β Sheet to α helix transition in the binding subunit of cholera toxin

Vol. 105, No. 2, 1982 March 30. 1982

BIOCHEMICAL

AND BIOPHYSICAL

S SHEET TO cr HELIX TRANSITION Randall

M. Robinson,

RESEARCH COMMUNICATIONS Pages 398-403

IN THE BINDING SUBUNIT OF CHOLERA TOXIN

Maher

M. Hamed, and Wayne L. Mattice

Department of Chemistry Louisiana State University Baton Rouge, Louisiana 70803 Received

January

26,

1182

dichroism spectra have been measured for the binding SUMMARY: Circular subunit of cholera toxin in water and in the presence of dodecyl sulfate. In water the protein has an appreciable amount of S structure and almost no Q helix. In the presence of dodecyl sulfate the spectrum undergoes drastic change over a time period of approximately four hours, and at equilibrium resembles that expected for a chain with an appreciable amount of a helix but no p structure. The change in helicity is in good agreement with that expected from our formulation of the configuration partition function for the The conformational change may have an important relationship binding subunit. to the means by which the binding subunit permits penetration of the active subunit into the cell.

The enterotoxin Subunit

of Vibrio

B, molecular

toxin

to the cell

weight membrane

The specificity

(448).

11,60& via

in the binding

hours

between

Experimental mechanical conformational

changes

produced

upon their

interaction

subunit

B of cholera

toxin,

membrane

thought

environment

bears

(11).

This

in water

a similarity

(9).

If

to that

398

2-4

(6). statistical

rationalize peptides

methods which

B has

lag may arise

and hormonal

(12-15).

lipid

in the

of about

and theoretical

theoretical

of anionic

0006-291X/32/060398-06$01.00/0 Copright 0 1982 by .-lcademir Pres, Inc. A II righis of reprodrrclion in 0n.vform reserved.

a lag

lipids

GM1

35 of subunit

is

opioid

of the

shown to reside

to successfully

anionic

binding

in the toxin

in endogenous

these

to exist

loss

AB5 (1,2).

ganglioside

Arginyl There

change studies

with

in the presence

the structure

(10).

have been combined

for with

(9).

and fluid

dichroism

structure

responsible

has been

a conformational

peptides

conformation

toxin

subunit

interaction

process

of the

circular

methods

specific

ganglioside

implicated

for

is

of the

been

the necessity

(3),

interaction

portion

from

has the

of this

oligosaccharide

binding

cholerae

(16)

When applied predict

is quite

different

the ganglioside provided

a

by anionic

from

GM1 - cell lipid,

to

Vol. 105, No. 2, 1982 the

BIOCHEMICAL

conformation

dichroism major

of subunit

studies

conclusions

B might

indeed

reported

here

prwide

expected

from

theory.

experimentally

is

period

toxin

between

AND BIOPHYSICAL

found

to take

binding

RESEARCH COMMUNICATIONS

change

experimental

confirmation

The conformational

place

on a time

and fluid

Circular

upon binding.

scale

for

change

comparable

the

observed to the

lag

loss.

METHODS Subunit B of cholera toxin from Vibrio cholerae and dithiothreitol were obtained from Sigma Chemical Co. Sodium dodecyl sulfate was from Matheson, Coleman, and Bell, Protein solutions investigated had an approximate concentration of 0.0187 mg/ml. Concentration was determined from the absorbance and extinction coefficient at 280 nm (17). Circular dichroism spectra were measured using a Durrum-Jasco recording spectropolarimeter calibrated with d-lo-camphorsulfonic acid (18). The theoretical and experimental basis for the statistical mechanical calculations has been described earlier (16,lg). The specific equations used are those numbered 1, 2, 4, and 6 in Robinson et al. (15) RESULTS Circular throughout having

dichroism the

[e]

of subunit

spectral

= -5000

range

B in water

studied,

with

deg cm* dmol-'.

agreement

with

spectra

indicates

the virtual

reported

a single

1) is negative minimum

Shape and intensity earlier

absence

(Figure

(9,lO).

of u helix

are

near

in reasonable

The spectrum

and the presence

215 nm

in water

of appreciable

B

structure. Upon addition period

of dodecyl

of several

the double

hours.

minimum in Figure

2.

of the change

times

as long

are

dodecyl

sulfate

is

dithiothreitol.

temperature disulfide

disulfide dependence is intact

time

in

intense,

Development

bond

is an increase

to attain

involved less

circular

dichroism

of [0]222

equilibrium.

of helicity

bond between

cysteinyl

of the equilibrium (d [0]222/dT

in the

therefore residues

399

ten

spectrum

in

of excess by the

9 and 86,

is also

in Figure

suggests

presence favored

is

but roughly

The equilibrium

spectra are

depicted

The profile

15X, is

is

of

spectrum

20 minutes,

the rearrangement.

Over a

and development

The equilibrium

in about

by about

changes

in intensity

dependence

has been completed

is required

steps

the

of the u helix.

1, and the

multiple

of an intact

There

characteristic

depicted Half

sulfate,

smaller

54 and 84 deg cm2 dmol-1

presence

The when the K-l).

The

Vol. 105, No. 2, 1982

BIOCHEMICAL

L 200

0

1

220

1.

Circular

dodecyl at 25' Fig.

2.

100

0

0

2

dichroism of subunit sulfate. Conditions C, respectively.

obtained

of about

33%.

Time,

B in (A) water, were pH 7.0 at

at equilibrium A helical

circular

dichroism

contents

of 8% in water

Experimental

RESEARCH COMMUNICATIONS

30'

200

minutes

and (B) C and

0.003 M pH 7.04

Time dependence of [a],,, for subunit B in 0.003 M dodecyl sulfate, pH 7.04, 25" C. The line through the points was calculated for two consecutive first-order reactions. Rate constants were kl = 0.21 -1 min and k2 = 0.0069 mine1 , and ellipticities of the three species, in order of their appearance, were -4400, -8700, and -13900 deg cm2 dmol-I.

of [0],2,

content

240

X, nm

Fig.

value

AND BIOPHYSICAL

content

in water.

circular

in dodecyl near

The matrix

zero

spectra

suggests

is deduced

calculation

and 38% in the presence dichroism

sulfate

change

from the

(15,16)

of dodecyl in

a helical

predicts

helical

sulfate.

the manner

predicted

by the

theory. DISCUSSION The theoretical in its

presence)

reported

the native

take

account

determination

presence

is

in FQure

for

should

prediction

state of long

(8% helix

in reasonable 1.

Agreement may be, range

agreement between

in part,

of dodecyl

which

Since

between

sulfate,

helicities

The calculation are

undoubtedly

state.

experiment

does not important

Greater and theory partition

38%

dichroism

and calculated

the configuration 400

of dodecyl

the circular

observed

of the native

to the agreement sulfate.

with

fortuitous.

interactions

of the conformation

be attached

in absence

significance in the function

in

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

0

Fig.

3.

reproduces

reasonable

to extract

propagation

probability

formation

kinase

dodecyl

sulfate

forming

a reverse

crosslinking

53-55,

much of the

would

make accessible from one another

negated.

The helical

disulfide

bond,

spectra

to excess

that

stabilizing

content

in harmony

would

with

(22,23)

Rupture

in which

then

for

turn

subunit

could

B in

bond

then

occur

of the disulfide

the helices

are

bond

sufficiently

interactions

decrease

upon reduction

of the

in

sequence

helix-helix

the response

for

and the disulfide

interaction

of the hairpin.

induced

probability

the Pro-Gly-Ser

of the hairpin

Helix-helix

28%) is

a reverse

model

with

hairpin,

predicted.

a high forms

A simple

the helix

of excess

presence

have

is

of two helical

(33% vs.

tripeptide

(20).

conformations

remote

in the

the

it

function

is clearly

structure

This

at the head

length

each

in

B

conditions,

Formation

Pro-Gly-Ser,

17-19)

these

for subunit the cysteinyl as well as

partition

3).

than

be a helical

turn

along

circular

are of the

dichroism

dithiothreitol. conformation

The cellular

an anionic

(Figure

(20,21).

the ends.

B.

under

configuration

intact

(residues might

The hairpin

helix

more helical

Residues

adenylate

(4-S),

this

bond is

dithiothreitol. turn

fraction

profile

shows slightly

reverse

100

as many as 30 residues

when the disulfide

subunit

the from

containing

Experiment

50

Computed helix propagation probability profile the presence of dodecyl sulfate. Locations of residues involved in disulfide bond formation, Fro-Gly-Ser (ES) sequence, are noted.

successfully

segments

RESEARCH COMMUNICATIONS

may be important

receptor

amphipathic

for

subunit

molecule. 401

to the biological B in vivo

In studies

is

activity

ganglioside

of cholera

of GM1

in canines,

Vol. 105, No. 2, 1982 Carpenter

and coworkers

the intestines filtrates

always

of Vibrio

an hour, rate

BIOCHEMICAL

has been observed cells

sulfate

Further

is study

(i)

reasons:

It

in a protein. disulfide conformational

after

scale

is

which

penetration

of fluid

of several

hours.

A lag period

of the

The molecule

constitutes for

may be related of subunit

for

a maximal minutes

tissues

and

observed

is warranted

a large

in

for

several

to CYhelix macrocycle,

interaction.

to the means by which A into

within

period.

a 8 structure

helix-helix

loops

of 15-60

change

transition

model

Sterile

in isolated

lag

through

and reached

of the conformational to that

loss

by jejeunal

two hours,

activation

an excellent

prwides

the onset

4-6 hours. cyclase

RESEARCH COMMUNICATIONS

absorbed

of the conformational

change

facilitates

period

usually

lasted

comparable

(ii) bond,

that

started

in adenylate The time

(24-29).

dodecyl

which

a lag were

secretion

hours,

observed

follows cholerae

and fluid

at four

(11)

AND BIOPHYSICAL

transition

closed (iii)

subunit

by a

The B

the cell.

ACKNOWLEDGEMENT This

work

was supported

by National

Science

Foundation

research

grant

PCM 78-22916. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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BIOCHEMICAL

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