The effect of sulfhydryl blocking groups on the thermal unfolding of αα tropomyosin coiled coils

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1279-1283

BIOCHEMICAL

Vol. 166, No. 3, 1990 February 14, 1990

The Effect of Sulfhydryl Blocking Unfolding of aa Tropomyosin Marilyn

Emerson

*Department

of Chemistry,

tHoward Hughes Washington Received

Holtzer*,

December

Alfred

Holtzer*,

Washington

Thermal

and Dan L.

University,

Medical Institute University School

18,

Groups on the Coiled Coils

St.

Crimminst

Louis,

Core Protein/Peptide of Medicine, St.

MO

63130

Facility, Louis, MO

1989

Equilibrium thermal unfolding curves from circular dichroism are given for tropomyosin and for aa tropomyosin blocked at Cl90 by a) carboxyamidomethylation; b) carboxymethylation. Although commonly assumed to be benign, these blocks in fact produce some weakening. All three substances are virtually completely a-helical at low T. Fraction helix vs T for parent protein is apparently monophasic (single inflection point). The curve for carboxyamidomethylated protein is very close to that of the parent, but is biphasic, with a small "pretransition". The curve for carboxymethylated protein is prominently biphasic, with a much larger pretransition. Some implications for the molecular model of these equilibria are discussed. aa

0 1990

Academic

Press,

Inc.

The native which

the

chains

one or more to form

are

cysteine

distinct

residues

Since

introduce

from

It

blockers

are

stress,

are

conformationally

(1).

reducing

not

the

reported

only

easily

benign.

(2,3).

or

either

side

coil

effects,

that

unfolds

In studies

of the by

blocking

the

are malodorous blocking

and stable of physical

have

molecule

carboxymethyl

attached A number

by

in

can be oxidized

must be prevented

solvent

agents

or other

they

cross-linked

oxygen

in

coiled

Many tropomyosins where

molecules

agent

has been

(2)

a-helical

positions

by ambient

absorbances

amidomethyl but

interior

non-crosslinked

appropriate

of choice.

a two-chain,

The resulting

reducing

interfering

the method

in

disulfide.

of sufficient

sulfhydryls.

is

and in register

crosslinking

therefore,

inclusion

molecule

in parallel

an inter-chain

in a manner latter,

tropomyosin

or

is (4)

sometimes

or carboxyunder

studies

thermal have

Abbreviations are; IAA, iodoacetic acid; IAM, iodoacetamide; Tm, reduced cardiac (au) tropomyosin; CAM-Tm, carboxyamidomethylated tropomyosin; CM-Tm, carboxymethylated tropomyosin; DTT, dithiothreitol; CD, circular dichroism. Aqueous solvent media are characterized by giving the millimolarity of each substituent as a subscript to its formula (or its standard abbreviated name) followed by the pH in parentheses. ODD6-291X/90 $1.50

1279

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 166, No. 3, 1990

been

based

extant

data

circular metry

least

base

in part

is

rather

dichroism

(6)

We choose thermal

cysteine

Experimental

small

call

rabbit

unfolding (C190)

upon

AND BIOPHYSICAL

that

assumption

and dates

(CD) instrumentation

experiments

point. its

at

BIOCHEMICAL

this

into

cardiac is well

in each

is

from

However,

since

back,

improved

some time

now available, we have

tropomyosin

documented

284-residue

(4,5).

question,

(aa)

RESEARCH COMMUNICATIONS

and recent

and because

calori-

reinvestigated

as our it

test

this

case,

has only

the

because

a single

chain.

Methods

The preparation and concentration determination tropomyosin has been described previously (7).

of rabbit

cardiac

(aa)

Reduced aa-tropomyosin was blocked at Cl90 using two different blocking reagents, iodoacetic acid (IAA , Sigma purified, packaged under nitrogen) and iodoacetamide (IAM, Sigma), as previously described (8). In this procedure, the final reaction medium is GdmCl ,,,oNaC1,,,TrisS,EDTA,(8.5)~ and the reactant concentrations are 0.5 mg/mL for protein and 0.5 mM for IAM or IAA. In our present experiments this corresponds to a molar ratio of blocker to protein sulfhydryl of 33. With either blocker, these conditions can be varied over wide limits without altering the product. Variation in protein concentration of 0.5-5 mg/mL and in blocker to protein-sulfhydryl ratio of 16:l to 3OO:l give the same product. Nor does it matter if the DTT (up to 30 mM) used for reduction is left in the solution, provided that a stoichiometric amount of blocker is used to overwhelm the DTT. The C190-blocked samples were characterized by NaDodSO,/PAGE (using 9% Laemmli gels), sulfhydryl titration (9), UV absorbance and CD spectroscopy, and amino acid composition analysis. Both blocked samples show a single No material was seen in the 66 kd electrophoretic band in the 33 kd region. region for any sample. The mobility of the IAM-blocked material is indistinguishable from that of unblocked, reduced parent a-tropomyosin; the mobility of the MA-blocked material is slightly less. Sulfhydryl titrations averaged 0.07 + 0.1 SH/chain for IAM-blocked samples and 0.03 f 0.1 for MA-blocked samples. These are within experimental error of the expected value of zero. UV absorbances and CD spectra of both blocked proteins in benign media at low temperature (< 3°C) were indistinguishable from parent. Amino acid analysis was employed to check for possible damage to methionines in the blocking (10). No differences were seen in the methionine (or any other residue) count in Nor was there any difference in parent and the two blocked proteins. methionine sulfone content after performic acid oxidation (10). CD measurements were performed using a Jasco J50OA instrument interfaced via Jasco IF50011 to a THE personal computer. Data collection and analysis have been described in detail, as have temperature control and measurement and the calculation of fraction helix from the ellipticity at 222 nm (7,ll). All denaturation curves reported here are completely reversible.

Results

and Discussion

The results temperature.

appear Experimental

in Figs. curves

1 and 2 as fraction for

reduced 1280

aa

helix tropomyosin

(from

CD) vs

(Tm) have

been

Vol.

166, No. 3, 1990

BIOCHEMICAL

published

in

some detail

agreement

with

those

our

new data

accommodates point)

the

at our

and,

1.10

at

current

level

points

The data define

points)

that

circles

for

in Fig.

(solid).

2, along

below

that

for

of both

of unfolding

(> 50°C)

in quantitative

spline

curve

curve).

through

The curve

has a single

concentration

(CAM-Tm) close

are

to those

distinctly,

inflection

in the

tropomyosin spline

CM-Tm fall

curves

(1.10

shown

as open

for

the parent,

biphasic

of the parent

with

parent

(i.e.

protein

very but

that

the

1 (dotted

monophasic

carboxymethylated

The data

in Fig.

are

of resolution.

CAM-Tm fall

is below

RESEARCH COMMUNICATIONS

measurements comparison,

at identical

a slightly,

The data

shown

tropomyosin

for

former

to facilitate

and appears

carboxyamidomethylated 1.

Our present

mg/mL is

data

Experimental

(7).

AND BIOPHYSICAL

curve range

but

(three

are

for

Tm (dotted)

on a drastically

biphasic

seems to be indistinguishable

squares

for in Fig.

the

inflection

40-50°C.

(CM-Tm)

and of CAM-Tm in the

mg/mL)

range

shown

and CAM-Tm curve,

30-50°C. in

the

as open

markedly

The last three

stage

cases.

1 .oo

2 2 2c

0.50

t; I

0.00

0.00

0

20

0

1

40

60

00

02

WC

0

20

40

60

trc

Figure

1. Fraction helix (from CD) vs T ("C) for Tm and CAM-Tm in NaCl,,,NaPi,, (7.4). Both are at 1.10 mg/mL. Dotted curve is spline through Experimental points (open squares) are for CAM-Tm. data for Tm. Size of squares indicates experimental error.

Figure 2. NaCl,,,NaPi,, for reduced Experimental

experimental

Fraction helix (from (7.4). All at 1.10 Tm. Solid curve is points (open circles)

CD) vs mg/mL. spline are

T ("C) for Tm, CAM-Tm, and CM-Tm in Dotted curve is spline through data through data for CAM-Tm (see Fig. 1). for CM-Tm. Size of circles indicates

error. 1281

80

Vol.

166, No. 3, 1990 From the

roughly,

empirical

but

not

differently as,

it.

distinct it

is

and chemical

effect

nature

-CH,COO- or

prominent

weakening

might

is

hydryls were

effect

are

already

two

and E218),

which

do not

In spite

of their

measurements represents

ture

leave

the

say)

If

the would

heptet

two

surrounding

with

as usual. which

the

turns

It accessible

within is

not

cost

experimental at all

to the

clear

molecule.

the

such

the

negative

charging

charge (and

on CM-

of the

sulf-

in helix

and 8.3.

Moreover,

negatively

charged

a

Because

on the parent

energy

that

this lesion

state

actually

are

attempted.

observing

Since

a population

minimum.

content

in parent

Tm

residues

coiled

is

introduced

at only

at somewhat

elevated

by complete acids small

and seemingly to form

(D137

be a decrease

on each

would

equilibrium

coil

temperaall

one site

of,

chain

(35"C, say,

one

immediately

the

chain

of,

would

remainder

coiled-coil content

three

per

temperature

provide

in helix

states

for

of unfolded

preferred

puzzling

at low

unfolding

eyelet

its

only

The results

the native

of amino

somewhat

of molecular

is

achievable

freedom would

structure.

no differences

results is

The resulting

sufficient

The result

is

terms

or so)

lesion.

loop-entropic

molecule

these

energy

seem to be nearly

(i.e.,

of little

free

of

any such destabilization.

a destabilizing

lowest

group

and seemingly

of the

further

positioned,

Gibbs

doubt

small can have

that

as large

a substantial

chain

a study

at pH 7.4

we are

global

no room for

substances. chain,

involved,

we thought

However,

in physical

are

per

is

(2,3).

produce

segment

matter.

simplicity,

when rationalization

site

rather

that

how a relatively

to see if

produce

CM-Tm unfolds

or a blocking

might

in order

interiorly

CAM-Tm unfolds

species

crosslink

performed

measurements

that

that

a pretransition

a large

in CM-Tm,

would

between

that

on such

clear

in crosslinked

so obvious

We therefore

parent

observed

CM-Tm displays

not

at pH 8.3

of the

there

is

is but

-CH,CONH, at a single

cause.

Tm as a control)

parent,

how a covalent

so much larger

be the

it

of N-ethylmaleimide

it

innocuous

then,

the

one seen

obvious

on unfolding,

the effect

like

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of view,

In fact,

from,

Although size

point

precisely,

from

but

BIOCHEMICAL

of

be the

structure at most,

3%,

strategy

is

error. why this It

"quarantine

seems unlikely 1282

the that

aggressor"

a carboxymethyl

or

not

Vol.

166,

No.

3, 1990

BIOCHEMICAL

carboxyamidomethyl neither

coiled

coil

and therefore

is

In any case,

these

preting coil

results

group

could

nor

random

misinterpreted experiments

obtained

AND

nucleate coil,

BIOPHYSICAL

an entirely that

is

as unfolded; re-emphasize

on blocked,

labelled,

RESEARCH

new local

of strong however, the

COMMUNICATIONS

structure,

positive this

is

CD at 222 nm not

caution

necessary

or even

site-mutated

impossible. in

intercoiled-

proteins.

Acknowledgments Supported United States Association. acid analyses.

by Grant GM-20064 from the Division of General Public Health Service and a grant from Muscular We thank Mr. R.S. Thoma for technical assistance

Medical Sciences, Dystrophy in the amino

References 1. Fraser, R. and MacRae, T. (1973) Conformation in Fibrous Proteins, pp. 419-468, Academic Press, New York. 2. Lehrer, S. (1978) J. Mol. Biol., 118, 209-226. 3. Holtzer, M.E., Askins, K., and Holtzer, A. (1986) Biochemistry, 25, 16881692. 4. Potekhin, S. and Privalov, P. (1982) J. Mol. Biol. 159, 519-535. 5. Yukioka, S., Noda, I., Nagasawa, M., Holtzer, M.E., and Holtzer, A. (1985) Macromolcules, 18, 1083-1086. 6. Sturtevant, J., Holtzer, M.E., and Holtzer, A. manuscript in preparation. 7. Holtzer, M.E., Holtzer, A., and Skolnick, J. (1983) Macromolecules, 16, 173-180. 8. Bracken, W.C., Carey, J., Holtzer, M.E., and Holtzer, A. (1988) Biopolymers, 27, 1223-1237. 9. Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70-77. 10. Glazer, A.N., DeLange, R.J., and Sigman, D.S. (1985) Chemical Modifications of Proteins, pp. 90-91, Elsevier Biomedical Press, New York. 11. Holtzer, M.E., Bracken, W.C., and Holtzer, A., Biopolymers, in press.

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