The Structure of TropomyosinAuthor(s): K. BaileySource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 141, No.902 (Mar. 11, 1953), pp. 45-48Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/82779 .

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Elastic properties and a-f-transformation of fibrous proteins Elastic properties and a-f-transformation of fibrous proteins 45 45

REFERENCES (Rudall)

Astbury, W. T. & Dawson, J. A. T. 1938 J. Soc. Dy. Col. 54, 6.

Astbury, W. T. & Dickinson, S. 1940 Proc. Roy. Soc. B, 129, 307.

Astbury, W. T. & Woods, H. J. I933 Phil. Trans. A, 232, 333.

MacArthur, I. 1943 Nature, Lond., 152, 38.

Pauling, L. & Corey, R. B. I95I Proc. Nat. Acad. Sci., Wash., 37, 261.

Rudall, K. M. 1952 Advanc. Protein Chem. 7, 253.

THE STRUCTURE OF TROPOMYOSIN

BY K. BAILEY, School of Biochemistry, University of Cambridge

In all muscles so far examined there is present an unusual protein to which no

function has yet been assigned. It occurs side by side with actin and myosin in the myofibril, and because of its similarity to myosin was named tropomyosin (Bailey 1948). Though soluble in water, it is not extracted from coarsely minced muscle by aqueous media; its extraction is facilitated by drying the minced fibre in ethanol and ether and finally extracting with strong salt solution. From fish

muscle, Hamoir (I95I) has been able to extract directly a nucleotropomyosin complex, and the extraction procedure given above might be explained by assuming that tropomyosin occurs in situ in association both with nucleic acid and with lipid. Of this there is no proof. Tsao has recently shown that tropomyosin can be brought out of fresh muscle mince provided the fibres are thoroughly disintegrated.

Compared with myosin, tropomyosin has a rather low molecular weight-53 000

against 850000-but in its amino-acid composition is very much like myosin except for the greater amounts of lysine and glutamic acid, which make it the most

highly charged protein known, excluding the protamines, which do not carry a mixed charge. No less than 45 % of the total residues consist of non-amidized acid plus base groups. Some of these points are illustrated in table 5. Isoelectric

TABLE 5. SOME COMPARATIVE AMINO-ACID DATA FOR TROPOMYOSIN AND MYOSIN

Results given as weight of amino-acid/100 g protein

tropomyosin myosin

tyrosine 3.1 3.4 alanine 8.8 6.5 valine 3.1 2.6 leucines 15.6 15.6

phenylalanine 4-6 4.35

proline 1*3 1.9 serine 4.4 4.35

arginine 7*8 7.4

aspartic acid 9 1 8. 9

glutarnic acid 32.9 22.1

lysine 15.7 11.9

REFERENCES (Rudall)

Astbury, W. T. & Dawson, J. A. T. 1938 J. Soc. Dy. Col. 54, 6.

Astbury, W. T. & Dickinson, S. 1940 Proc. Roy. Soc. B, 129, 307.

Astbury, W. T. & Woods, H. J. I933 Phil. Trans. A, 232, 333.

MacArthur, I. 1943 Nature, Lond., 152, 38.

Pauling, L. & Corey, R. B. I95I Proc. Nat. Acad. Sci., Wash., 37, 261.

Rudall, K. M. 1952 Advanc. Protein Chem. 7, 253.

THE STRUCTURE OF TROPOMYOSIN

BY K. BAILEY, School of Biochemistry, University of Cambridge

In all muscles so far examined there is present an unusual protein to which no

function has yet been assigned. It occurs side by side with actin and myosin in the myofibril, and because of its similarity to myosin was named tropomyosin (Bailey 1948). Though soluble in water, it is not extracted from coarsely minced muscle by aqueous media; its extraction is facilitated by drying the minced fibre in ethanol and ether and finally extracting with strong salt solution. From fish

muscle, Hamoir (I95I) has been able to extract directly a nucleotropomyosin complex, and the extraction procedure given above might be explained by assuming that tropomyosin occurs in situ in association both with nucleic acid and with lipid. Of this there is no proof. Tsao has recently shown that tropomyosin can be brought out of fresh muscle mince provided the fibres are thoroughly disintegrated.

Compared with myosin, tropomyosin has a rather low molecular weight-53 000

against 850000-but in its amino-acid composition is very much like myosin except for the greater amounts of lysine and glutamic acid, which make it the most

highly charged protein known, excluding the protamines, which do not carry a mixed charge. No less than 45 % of the total residues consist of non-amidized acid plus base groups. Some of these points are illustrated in table 5. Isoelectric

TABLE 5. SOME COMPARATIVE AMINO-ACID DATA FOR TROPOMYOSIN AND MYOSIN

Results given as weight of amino-acid/100 g protein

tropomyosin myosin

tyrosine 3.1 3.4 alanine 8.8 6.5 valine 3.1 2.6 leucines 15.6 15.6

phenylalanine 4-6 4.35

proline 1*3 1.9 serine 4.4 4.35

arginine 7*8 7.4

aspartic acid 9 1 8. 9

glutarnic acid 32.9 22.1

lysine 15.7 11.9

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K. Bailey (Discussion Meeting)

point and salting-out range are similar to myosin, and though fairly soluble in water alone above pH 7, its inherent globulin properties can be shown at pH 6 when the addition of salt increases its solubility.

Three properties of tropomyosin led us to undertake a fairly detailed molecular

study: (1) If a solution of tropomyosin in salt is dialyzed, a somewhat viscous solution

forms one which is very viscous indeed, a solution of 0.8 % protein concentration with a relative viscosity of about 3 in 0-5 M-KC1 rising to one of 50. Such solutions show double refraction of flow, and when dried down on a glass plate, the resulting film, after washing off on to a suitable support, is found to consist of fibrils, 3000 to 6000A long and about 200 to 300A broad (Astbury, Reed & Spark 1948). These aggregates break up immediately on addition of salt, showing that the fibrils are formed largely by an electrostatic mechanism.

(2) If salt is added to ionic strength 0-4, the protein can be obtained in the form of fragile birefringent crystals, which contain as much as 90 % water. All attempts to obtain crystals by salting-out techniques have been unsuccessful.

(3) If the crystals, or solutions of tropomyosin, are dried down on a glass plate, and the resulting films examined by X-rays, the wide-angle pattern is of the typical a-keratin type. In this, tropomyosin again resembles myosin, but is the first

protein of the so-called k.-m.-e.-f. group to be crystallized. The questions which these properties invoke are likewise three in number. First,

what is the particle weight of the units which interact with each other to give fibrils in absence of salt? Secondly, what is their shape? And thirdly, does each consist of one or more than one polypeptide chain?

Particle weight

This investigation was carried out by Dr T.-C. Tsao with the kind co-operation of Mr G. S. Adair (Tsao, Bailey & Adair 195I). The average particle weight by osmotic pressure measurements was studied first of all at pH 6.5 from ionic

strength 0-1 to 1 1, and the results (table 6) show very clearly that, over the range where relative viscosity rises steeply, the particle weights also increase; but any increase in salt concentration above I = 0-3 causes only a small decrease, showing

TABLE 6. PARTICLE WEIGHT AND SHAPE OF RABBIT-TROPOMYOSIN

PARTICLES IN SOLUTION

mediuim axial ratio

average (unhydrated) solvent and particle weight viscosity from Simha

pH ionic strength (osmotic pressure) increment equation

6-5 salt (0.1) 135000 197 53 6.5 salt (0-2) 111000 141 44 6-5 salt (0-3) 72000 ' 99 35 6.5 salt (0-6) 67000 83 32 6.5 salt (1-1) 64500 80 31 6-5 urea (0-3) 53100 74 30 2*1 HC1 (0-3) 52700 74 30

12 NaOH (0-2) 61400 55 25

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The structure of tropomyosin

that a limiting value of particle weight is being attained. It should be noted that in this range only can tropomyosin be crystallized.

The particle weight of tropomyosin was next investigated in media which are known generally to depolymerize proteins into their subunits if these are not held

together by covalent bonds of the disulphide type. Both in concentrated neutral urea, and in acid at pH 2, the particle weight was identical (53000) and rather lower than in the salt solutions at pH 6-5; in alkali at pH 12 the particle weight was of the order of magnitude found for the limiting value in salt solution. It is not altogether clear why this latter does not actually attain the value found in urea or acid, nor why alkali is less efficient than acid in the disaggregation process; but it seems clear that the true particle weight is 53 000, that this is the 'monomer' concerned in aggregation, and that if it is a composite molecule its subunits are not readily liberated.

Shape Under these various conditions also, viscosity measurements were carried out

as a function of protein concentration. It was thus possible to determine the in- trinsic viscosity and from this to calculate the viscosity increment. In urea and in acid the values were again identical (74), and a little higher in strong salt solution at pH 6-5 (80). Accepting the Simha equation it was then possible to find the asymmetry, and under conditions where only the monomeric form exists in solution, this works out at 30 for an unhydrated molecule. The trend of the viscosity data (table 6) shows quite clearly that the polymerization which occurs as salt is removed is predominantly an end-to-end process; but the electron microscope studies show that at the stage where fibrils can be seen, side-to-side aggregation has also occurred. It seems probable that the initial aggregation, which, as we have noted, is electrostatic in character, is caused by virtue of a region in which

positive charges predominate on one end of the molecule and negative on the other. As the aggregates lengthen, the tendency for side-to-side aggregation will become increasingly great, especially on drying.

It is possible from analytical data and X-ray diffraction to make some predic- tions as to the detailed structure of tropomyosin.

These, however, are based on two major assumptions: first, that all the amino- acids are disposed in the a-configuration, and secondly, that the number of amino- acids in the fold is approximately known. At the time of the investigation, the calculation was made for a structure based on the Astbury model. Since the average residue weight is 116.4 (Bailey I948), the particle of 53000 molecular weight con- tains 455 residues. Supposing the molecule were one long a-chain, the length would be 455 x 5-11/3 = 755A. This, with an asymmetry of 78 (the cross-section of the chain being 10 x 9-5A), is not in keeping with the viscosity data. Two chains, side

by side with an average width of 14-5A, would have an asymmetry of 26, and three chains one of 13. These figures would seem to rule out all but the double-chain model. If, however, the Pauling-Corey model is considered, the agreement would be less good, giving an asymmetry of about 19 for two spirals side by side.

The double-chain model seemed very feasible from certain chemical considera- tions. Sanger's method may be used to characterize the N-terminal amino-acid

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K. Bailey (Discussion Meeting)

and to estimate it. As there do not seem to be any subunits in the particle of 53 000, one might expect to find just one terminal amino-group; or, if the particle consisted of two chains joined by covalent links, two terminal groups. Actually, no N-terminal acid could be identified, suggesting that the molecule might consist of a cyclic polypeptide, and such a structure conforms very well with a model based on a double ac-chain.

No terminal amino-acid has been identified in ovalbumin, and, interestingly enough, in myosin itself, and if this can be taken to imply that these proteins are built up from cyclopeptides, then tropomyosin is not unique. The case for cyclo- peptide structures would be very much strengthened if these proteins contained no a-carboxyl groups, and Dr R. H. Locker has begun such an investigation, using carboxypeptidase as a means of splitting off in succession the amino-acids from the C-terminal ends, if such exist. There seems no doubt that in the case of tropo- myosin, isoleucine, in an amount consistent with one group per molecule of 53 000, is present as a C-terminal acid.

This finding certainly raises difficulties, but it is equally difficult to see how the deductions made so far can be greatly in error. The cyclic nature of the chain seems to be supported by the extreme resistance of tropomyosin towards denatura- tion, which could be explained if the molecule is restrained from opening out in spite of the high net charge induced at extreme pH values. The general shape of the molecule has also been confirmed from dissymmetry measurements of the light scattered at 45 and 135? for tropomyosin at pH 6.5 and ionic strength 1-1 (Dr P. Doty, personal communication). The experimental dissymmetry factor. 1.06 compares very favourably with that for a rod of 400A length (1-05). The newer chemical evidence is thus not sufficient in itself to cast grave doubt upon the correctness of the model, but it may indicate the existence of a branching point; for example, a cyclic chain could possess a branch at any (o-carboxyl group, or, alternatively, the cyclopeptide could be closed through an ow link, and the branch could occur at an a-carboxyl.

In conclusion, the present study tends to confirm that the ac-fold, whatever its precise configuration, contains about three residues per fold; and it suggests that the possibility of cyclopeptide structures must seriously be considered. Above all, chemical evidence points to complexities which could not readily be discovered by physical methods.

REFERENCES (Bailey)

Astbury, W. T., Reed, R. & Spark, L. C. 1948 Biochem. J. 43, 282. Bailey, K. I948 Biochem. J. 43, 271. Hamoir, G. I95I Biochem. J. 48, 146. Tsao, T.-C., Bailey, K. & Adair, G. S. 195I Biochem. J. 49, 27.

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