TN-C determined

Constant

fluorescence

5

x

Mg++,

Therefore

added.

Mg++ can induce

intensity

induced

1 is

contaminating

when MgC12 is

change

in fluorescence

of Mg++

in

state,

in the -Ca++ state. the -Mg ++ state.

Ca '+

However

rotation

transition Change

[m'1233 only

change

in

-Ca++

no EDTA

This

a conformational

by Mg++ is only

and 80 % of the The apparent from

the

36 %

change

binding

midpoint

of the

M-1 .

lo3

of Ca++ to TN-C by Mg++ . intensity

900

of tyrosine

We

in TN-C as a

BIOCHEMICAL

Vol. 49, No. 4, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-NJ -=v

0.7

+/(

I

06 s-

*.+Ca

state

4/‘k

f 0.5

/+ If

0

I MgCl2

2

3

added

7

(mM)

Fig. 1.

9

0

7 PC0

6

5

Fig. 2. Figure 1. The effect of Mg++ on TN-C. o - 0, relative fluorescence intensity, l - l , reduced mean residue optical rotation at 233 nm. Protein concentrations are 0.16 mg/ml for the fluorescence measurements and 0.2 mg/ml for the optical rotation measurements. Temperature 30°C. The initial decrease and final increase in fluorescence intensity and optical rotation are due to the addition of 4 mM EGTA and 4 mM CaC12 respectively. Vertical bars represent standard deviations. Figure 2. The change in the fluorescence intensity of the tyrosine residues in TN-C as a function of the Ca++ concentration (pCa). The' fluorescence intensities are expressed as a fraction of the Ca++-' induced change in fluorescence intensity. 0 - 0, without MgC12, l - a, in the presence of 2 mM MgC12. Protein concentration is 0.09 mg/ml. Temperature 300C. Vertical bars represent standard deviations.

function

of the

of Mg++

(see Fig.

between

the

while

Ca++ concentration 2).

binding

constant

binding

constant

8.0

the

The fluorescence

pCa values

between

in

8.5

and 5.0

and 6.0 in

the

presence

intensity in

the

presence

absence of Mg++.

and absence changes of Mg++ The apparent

of Cat+ to TN-C is 4 x lo7 M-l in the absence of Mg++ and 6 x lo6 M-1 in the presence of Mg++. The apparent of Cat+

to TN-C is

901

shifted

to a lower

value

Vol. 49, No. 4,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Wave

Longlh

( nm

)

Figure 3. Circular dichroism spectra of TN-C. Continuous lines represent experimental data. Dashed lines represent reconstituted spectra calculated from the data shown in Table I. Protein concentration is 0.25 mg/ml in 10 mM Na-cacodylate buffer, pH 7.3. Temperature 27OC. The o tical path length is 1 nun. 1.6 mM EGTA is present in the -Ca P+ state, no EGTA in the +Ca++ state. 1.6 mM EGTA and 2mM MgC12 is present in the +Mg++ state. Vertical bars represent errors due to line width. by Mg++. the

The binding

one obtained

experiments, achieve

before4.

we used only

the

Also,

fluorescence in

the

assume

circular

This

4 x 10'

is

M-l

because,

is hihger

2, the

is

too

low to

(see Method).

binding the

than

in previous

0.5 mM EGTA which

of Ca++ to TN-C is weakened

Ca++ -induced

intensity

is smaller in ++ of Mg . These results

change

the presence

in

the

of Mg++ than

can be understood

if

we

that

(1) the Mg++ -induced conformational change is not as the Ca ++ -induced one, and that (2) Mg++ and Ca++

for

the

Determination structure

Fig.

at low pCa,

absence

so large compete

of

-Ca ++ state

complete

As shown in by Mg++.

constant

same binding

of the in

the

dichroism

sites.

Secondary

conformation

Structure.

The secondary

of TN-C was calculated

measurements

902

(see Fig.

3 and Table

from I).

The

BIOCHEMICAL

Vol. 49, No. 4, 1972

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

”

I

(TN-Cl-(TN-T) IO-

V,+Ca

0.9-

II +v--0

51 ate

0

I 11 v-G-0 a 1.0-

stat

e

*,+Ca

state

I 0

I I MgC12

(TN-C)-(TN-T)-(TM~~~)

08I 2 added

(mM

)

Figure 4. The effect of Mg++ on the complex of TN-C and TN-T, and the complex of TN-C, TN-T and TMDNS. o -0, corn lex of TN-C and TN-T, l - l , complex of TN-C, TN-T and TMDi s. The protein concentration is 0.23 mg/ml for the complexof TN-C and TN-T. 0.2 mg/ml TMDNS and 0.1 M KC1 is present in the complex of TN-C, TN-T and TMDNS. Other conditions are the same as in Fig. 1. The addition of CaC12 to the complex of TN-C, TN-T and TMDNS made the complex unstable and often caused precipitation to occur. Therefore, we could not observe the reversibility of the fluorescence intensity of DNS bound to TM.

Table

I Secondary

structure

*helix(%) -Ca++

-Mg++

-Ca++,+Mg++ +Ca ++ -Mg++ +Ca

++

Table

+Mg I.

++

from

results

indicate

simple

helix-coil

Propagation

random-coil(%)

S-structure(%)

36

14

50

48

14

38

52

12

36

52

12

36

The composition

calculated

of TN-C

the

of the

data that

of the

secondary

shown in Figure the

change

in

structure

of TN-C

3.

conformation

of TN-C are

transitions. Conformational

Change.

903

The Ca++-induced

Vol. 49, No. 4, 1972

BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in TN-C can be propagated to TM via TN-T 14 . Therefore, we examined whether the Mg ++ -induced change could also be change

propagated is

to TN-T

absent

and to TM. TN-T

in TN-C.

Therefore

we can obtain

in

of TN-C and TN-T.

complex

fluorescence

of DNS labelled

information Fig.

of TM in the

4 shows that

change

neither

concluded

the

tryptophan,

when we observe

of tryptophan, the

contains

information

the

of TN-T

Likewise,

of TN-C,

Mg++ -induced

change

fluorescence selectively

by observing

to TM, we can obtain complex

which

TN-T

the

selective and TM DNS .

in TN-C induces

a

in TN-T nor in TM. Therefore, it can be the Mg ++ -induced change in TN-C has no

that

physiological

meaning.

CONCLUSION only

Ca ++ but

in TN-C,

but

Not change induced

one.

change

cannot

it

Unlike

this the

also

a conformational

change is not so large as the Ca++++ Ca -induced change, the Mg++ -induced

be propagated

has no physiological

Mg++ can induce

to other

muscle

proteins.

Therefore

meaning.

ACKNOWLEDGEMENT The authors wish to present their thanks to Prof. F. Oosawa, Prof. S. Asakura and Dr. K. Mihashi for their stimulating discussions. This work was performed while one of us JPvE held a postdoctoral fellowship of the Japan Society for the Promotion of Science. REFERENCES 1. 2. 3. 4.

L. V. Heilbrunn, The Dynamics of Living Protoplasma, Acad. Press, New York, (1956). S. Ebashi and M. Endo, Progr. Biophys. Mol. Biol. 18, 123, (1968). T. Wakabayashi and F. Ebashi, J. Biochem. 69, S. Ebashi, 441, (1971). J. P. van Eerd and Y. Kawasaki, Biochem. Biophys. Res. Comm.

5. 6. 7. 8.

47,

859,

(1972).

A. C. Murray and C. M. Kay, Biochemistry, 11, 2622, (1972). L. B. Nanninga, Biochim. Biophys. Acta, 54, 338, (1961). S. Ebashi, A. Kodama and F. Ebashi, J. Blochem. 64, 465, (1968). M. L. Greaser and J. Gergely, J. Biol. Chem. 246, 4226, (1971).

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BIOCHEMICAL

Vol. 49, No. 4,1972

9.

10.

K. Weber and M. Osborn, J. Biol. N. Greenfield and G. D. Fasman, (1969)

11. 12. 13. 14.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Chem. 244, Biochemistry,

4406, (1969). 8, 4108,

.

G. Schwarzenbach, H. Senn and G. Anderegg, Helv. Chim. Acta, 40, 1886, (1957). G. Schwarzenbach, Die komplexometrische Titration, (1955). A. G. Gornall, C. J. Bardawill and M. M. David, J. Biol. Chem. 177, 751, (1949). for publication. Y. Kawasaki and J. P. van Eerd, Submitted

905