Structural studies on the freezing-point-depressing protein of the winter flounder Pseudopleuronectes americanus

Vol. 74, No. 2, 1977

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STRUCTURAL STUDIESONTHE FREEZING-POINT-DEPRESSING PROTEIN OF THEWINTERFLOUNDER PSEUDOPLEURONECTES AMERICANLISt V.S. Ananthanarayanan* and Choy L. Hew Department of Biochemistry Memorial University of Newfoundland St. John's, Newfoundland, Canada Received

November 29,1976

SUMMARY The freezing-point-depressing protein from the winter flounder, Pseudopleuronectes americanus has been shown from circular dichroism measurements to possess a large proportion (-85%) of the o-helical conformation in aqueous solution (pH 8.0) at -loC. The helical content decreases as the temperature is raised. Viscosity data at -1oC indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this freezing-point-depressing protein as well as its primary structure (reported elsewhere), are found to be different from its counterpart glycoproteins isolated from the Antarctic fish. INTRODUCTION The survival

of certain

marine fishes under sub-zero temperatures (< -1oC)

has been shown to be due to the presence of an unique class of proteins that depress the freezing the proteins

point of their

serum [1,2].

isolated from an Antarctic

Detailed studies madeon

fish Trematomusborchgrevinki

shown that these proteins are a family of glycoproteins weights, all consisting of repeating alanyl-alanyl-threonyl with a disaccharide moiety attached to the threonyl

have

of different

molecular

tripeptide

residue [1,2].

units

Hew et al.

(3) and Dumanand DeVries [4,5] have demonstrated the presence of similar freezing-point-depressing

proteins

(FPDP) in the winter flounder Pseudopleuronectes

iContribution No. 246 from the Marine Sciences Research Laboratory, Memorial University of Newfoundland. *Permanent address: Molecular Biophysics Unit, Indian Institute of Science Bangalore, India. Abbreviations

- FPDP(freezing-point-depressing

Copyright 0 1977 by Academic Press, Inc. AN rights of revroduction in anv form reserved.

685

protein)

CD (circular

dichroism)

Vol. 74, No. 2, 1977

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americanus which resides in the northern Atlantic

coast of America and Canada.

Flounder's FPDPhas a molecular weight of about 10,000 and contains eight amino acids in its composition. this protein

The N-terminal 28 amino acid sequence of

has been determined [6].

Flounder's FPDPis not a glycoprotein

and does not possess the repeating tripeptide structure

of the Antarctic

fish proteins

between these two classes of proteins 60%) of alanyl residues in their The freezing-point-depressing Antarctic

different

11,2].

However, one commonfeature

is the relatively

large content (over

amino acid composition. activity

of the glycoproteins

from the

fish has been ascribed to the presence of the sugar moieties as

well as to the extended configuration proteins

sequence seen in the primary

[1,2].

of the polypeptide

chain in these

Inasmuch as the FPDPfrom the winter flounder possesses a

primary structure,

it was of interest

to us to find out the

similarity

or otherwise of the secondary and tertiary

proteins.

Our results presented below show that the FPDPfrom the flounder

has a relatively

large a-helical

asymmetric (rod-like)

structures

content (~85%) and possibly possesses an

shape in contrast to the random coil,

reported for the Antarctic

of these two

fish glycoproteins

extended conformation

[1,2].

Experimental FPDpwas isolated and purified according to the procedure described elsewhere [3,6]. The lyophilized protein was dissolved in 0.05M NH4HC03to yield concentrations between 0.05 and 0.5% (w/v). The protein concentration was determined from the optical density of the solution of 230 nm which was calibrated by meansof micro-kjeldahl nitrogen analysis (7). The pH of the protein solutions was 8.0 : 0.1. Circular dichroism (CD) measurementswere made using a Jasco J-20 spectropolarimeter. Water jacketed cells of 0.1 and 0.01 cm path lengths were used. Temperature control was achieved by circulating thermostated water from a Lauda K2-R bath through the cells. The CD data are expressed in terms of the ellipticity, [e], in deg.cm2.dmole-1, using a mean residue Viscosity measurementson a 0.25% weight of 86.4 for the protein (3,6). protein solution were made using Ostwald-and ubbelohde-type viscometers. RESULTS AND DISCUSSION The CD spectra of FPDPin 0.05M NH4HC03(pH 8.0) obtained at -lo, 80°C are shown in Fig. 1. for model polypeptides

25' and

Comparison of these spectra with those obtained

and other proteins 8,9] reveals that the spectrum at

686

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BIOCHEMICAL

eo-

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

60-

40-

20-

o-

-2o-

-4oI

1

I

200

I

WAVELENGTH,

Fig.

-1'C

arises

from a predominantly

resembles

in essentially spectra

with

very

those

the

overlap

to those

calculated

for

little,

was made using

obtained

random

as well

An estimate

if

coil

a-helical for

240

nm

form.

mixtures

any,

content

is

the

ellipticity

at 80°C,

and proteins

at which

[13] at this

of the a-helix

all

wavelength

and random

coil

=

[lo]

the three correspond conformation

[8,9]. of the protein

at the

three

temperatures

- 4000 x 100

29,000 at 208 nm.

at

the

[8]: [0]2QB

[O],os

of

while

[8,9]

The wavelength

of the Bstructure

of the a-helical the equation

conformation,

polypeptides

as the magnitude

% a-helix where

I

230

CD spectra of flounder FPDP in 0.05M NH4HC03 (pH 8.0) the indicated temperatures(oC).

1

spectrum

I

220

210

The FPDP is

found

to possess

87%

BIOCHEMICAL

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a-helix

at -l'C,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

47%at 25OCand 9% at 800C. These estimates should be taken

as approximate owing to the limitations It is, however, interesting

data [8,9]. helical

involved

conformation in the protein

of its biological

function.

in the use of the model compound

to note the preponderence of the a-

at -1'C which is close to the temperature

This conformation appears to be stable since we

find that taking the protein

solution

to the higher temperatures (up to about

1OOoC)and cooling it back to lower temperatures restores the CD spectra back Changing the pH of the solution from 2 to

to those obtained before heating. 10 did not change the CD spectrum. predominantly a-helical

It

is worthwhile mentioning here that a

conformation has been observed earlier

solutions of polypeptides containing

large proportions

randomly in binary copolymers,especially An insight from viscosity

into the tertiary

of FPDPwas sought to be obtained

(0.25%) solutions of the protein

The values of the reduced viscosity,

at -lo and ZO'C.

temperatures are, 5 -+ 1 and 11 + 1 respectively. with the intrinsic

viscosity

any concentration

an asymmetric (rod-like) temperature,

Comparison of these values

et. al. Antarctic

dependence for the moment) is compatible with

hydrodynamic shape of the FPDPmolecule at this

while at the higher temperature (20°C), it might exist

viscosity

fish.

in a more

In contrast,

an

of about 20 was obtained at 0.5'C and 17'C by DeVries

[13] for the glycoprotein

that the Antarctic

of molecular weight 21,000 isolated

from the

On the basis of this and the CD data, these authors concluded fish glycoproteins

as extended random coils. structure

acid [12]

that the value of n*at -1'C

expanded shape due to the admixture of the random coil. intrinsic

(pH 8.0)

nr, at these

data obtained with poly-L-glutamic

(of molecular weights 5000 - 10,000) indicate (ignoring

of alanine distributed

at low temperatures [ll].

structure

measurementson dilute

in aqueous

of flounder's

shape, is most likely

The entirely FPDPi.e.,

have no ordered structure different

high a-helical

and exist

secondary and tertiary content and an asymmetric

to arise out of the absence of conformational

ments imposed by the disaccharide moieties present in the Antarctic proteins. 688

requirefish

Vol. 74, No. 2, 1977

BIOCHEMICAL

Both the flounder's the freezing colligative

FPDPand the Antarctic

fish glycoproteins

point of water by a mechanismdifferent properties

are different and tertiary

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in their structures

large proportion

of solute molecules (1,2,5).

depress

from the usual Although these proteins

amino acid sequence as well as in their

secondary

as reported in this communication, the presence of

(over 60%) of alanine residues in both proteins may lead us

to detect a commonstructural

basis for their

function.

A detailed

study of

the physical chemical aspects of this problem is now in progress in our laboratory. ACKNOWLEDGEMENTS This work is aided by a grant from the Banting Research Foundation (to C.L.H.).

We wish to thank Professor C.C. Bigelow for initiating

reported here and for helpful

discussions.

V.S.A.

the work

thanks the Canadian

CommonwealthResearch Fellowship Committee for the award of a Fellowship for 1976-77. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Feeney, R.E. (1974). Amer. Scientist 62: 712-719. DeVries, A.L. (1974). Biochemical and Biophysical Perspectives in Marine Biology, Vol. 1, 290-327. Academic Press, New York. Hew, C.L. and Yip, C. (1976). Biochem. Biophys. Res. Comm.71: 845-850. Duman,J.G. and DeVries, A.L. (1974). Nature 247: 237-238. Duman,J.G. and DeVries, A.L. (1976). Comp. Biochem. Physiol. 54B: 375-380. Hew, C.L., Yip, C.C. and Fletcher, G. Submitted for publication. Lang, C.A. (1958). Anal. Chem. 30: 1692- . Greenfield, N. and Fasman, G. (1969). Biochem. 10: 4108-4116. Chen, Y-H., Yang, J.T. and Chan, K.H. (1974). Biochemistry 13: 3350-3359. Cartijo, M., Panipjan, B. and Gratzer, W.B. (1973). Intl. J. Peptide Protein Res. 5: 179-186. Platzer, K.E.B., Ananthanarayanan, V.S., Andreatta, R.H. and Scheraga, H.A. (1972). Macromolecules 5: 177-197. Morcellet, M. and Loucheux, C. (1976). Biopolymers 15: 1857-1862. DeVries, A. L., Komatsu, S.K. and Feeney, R.E. (1970). J. Biol. Chem. 245: 2901-2908.