Purification and properties of native titin

J. Mol. Biol. (1984) 180, 331-356

Purification and Properties of Native Titin J. TRINICK, P. KNIGHT AND A. WHITING

Muscle Biology Division Agricultural and Food Research Council Langford, Bristol BS18 7D Y, U.K. (Received 18 November 1983, and in revised form 24 May 1984) A procedure has been developed for the extraction and purification of the massive myofibrillar protein titin without exposing it to denaturing conditions. The form of the molecule that has been isolated is soluble at high ionic strength and alkaline pH, but precipitates in low salt or at pH values below 7. Sedimentation velocity experiments indicate that titin is a highly asymmetric molecule with a sedimentation coefficient of 13.4 S. This asymmetry is confirmed by electron microscopy of rotary-shadowed specimens, which shows string-like structures of diameter 40 A and lengths up to 8000 A. Significant differences were observed depending on whether the electron microscope specimens were prepared by spraying or by layering of the titin onto a mica substrate; we tentatively attribute these differences to elasticity in the titin, revealed by the high shearing forces that accompany spraying. In accord with this, the circular dichroism spectrum of titin indicates that its secondary structure is largely random coil, a conformation characteristic of elastic proteins such as elastin. R'egative staining of titin again shouts long string-like structures, but these can now be seen to have an appearance similar to a string of beads, where the spacing between successive beads is about 40 A. Very similar beaded strings have been observed also associated with negatively stained separated native thick filaments; these are found running alongside the cross-bridge regions and in coils near the filament ends. Since the periodicity of the strings is similar to that of endfilaments, recently identified structures at the tips of thick filaments, it is likely that end-filaments are formed from titin. Titin comprises approximately 9% of the myofibrillar mass, which means that it is the third most abundant protein in muscle. The possible role of titin in forming elastic filaments within myofibrils is discussed.

1. Introduction Titin is a protein of v e r y high chain molecular weight ( ~ 10 6 Mr) and is a m a j o r c o m p o n e n t of v e r t e b r a t e skeletal muscle myofibrils. I t was discovered b y W a n g et al. (1979), who a n a l y s e d myofibrils using sodium dodecyl s u l p h a t e / p o l y a e r y l a m i d e gels with p a r t i c u l a r l y large pores t h a t allowed the e n t r y of polypeptides too big to penetrate n o r m a l gels. The solubility of titin in benign solvents a p p e a r e d to be e x t r e m e l y low and it was purified only in its d e n a t u r e d state after dissolution of 331 0022 2836/84/340331-26 $03.00/0

© 1984 Academic Press Inc. (London) Ltd.

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whole myofibrils in hot sodium dodecyl sulphate; consequently, the properties of the native molecule were unknown. Reaction to myofibrils with fluorescent antibodies to this denatured preparation produced a complex and somewhat variable labelling pattern. Binding was most strongly and consistently observed at the junctions of the A and I-bands, but it was also seen at the M and Z-lines and sometimes the whole A-band fluoresced. I f the myofibrils were first treated with 0"6 M-KI to remove most of the thick and thin filament proteins, strong labelling was seen on either side of the Z-line and weaker fluorescence was seen throughout the sarcomere. On the basis of these data, it was suggested (Wang et al., 1979) t h a t titin might form elastic connections within the myofibril, possibly analogous to connections thought to join the ends of thick filaments to the Z-line in insect flight muscle. More recently, the idea was put forward (Wang, 1982a) t h a t titin is largely separate from thick or thin filaments, and t h a t with nebulin, another high molecular weight protein responsible for the N2 line (Wang & Williamson, 1980), it forms a set of elastic filaments joining successive Z-discs. Independently, M a r u y a m a and his colleagues have studied the properties of a preparation called conneetin, which they also suppose forms elastic filaments inside myofibrils (e.g. see M a r u y a m a et al., 1977). This was isolated as the insoluble residue remaining after exhaustive extraction of myofibrils b y a succession of harsh solvents. I t is now a p p a r e n t t h a t preparations of connectin contain titin, although they also contain other proteins (Maruyama et al., 1981,1983). Once again, however, the rigour of the extraction conditions will have completely denatured the titin. We became interested in titin because of its suggested location at the junction of the A and I-bands. This is also the location of newly discovered structures called end-filaments, which are a b o u t 850 A long and 50 A wide, and which show a periodicity of 43 A (Trinick, 1981). In order to compare the structure of end-filaments with t h a t of titin, we needed to purify titin in its native form. Contrary to expectation, we have found it straightforward to isolate a large fraction of the titin in myofibrils using as a starting point a simple high salt extract. We present in this p a p e r details of the purification procedure and some properties of the native protein. 2. Materials a n d M e t h o d s (a) Preparation of washed myofibrils Washed myofibrils were prepared from strips of rabbit psoas muscle that had been incubated overnight at fixed length and at 0°C in an EGTA-Ringer solution, as described by Knight & Trinick (1982). After incubation, the strips were cut into short segments and homogenized in ice-cold rigor buffer (0.1 M-KC1, 2mM-MgC12, 1 mM-EDTA, 0'5m~dithiothreitol, l0 mM-imidazole. HC1, pH 7"0 at 0°C) for 30 s at top speed in an MSE homogenizer. This suspension was washed 4 times by cycles of centrifugation at 2000 g for 5 min followed by resuspension in l0 vol. rigor buffer. The protein concentration of the myofibrils was estimated from the absorbanee at 280 nm of a portion dissolved in warm 1%0 value of 7 (Sutoh & Harrington, 1977). (~40°C) 1% (w/v) SDSt, assuming an E2s t Abbreviation used: SDS, sodium dodecyl sulphate.

NATIVE T1TIN

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(b) Pilot experiments on the extraction of titin from myofibrils Conditions for the extraction of titin were explored through a series of small-scale trails conducted as follows. Plastic centrifuge tubes (capacity 1.5 ml) containing 0.2 ml of a 1% (w/v) myofibril suspension in rigor buffer were centrifuged for 30 s in an Eppendorf bench centrifuge (nominal force 10,000 g) operating in the cold. After removal of the supernatants, the pellets were resuspended in 0.4 m] of cold rigor solutions supplemented with various concentrations of KCI or other ions. At appropriate times, the soluble and insoluble fractions from these extractions were separated by further centrifugation for 30 min at 10,000 g. The pellets were then resuspended in 0.4 ml of 100 mM-Tris. HC1 (pH 8'0 at 4°C) and dialvsed against this buffer~ together with the supernatants, prior to analysis by SDS/polyacrylamide gel electrophoresis. (c) Preparation of purified native titin A volume of fresh myofibri] suspension containing approximately l g of protein was centrifuged for 5 rain a~ 5000 g and the supernatant removed. To the pellet was added a quantity of an ice-cold extracting solution r0.6 M-KC1, 2 mM-MgC12, l m.~I-EDTA. 0'5 mMdithiothreitol, l0 mM-imidazole. HC1. pH 7 a~ 0°C sufficient to bring the final protein concentration of the suspension to about 5 mg ml. After thoroughly dispersing the pellet with a glass rod [which took about 20 sl. the suspension was immediately centrifuged for 1 h at 15,000 g. The clarified extract was then carefully removed with a large syringe, since the upper portion of the pellet was easily dislodged. I t was then dialysed for several hours agains~ 2 vol water, following which precipitated myosin was removed by centrifugation at 6000 g for 30 rain. The supernatan~ was dialysed against 2 changes of 0.1 ~-KC]. 1 mMEDTA, 0.3 mM-dithiothreitol, 50 mM-Tris- HC1 (pH 7'9 at 4°C~ and pumped onto a column (i.6 cm × 42 cm) containing DEAE-cellulose W h a t m a n DE52) equilibrated in this buffer. The flow-rate was 30 ml h and 8-ml fractions were collected Bound protein was eluted by an approximately linear salt gradient running from 0"1 M to 0.4 M-KC1 in a total volume of 2 ]. The titin peak fractions were concentrated by the addition of solid ammonium sulphate (Schwarz-Mann. Special Enzyme Grade~ to 35% of saturation followed by centrifugation at 8000g for 30 rain. after which the pellets were resuspended and dialysed overnight against 0"5 M-KC1, 1 mM-EDTA, 0'3 mM-dithiothreitol. 50 mu-Tris. HCl (pH 7-9 at 4°C~. The dialysed protein was then passed through a gel filtration column 11.6 cm × 84 cm) containing Bio-Ge] A50-M equilibrated in the dialysis buffer and run at a rate of 15 m] h. The titin-containing fractions were again concentrated by the addition of solid ammonium sulphate to 350/o of saturation.

(dl Preparation of separated native thick filaments Washed separated native thick filaments were prepared as described by Trinick (1982).

(e) Sodium dodecyl sulphate/polyacrylamide gel electrophoresis The procedure for gel clectrophoresis in the presence of SDS at low acrylamide concentrations was essentially that described by Wang et al. (1979), the only difference being that the gels and electrode buffers contained 100 mM-Tris-bicine rather than Trisglycine. Gels 10 cm × 0-5 cm containing 3-2 (w/v) acrylamide (acrylamide/bis = 30:0.8, w/w) were cast in glass tubes 15 cm long. Proteins were dialysed against 100 m~I-Tris. HC1 (pH 8-0) at 4°C, and then treated at 100°C for l min in the presence of 1% (w/v) SDS, 70 mM-2-mercaptoethanol. Before loading, samples were diluted with 0-25 volume of 50% (v/v) glycerol, 1% (w/v) bromophenol blue. Electrophoresis was performed until the bromophenol blue reached the end of the gel: 50 V until the sample entered the gel and 100 V thereafter. Gels were stained overnight in a solution containing 0-025% (w/v) Coomassie brilliant blue (G-250) dissolved in 50% (v/v) methanol, 10% (v/v) acetic acid and then destained in a solution containing 10°/o (v/v) methanol, 10% (v/v) acetic acid.

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J. T R I N I C K , P. K N I G H T AND A. W H I T I N G (f) Quantification of the amount of titin present in myofibrils

The amount of titin present in myofibrils was estimated from densitometry of SDS/polyacrylamide gels loaded with whole myofibrils. Gels were calibrated by using the myosin heavy chain as a reference and by assuming that myosin forms 55% of myofibrillar protein (Huxley & Hanson, 1957). The ability of the myosin heavy chain and titin to take up the Coomassie stain was estimated from gels loaded with known amounts of titin and myosin. The amounts of titin put on these gels were determined from absorbance measurements at 280 nm, corrected for scatter by subtraction of the absorbance at 340 nm. The Egso ~o~;, value used for titin was 13.7 (mean of 4 measurements). This was obtained using a synthetic boundary cell in the analytical ultracentrifuge, assuming 40 fringes for a 1% (w/v) solution in a 12 mm path-length cell. The E2so 1% value for myosin was taken to be 5-0. Densitometry was performed on a Joyce Loebl MIII CS scanning microdensitometer operating with an orange filter. (g) Analytical ultracentrifugation Sedimentation velocity experiments were carried out in a Beckman Model E ultracentrifuge using an An-D rotor and 12 mm double-sector cells fitted with Kel-F centre pieces. Sedimentation patterns were observed using schlieren optics, a rotor speed of 52,000 revs/min and temperatures near 20°C. The partial specific volume was taken to be 0"730 ml/g. (h) Circular dichroism spectra Spectra were recorded on a Cary model 61 spectropolarimeter with a circular dichroism attachment, using 0-1 and 0.2 cm path-length cells and a protein concentration of about 1 mg/ml in 0"5 M-KC1, 50 mM-potassium phosphate (pH 7'5 at 5°C). The percentage a-helix was calculated from [0120s, the mean residue ellipticity at 208 nm using the empirical relationship given by Greenfield & Fasman (1969): % helix -

[012s0 - 4000 33,000 - 4000'

where 33,000 deg. cm2/dmol is the ellipticity value of 100% helix and 4000 deg. cm2/dmol is the contribution from random coil at this wavelength. The mean residue mass was taken to be 115. (i) Electron microscopy (i) Spraying and shadowing Titin was first prepared for electron microscopy using the spraying and shadowing procedure described by Tyler & Branton (1980), as modified from the method of Elliott & Offer (1978). Samples were dialysed against 0-4 M-ammonium acetate (pH 7.2) and then diluted to a concentration of about 0-1 mg/ml with a solution containing 0.4 M-ammonium acetate, 50% (v/v) glycerol before spraying onto freshly cleaved mica. After drying in vacuo for 15 rain (ultimate pressure 1 × 10 -3 Pa) specimens were rotary shadowed with platinum at an angle of 6o and then coated with carbon. Replicas were floated off on a surface of distilled water and collected on copper grids. (ii) Layering and shadowing Specimens of titin were also layered, rather than sprayed, onto mica in preparation for shadowing. Here, a drop of the titin solution in the desired buffer (which was not necessarily volatile) was applied to a piece of mica at a concentration of about 0.01 mg/ml and allowed to run evenly over its surface. Excess liquid was removed by holding the mica in a pair of forceps and drawing an edge across a filter paper. Without being allowed to

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dry~ the mica was then washed with several drops of the buffer in which it was applied, excess liquid being again drawn off on the filter paper before the application of each successive drop. The washing procedure was then repeated using a solution containing 50% (v/v) glycerol, 0.4 M-ammonium acetate. At this stage, the thickness of the liquid on the mica was about 0.2 ram. Prior to drying in vacuo, the mica was clamped at a corner to the periphery of an 8 cm diameter disc mounted on the spindle of a bench centrifuge and spun at about 1000 revs/min for 10 s. This had the effect of throwing off further excess liquid, after which interference colours could be seen in the remaining layer. The mica was then placed in a vacuum coating unit and dried, and coated with platinum and carbon as described above. We are indebted to Drs A. Elliott and G. Offer for suggesting this method. (iii) Negative staining Negative staining was performed using ultraviolet light-treated grids, as described by Knight & Trinick (1984). :For this purpose, 400-mesh copper grids coated with a layer of carbon about 100 A thick were irradiated for 40 min by an ultraviolet lamp (type 1~51, UV Products I n c , Pasadena, Calif.) placed about 10cm away. The grids, which were hydrophilic after this procedure, were generally used within an hour of irradiation. (iv) Conditions of observation Electron microscopy was performed using a Philips EM 400T electron microscope operating at 80kV with a 30pro objective aperture, a liquid nitrogen-cooled antieontaminator and a low-dose attachment. Micrographs of separated native thick filaments were recorded under conditions of relatively low electron dose (~100e/A 2, calculated from charts of screen current versus exposure time issued by Philips); dose was not controlled when recording or examining other specimens. Length measurements were made by calibrating the microscope magnification with a paramyosin standard (Elliott et al., 1976), assuming a striation spacing of 144 A.

3. Results (a) Extraction, purification and 8olubility of native titin Hitherto, titin has been considered an insoluble protein t h a t could be dissolved only in d e n a t u r i n g solvents. Figure 1 shows S D S / p o l y a e r y l a m i d e gels of the soluble and insoluble fractions o b t a i n e d after e x t r a c t i o n of fresh psoas muscle myofibrils at progressively higher ionic strengths. The gels d e m o n s t r a t e t h a t while v e r y little titin is released at a KC1 c o n c e n t r a t i o n of 0"4 M, an appreciable fraction of it (30 to 40%) is e x t r a c t e d at 0-6 M. Nebulin is n o t e x t r a c t e d b u t some m y o s i n and m o s t of the C-protein are dissolved. Titin usually migrates on such gels as a doublet, b u t on occasion we have observed only a single band; where the doublet is seen, we assume the leading b a n d to be due to proteolysis. Similar b e h a v i o u r was also observed b y W a n g et al. (1979). D u r i n g purification, the intensity of the leading b a n d increases while t h a t of the trailing b a n d declines, to the e x t e n t t h a t the final purified protein generally appears as a single b a n d (Fig. 6). N e a r l y half of the titin present in myofibrils is released at a KC1 c o n c e n t r a t i o n of a b o u t 0.6 M, b u t this p r o p o r t i o n is raised only slightly if the ionic s t r e n g t h is further increased or the e x t r a c t i o n prolonged. Thus Figures 1 (lanes g and h) and 2 d e m o n s t r a t e t h a t KC1 c o n c e n t r a t i o n s up to 2 ~ or lengthening of the e x t r a c t i o n time from a few seconds to 70 minutes still dissolves only a b o u t half the titin present. A demerit of these more e x h a u s t i v e extractions is t h a t t h e y also p r o m o t e release of u n w a n t e d proteins such as myosin.

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J. T R I N I C K , P. KNIGHT AND A. W H I T I N G Titin

Nebulin

- -

Myosin

a

b

c

d

e

f

g

FIG. 1. SDS/polyacrylamide gels demonstrating the extraction

h of

titin at high ionic strength.

Lane a, 100 #g myofibrils; lane b, resuspended pellet after centrifugation of myofibrils in buffer containing 0-1 M-KC1(0.2 ml of a 1% suspension centrifuged for 30 s at 1000 g; pellet resuspended in 0-4 ml of 100 mM-Tris. HCl (pH 8) and dialysed against this), 30 #l of sample applied to the gel. Lanes c and d, residue and extract, respectively, after extraction of myofibrils with buffer containing 0.4 MKC1 (for details, see Materials and Methods, section (b)), 30 #1 of sample was applied to each gel. Lanes e and f, as c and d but extracted with 0.6 M-KC1. Lanes g and h, as c and d but extracted with 2 M-KC1.

Titin could be e x t r a c t e d using a simple solution containing 0"6 M-KC1, 10 mMimidazole- HC] (pH 7'0). Pilot experiments showed t h a t the a m o u n t of it t h a t was released was unaffected b y the addition of Mg 2+ (1 raM), E G T A (2 raM), p h o s p h a t e (10 raM), or dithiothreitol (2 raM). The a d v a n t a g e of e x t r a c t i n g titin from washed myofibrils, r a t h e r t h a n from whole muscle, is t h a t m o s t of the soluble proteins have been r e m o v e d and the extraction of m y o s i n is limited because it is b o u n d to actin. A significant a m o u n t of myosin is nevertheless liberated from myofibrils b y 0-6 ~-KC1, p r o b a b l y because the a c t i n - m y o s i n interaction is weakened at high ionic strength. Most of the m y o s i n can, however, be conveniently r e m o v e d b y reducing the ionic strength to a b o u t 0.2; here, virtually all the m y o s i n precipitates and can be separated b y centrifugation. The m a j o r i t y of the titin ( ~ 8 0 ~ o ) stays in the s u p e r n a t a n t (Fig. 3).

NATIVE TITIN

a

b

c

d

e

f

337

g

h

FIG. 2. SDS/polyaerylamide gels showing the effect of time o[1 titin extraction. Lanes a and b, residue and extract obtained after centrifugation of myofibrils immediately followingtheir suspension in buffer containing 0.6M-KC1. Lanes c and d, as a and b but extracted for 10min before centrifugation. Lanes e and f, as a and b but extracted for 30 rain. Lanes g and h, as a and b but extracted for 70 rain. In all cases, the sample volume was 30/li.

The principal proteins contaminating the titin at this stage are C-protein (134,000 Mr; Offer et al., 1973) and a protein having a chain weight in the region of 40,000 to 50,000 Mr, presumably actin or ereatine kinase (Fig. 3(e)). The bulk of these are removed by ion-exchange c h r o m a t o g r a p h y in DEAE-eellulose, from which a typical elution profile is shown in Figure 4. SDS/polyacrylamide gels of various column fractions (Fig. 5) demonstrate that most of the contaminating proteins are unretarded by the ion-exchange column, while most of the titin is bound and eluted in a broad peak at a KC1 concentration of about 0.13 M. The final stages in the purification procedure involve concentration of the titin by precipitation with ammonium sulphate followed by passage through a gel filtration column (Bio-Gel A 50-M). The protein is eluted from this column in a single peak and is again concentrated by addition of ammonium sulphate to 35~o of saturation. A number of low molecular weight contaminants present in the titin preparation in small amounts are removed by the gel filtration column. Figure 6

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J. TR1NICK,

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Titin

Myosin

-

-

o b c d e

Fig 3

FIG. 3. SDS/polyacrylamidc gels demonstrating the separation of titin from myosin by low ionic strength precipitation. Lane a, extract after brief treatment of myofibrils (5 mg/ml) with buffer containing 0.6 M-KCI, load 60 pl. Lanes b and e, precipitate formed by dialysis of the extract against 2 vol. water. The precipitate was collected by centrifugation (20 rain at 5000 g) and resuspended in a volume of 100 mM-Tris • HC1 (pH 8) equal to that of the original extract before dialysis against the Tris buffer. The volumes applied to the gel were 20 ttl and 100 #1, respectively. Lanes d and e, supernatant after removal of precipitated myosin, loads were 20 #l and 100 #l: respectively.

m

0.4

E c

o

oo

0-2

~

0-3

oJ

:~ c

c

o za

0'I

,0,2

y

o I

I

I

20

40

60

I - 0.~ 80

Froct'ion n u m b e r

FIG. 4. Column chromatography of titin on DEAE-cellulose. Titin was eluted in the second peak at a KC1 concentration of about 0-13 M after the application of a linear salt gradient; other details were as described in Materials and Methods.

NATIVE T I T I N

o

b

c

ci

e

f

339

g

h

Fig 5

FIG. 5. SDS/polyacrylamide gel electrophoresis of column fractions after chromatography of titin on DEAE-cellulose. (See also Fig. 4.) Lane a, before ion-exchange chromatography, the load was 100 ttl of extract supernatant after myosin removal. Lanes b and h, samples (100/ll) from across the peaks of the chromatogram seen in Fig. 4. Lane b, fraction 5; c, fraction 16; d, fraction 20; e, fraction 25; f, fraction 37; g, fraction 40; h, fraction 53.

shows gels of the purified titin at this stage; the yield is a p p r o x i m a t e l y 20 m g per g r a m of myofibrillar protein. A l t h o u g h titin does n o t precipitate with m y o s i n on reduction of the ionic s t r e n g t h of the initial e x t r a c t to 0.2, its solubility properties are b r o a d l y similar to those of m y o s i n and C-protein in t h a t it is soluble at high ionic s t r e n g t h and aggregates in low salt. Precipitation as disordered aggregates is seen when the KC1 c o n c e n t r a t i o n is reduced to 0'05 M. (b) Sedimentation velocity experiments Figure 7 shows sehlieren p a t t e r n s o b t a i n e d during sedimentation velocity analytical ultraeentrifugation of the purified titin. This typical p r e p a r a t i o n shows two peaks, indicating the presence of at least two sedimenting species; we have,

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J. TRINICK, P. KNIGHT AND A, WHITING

(1

b

Fig 6

FIG. 6. SDS/polyacrylamide gels of purified titin. Loads were 4 #g and 80 #g in lanes a a n d b, respectively.

however, on occasion observed a third peak moving more slowly t h a n the other two. The leading peak in Figure 7 is diffuse, while the slower b o u n d a r y is hypersharp. The m a j o r i t y of the protein (70 to 80%, estimated from the relative areas of the peaks from a run at low concentration) sediments at the rate of the hypersharp boundary. The leading peak could be due to small quantities of aggregated contaminants but is more likely the result of aggregation of the slower moving material. I f this is the case, then the aggregation is not a rapid equilibrium of a monomer and its polymer, since the schlieren trace can be seen to fall to zero between the two peaks; moreover, the relative areas of the peaks were not significantly different when the preparations were run at a lower concentration. Figure 8 shows the concentration dependence of the sedimentation coefficient (S2o,w). When extrapolated to zero concentration, the sedimentation coefficient of the major hypersharp b o u n d a r y was 13.4 S; t h a t of the leading diffuse peak was about 40 S. The hypersharp nature of the 13.4 S b o u n d a r y indicates t h a t the

NATIVE

TITIN

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(a)

(b)

FIG. 7. Sedimentation velocity patterns of purified titin. The solvent was 0.5 M-KC1, 50 mMTris' HC1 (pH 7"9), 2 m~-MgC12, 1 mM-EDTA, 0-3 mM-dithiothreitol. The protein concentration was 2-0 mg/ml. (a) and (b) were taken 12 rain and 48 min, respectively, after reaching the operating speed of 52,000 revs/min. The bar angle was 55 °.

sedimenting species is highly asymmetric. In accord with this, the sedimentation coefficient shows a relatively strong dependence on concentration. Calculation of ks, the constant characterizing this dependence, from the equation: 1

l+ksC

8

8o

yields a value of 93 ml/g. This can be compared with values of less than l0 ml/g for globular proteins (Creeth & Knight, 1965) and approximately 50 ml/g for myosin, myosin rod and LMM (calculated from Lowey et al., 1969). 14

15

v

3

m

12

II

I0

I o

I

1 2 Concentration (mg/ml)

3

FIC. 8. Concentration dependence of S2o,w of the material giving rise to the hypersharp pe&k in titin preparations,

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J. T R I N I C K , P. K N I G H T AND A. W H I T I N G

The majority of sedimentation velocity runs were performed in 0"5 M-KCI, 50 mM-Tris-HC1 (pH 7"9 at 4°C), but we have also performed a few runs under different conditions. (Note that, since the runs were actually performed at about 20°C, the pH will have been somewhat lower at about 7.4.) Increasing the KC1 concentration to 2 M did not significantly change the observed pattern; reducing it to 0.2 ~, on the other hand, resulted in an approximately 50% increase in the rate of sedimentation of the hypersharp boundary. Increasing the pH to 9 appeared to have no effect on the relative amounts of the boundaries or on their sedimentation coefficients, while precipitation occurred at values below neutrality. (c) Circular dichroism spectra Figure 9 shows that purified titin exhibits only weak circular dichroism above 200 nm. In particular, the values of negative elliptieity at 208 nm and 222 nm, where e-helix produces large values, are very low. Since the elliptieity at 208 nm is only about - 1000 deg. cm2/dmol and is less than that due to a protein consisting wholly of random coil ( - 4 0 0 0 deg. em2/dmol; Greenfield & Fasman, 1969), the e-helix content of titin is close to zero. Comparison of Figure 9 with spectra from polypeptides containing all random coil or all fl-conformations (Greenfield & Fasman, 1969) suggests that titin contains very little fl-strueture and that its spectrum most clearly resembles that of 100°/0 random coil. (d) Quantitation of the amount of titin present in myofibrils The amount of titin in myofibrils was estimated from densitometry of SDS/polyacrylamide gels of whole myofibrils by comparing the-quantities of protein in the titin and myosin heavy chain bands. These quantities were obtained from standard curves constructed using data from gels loaded with known amounts of titin and myosin. The curves showed t h a t a given mass of titin bound about half as much stain as did the same mass of myosin. The mean myosin to titin mass ratio in myofibrils was found to be 6.5, although this figure Wavelength (nm) 210 I

I

220 I

I

2:30 I

E-

b 0

"0

FIO. 9. Circular dichroism spectrum of purified titin (smoothed trace of experimental curve).

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varied between 5 and 9, which is a wider range than would have expected from the precision of the method. If myofibrils contain 55% myosin (Huxley & Hanson, 1957), the amount of titin present is therefore 8"5+3~o of total myofibrillar protein.

(e) Electron microscopy of native titin (i) After spraying and shadowing In the first instance, electron micrographs of titin were obtained by rotary shadowing of preparations dried in vacuo after spraying onto mica in the presence of glycerol. Such specimens (Fig. 10(a)) reveal two types of object; extremely long string-like structures that follow a very tortuous path, suggestive of considerable flexibility, and much smaller structures with a globular appearance. Frequently, the two are found in association with one another in the form of a globular region with one or two tails. Although it seems likely (see below) that the globular structures are formed by coiling up of the strings, measurements from the micrographs did not reveal a strong correlation between the diameter of the globular regions and the shortness of their tails. The widths of the strings was found to be 40( + 8) A. This value was estimated by shadowing specimens in the presence of myosin and correcting for metal thickness (~ l0 A) by assuming that the myosin tail is 19 A in diameter (Elliott et al., 1968). The lengths of the strings were extremely variable, ranging between about 1000 A to over 8000 A. Histograms of these length values did not reveal any preferred value but showed a broad maximum with a mean at about 5000 A (Fig. ll). A curious appearance seen not uncommonly in micrographs of the sprayed preparations is that of irregular gaps between aligned sections of the string-like structures. On rare occasions, very thin elements can be seen connecting such aligned sections (Fig. 10(b)); more often though, nothing can be discerned in the gaps (Fig. 10(c)). We assume that the connections are always present but they are at the limit of detectability of the shadowing method. (ii) After layering and shadowing Some reduction in the complexity observed in the specimens obtained by spraying was achieved when the titin solution was layered on to mica and the bulk of the liquid then removed by centrifugation. Micrographs of such preparations (Fig. 12) exhibit string-like structures similar to those seen after spraying, but very few of the globular structures are seen. A further difference is that the aligned sections of the strings with gaps between them are no longer found. We suppose that these differences arise due to differences in the shearing forces that the preparations experience. We imagine that shearing forces are relatively large during spraying, both when the specimen is atomized and when droplets impinge on the mica, spread out and then retract. (iii) After negative staining We have also been able to see titin by negative staining, using grids coated with a thin film of carbon and irradiated with ultraviolet light before staining in the

FIG. 10. Electron microscopy of purified native titin prepared by spraying followed by rotary shadowing. Magnification, 66,000 x . (a) Usual appearance of a field of molecules. (b) Titin strands showing aligned sections joined by thin connections. These are best seen by viewing the photographs obliquely in the direction of the arrow. (c) As (b) but with no thin connections.

NATIVE TITIN

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28 --

24 --

2O

16

E

8

4

0

2000

4000 Length

6000 (~ )

8000

FIG. l l i H i s t o g r a m oftitin lengths a~er spraying.

FIG. 12. Elee~ton microscopy of purified native titin prepared by layering and rotary shadowing. Magnification, 66,000 x. 13

346

J. TRINICK, P. KNIGHT AND A. WHITING

conventional manner. The use of negative staining rather than shadowing results in a substantial increase in resolution and allows substructure to be discerned within the titin strands. Figure 13 shows two micrographs of such a stained specimen. Long string-like structures reminiscent of those seen after metal shadowing are frequently visible, although the exact course of the strings is often difficult to trace, making length measurements difficult. The diameter of the strings at their widest point is 35( __4) A and is similar to that determined by shadowing. They usually appear to be composed of regularly spaced particles roughly circular in profile and having a diameter equal to the maximum width of the string. The spacing of successive particles is 40( +_3) A. (iv) Negative staining of intact native thick filaments String-like structures having a beaded appearance can be found also associated with separated native thick filaments after negative staining (Fig. 14(a) and (b)). Since their appearance and dimensions are very similar to those of purified titin, we conclude that they are titin strands. As with the micrographs of purified titin, it is only in optimally stained areas that extremely small details, such as titin substructure, can be seen clearly. Interpretation is therefore often difficult, but the following points can be made. The titin strings are found in two locations: coiled up at the ends of the filaments (Fig. 14(a)) and running alongside the myosin heads in the cross-bridge regions (Fig. 14(b)). Both these appearances can be found in a single half-filament. In the coils of titin at the ends of a filament, several strings ( ~ 4) can sometimes be found running alongside one another in parallel (e.g. Fig. 14(a) top left). This behaviour appears to result from the strings folding back on themselves, although free ends can sometimes be found. Coils of titin can be found at both ends of the same filament. We have only ever seen one strand running alongside the cross-bridge region at any point (Fig. 14(b)), although we have seen single strings alongside the bridges in both halves of the same filament. These strands can extend over distances of 4000 to 5000 A and sometimes appear to run into the coils at a filament end (e.g. Fig. 14(b), second from right). Their distance from the filament axis is usually about 500 A; that is, of the order of 100 A beyond the point where myosin heads usually terminate in such micrographs (Triniek & ElliotL 1979; Knight & Trinick, 1984). 4. D i s c u s s i o n

The fact that titin can now be extracted and purified without exposure to denaturing conditions opens the way to a full characterization of its structure and function. We have presented here some basic properties of the native protein as a start towards this goal, and these show titin to have unusual secondary and tertiary structure that are compatible with the idea t h a t it functions as an elastic component of the myofibril. Although there are a number of properties of the native molecule that we do not fully understand, the material t h a t we isolate provides a relatively tractable preparation with which to work, and its

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FIG. 13. Electron microscopy of native titin prepared by negative staining. Magnification, 150,000 x.

348

J. T R I N I C K , P. K N I G H T AND A. W H I T I N G

(a) FIG. 14. Electron microscopy of negatively stained separated native thick filaments showing titin strands. Magnification, 150,000 ×. (a) Strands emanating from filament ends. (b) Strands running alongside cross-bridge regions. p u r i f i c a t i o n is easily a c h i e v e d t h r o u g h c o n v e n t i o n a l p r o c e d u r e s such as ionic strength manipulation and column chromatography. (a)

Solubility of titin

B e t w e e n a t h i r d a n d a h a l f of t h e t i t i n p r e s e n t in m y o f i b r i l s can be e x t r a c t e d

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(b) l?l~. 14.

rapidly simply by raising the KCI concentration to a value of about 0"6 ~. Surprisingly, this fraction rises only slightly with further increases in ionic strength or with prolongation of the extraction time, implying that titin can take two forms, soluble and insoluble. We have considered two possible explanations for this behaviour. One possibility is that the insoluble form of titin is stabilized by covalent crosslinks, and indeed Fujii et al. (1978) have reported that connectin preparations contain lysine-derived cross-links of the type found in collagen. Were this idea to

350

J. TR1NICK, P. KNIGHT AND A. WHITING

be correct, the cross-links in the residue would have to be broken under the dissociating conditions used prior to electrophoresis, in order to generate the same gel pattern as the soluble titin. We have, however, been unable to detect lysinonorleucine cross-links in denatured titin purified in the presence of SDS by the method of Wang (1982b); (Whiting & Trinick, unpublished data; see also Robins & Rucklidge (1980) and Fujii & Maruyama (1982) for discussion of this point). An alternative explanation concerns the effect of proteolysis. Wang (1982a) has reported that titin is not extracted by 0.6 M-KC1, but that it is subject to proteolysis by endogeneous proteinases. I t could therefore be argued that the protein extracted in this work had been degraded proteolytically, and that the insoluble form was that which had not been cleaved. The fact that titin generally migrates as a doublet on SDS/polyacrylamide gels is indicative of proteolysis; but if only degraded molecules were soluble, one would expect to find only the leading band of the doublet in the extracts, and mainly the trailing one in the residues. In fact, the intensities of the two bands are roughly similar in the extract and residue, demonstrating that it is possible to extract undegraded molecules. Since the data presented here suggest that the longest titin strands are likely to contain several polypeptide chains of weight ~ 10 6, what is perhaps more likely is that proteolysis at one or both ends of such a strand causes liberation, but leaves undegraded molecules elsewhere in the strand. (b) Secondary structure Amino acid analyses of titin purified in the presence of SDS showed it to have a relatively high proline content (8 to 9~o), implying that it might well not contain much a-helix (Wang, 1982a,b). This suggestion is confirmed by the circular dichroism spectrum of the native molecule, which shows a value of negative ellipticity at 208 nm, indicating little or no a-helix. The circular dichroism spectrum most closely resembles that of a structure entirely in random coil configuration. This result may be important in view of the fact t h a t a high random coil content is characteristic of elastic proteins, such as elastin (Aaron & Gosline, 1981), and is compatible with the idea that titin forms elastic filaments within the myofibrils (Wang, 1982a).

(c) Amount of titin in myofibrils Titin was estimated to form approximately 9% of the total myofibrillar protein, suggesting that it is the third most abundant protein in muscle; the next most common being troponin and tropomyosin, each of which forms 6.5°/o of the myofibril. However, this value should be treated with some caution, since there was appreciable variability in the results. In their 1979 paper, Wang et al. estimated the titin content of myofibrils in three different ways, although all the values they obtained also included the contribution of the "band 3" protein (now known as nebulin, Wang & Williamson, 1980), so exact comparisons with the present data are difficult to make. Using the method of Lowry et al. (1951) to determine the amounts of

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protein in column fractions after gel filtration chromatography of whole myofibrils in SDS, titin and nebulin together appeared to form 10 to 15% of the myofibril. Higher estimates of 15 to 22% were obtained on the basis of absorbanee measurements at 280 nm on the same column fractions, although it is not clear what extinction coefficients were used in the calculations. This range is likely to be an overestimate, partly because we have now shown titin to have the relatively high extinction coefficient of 13.7; but also because, in our hands, the leading fractions of the titin peak after such chromatography have absorbanee values at 260 nm higher than those at 280 nm, suggestive of significant nucleic acid contamination (Whiting & Trinick, unpublished data). The final range quoted by Wang et al. (1979) was 5 to 8%, and was based on comparison of the staining intensities of the titin and nebulin bands with those of all other bands after SDS/polyaerylamide gel eleetrophoresis of whole myofibrils and is close to the values quoted here. Since the electron microscope data presented here suggest that titin is associated with thick filaments, it is useful to use the myosin to titin mass ratio of 6.5 to calculate approximately how many molecules of titin of mass 106 there are per thick filament. From sequence studies, myosin is known to have a molecular weight of 520,000 (M. Elzinga, personal communication) and there will therefore be about 13 myosin molecules for very titin chain. Since there are 294 myosin molecules in a thick filament (Craig & Offer, 1976; Luther & Squire, 1978), the number of titin molecules per filament will be 24. This value may, however, be an overestimate as the chain weight of titin may be significantly higher than the value of 106 generally used. Wang (1982b) reported that titin comigrates on SDS/polyacrylamide gels with a cross-linked hexamer of the myosin heavy chain. Since the molecular weight of the heavy chain is 220,000 (M. Elzinga, personal communication), the chain weight of titin may in fact be about 1.3 x 106. -Use of this higher value reduces the estimate of the number of titin molecules per thick filament to about 18. (d) Shape of titin observed in the electron microscope after shadowing That titin is a highly asymmetric molecule can be inferred from the hypersharp character of the main boundary seen in sedimentation velocity experiments, and from the high dependence of its sedimentation coefficient on concentration. This asymmetry is confirmed by electron microscopy of sprayed and shadowed titin specimens, which shows long string-like structures. What is not clear, however, is why the lengths of these strings should be so heterogeneous, although we note that similar heterogeneity has been referred to in a brief report by Wang & Ramirez-Mitchell (1983). The longest of the strings (~8000 A) are too long to be composed of a single polypeptide chain of l06 Mr, since a cylindrical molecule of this weight and a diameter of 40 A would have a length of about 1000 A. In view of the relative invariance of the width of the strings, an end-to-end polymerization seems possible; but this would probably give rise to peaks in the length histograms at integral multiples of a particular value and such behaviour is not apparent, possibly due to proteolysis.

352

J. TRINICK, P. KNIGHT AND A. WHITING

Two unusual features of the micrographs of the sprayed titin are an apparent propensity of the string-like structures to roll up into a more compact form and the separation of the strings into aligned sections, which can occasionally be seen to be joined by very thin connections. We assume that these appearances are due to strong shearing forces experienced during spraying, since both are absent if a method involving layering onto mica is used. I t seems possible that the aligned sections are due to elasticity in the titin but, if this is the case, the behaviour is different from rubber, where the extension per unit length is invariant. Here, in contrast, it would appear that the structure has yielded at some points and remained unextended elsewhere. Since the very thin connections that can sometimes be seen to join these aligned sections are less than half the width of a myosin tail (eoiled~coil of 2 e-helices), it is likely that they consist of a single, extended polypeptide chains. Although considerable simplification results from layering the titin rather than spraying it, the lengths of the string-like structures remain very heterogeneous. A number of attempts have been made to find conditions under which dissociation into smaller more uniform structures would occur, and the effects of raising both the p H and ionic strength have been monitored in the analytical ultracentrifuge and electron microscope. To date, these experiments have been unsuccessful, but it may well be possible in the future to induce dissociation with a suitable solvent.

(e) Substructure of titin observed by negative staining While the gross details of the shape of titin can be seen in micrographs of shadowed specimens, little fine structure is visible in the strands, since the resolution attainable with this technique is relatively low, probably ~ 5 0 A. Negative staining, on the other hand, is capable of appreciably higher resolution and, after treatment with uranyl acetate, the titin strands can be seen to have an appearance similar to a string of beads; the repeat distance between successive beads being approximately 40 A. Whether this appearance is due to repeating domains or merely to particularly strong binding of stain at regular intervals is not clear. In either case, the repeating appearance probably represents some form of periodicity in the primary structure of the molecule. The beaded appearance of negatively stained strands of purified titin allows its identification relatively unambiguously in more complex systems. String-like structures were seen emanating from the ends and running along the sides of shadowed separated thick filaments by Triniek & Elliott (1982), but their origin was then unclear and it seemed possible that they were DNA or RNA, since nucleic acids can often be found associated with purified myofibrils. In negatively stained filaments, however, these strands take on a beaded appearance strongly reminiscent of the native titin. (f) Relationship of titin to end-filaments Structures termed end-filaments about 850 A long and having a periodicity of 42 A have been identified at the ends of thick filaments by Trinick (1981). They

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were first seen in thick filaments that had been "frayed" into three sub-filaments by exposure to very low ionic strength, but more recently they have also been observed projecting beyond the ends of unfrayed thick filaments (Craig & Knight, 1983; Kensler & Stewart, 1983). Since the end-filament periodicity is very similar to that of negatively stained titin strands, and since the two are found in very similar locations, it is likely that end-filaments are composed of titin. It may also be significant t h a t at 850 A, the length of the end-filament is similar to the value of about 1000 A that can be calculated as the length of a cylindrical molecule of 106 M r and diameter 40 A. The appearances of end-filaments and titin strands are, however, somewhat different and the end-filaments appear slightly thicker (48(+ 10) A; Triniek, 198l) and straighter, and they often give the impression of two or three beaded strands packed in register. If end-filaments were to be composed of more than one titin strand, then the most likely number is three, since thick filaments have 3-fold rotational s y m m e t r y (Luther & Squire, 1978). Although we have shown that there are several separate titin strands emanating from the ends of the thick filaments, their exact number is difficult to count and they sometimes appear folded back on themselves. W h y the titin strands should sometimes be separate but at other times appear aggregated into end-filaments is not clear. I t is not the case that end-filaments form purely as the result of exposure to very low ionic strength, since observations showing them on unfrayed thick filaments (Craig & Knight, 1983; Kensler & Stewart, 1983) were made on filaments stained after being held under roughly physiological ionic conditions. If the number of titin strands forming an end-filament is three, and if, as seems likely, these strands extend back from the tips of thick filaments towards the Mline, then the figure of 24 titin molecules per thick filament previously calculated suggests that the titin could extend over 0-8 #m in the sarcomere. Interestingly, 0.8 #m is about the maximum strand length observed in mierographs of shadowed native titin. If the titin strands are elastic, 0.8 #m would be the rest length.

(g) Function of titin and its relation to putative

elastic filaments in myofibrils The suggestions by Maruyama et al. (1977) and Wang (1982a,b) of elastic connections within myofibrils are but two examples of several proposals concerning such connections, based both on theoretical grounds and on experimental data. Elastic filaments have been invoked to explain the passive elasticity of muscle, much of which is now thought to reside inside myofibrils, and particularly to account for the integrity and elasticity of myofibrils after extraction of their A and I-bands (Hanson & Huxley, 1955). In insect flight muscle, connections between the ends of thick filaments and the Z-line are relatively well-established (Pringle, 1977), but in vertebrate skeletal muscle such linkages, in this or any other configuration, have yet to be generally accepted. A likely reason why the idea of elastic filaments in vertebrate muscle has not gained wider acceptance, despite a considerable weight of experimental evidence, is that there is as yet no concensus as to their precise arrangement. In the sliding

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J. TRINICK, P. KNIGHT AND A. WHITING

filament theory as it was originally formulated (e.g. see Hanson & Huxley, 1953), there were proposed extensible S-filaments joining the ends of thin filaments in each half sareomere, but this idea was not pursued, for want of supporting evidence. McNeill & Hoyle (1967) and Waleott & Ridgway (1967) found evidence of superthin (25 A) filaments in micrographs of a number of vertebrate and invertebrate muscles and these were found in both the A and I-bands, suggesting connections running from Z-disc to Z-disc. From observations of myofibrils labelled with antibodies to connectin, Maruyama and co-workers (e.g. see Maruyama et al., 1980) have suggested a similar but slightly different arrangement consisting of a net-like array of elastic filaments running throughout the sareomere but not necessarily parallel to the filament axis. It is difficult, though, to see how such an arrangement could not interfere with force generation. Some of the strongest evidence of elastic filaments comes from the work of Locker & Leet (1975), who showed connections which they termed gap filaments bridging the gap between the ends of the A and I-bands in highly stretched muscle. These appeared to run from the ends of thick filaments to the Z-line, similar to the arrangement proposed in insect flight muscle. A functional advantage of such a system could be to maintain the A-band centrally in the sareomere, the mechanism of which is not otherwise obvious (see also Magid, 1983). Locker & Leer (1976) were also able to see what appeared to be gap filaments extending from the ends of thick filaments that had been partly depolymerized down to a length of about 0.4 pm by exposure to an elevated salt concentration. This suggested that the gap filaments did not terminate at the edges of the A-band but extended into the thick filaments. They were not, however, able to decide whether the gap filaments entered the core of the thick filaments or ran along their surface. One unusual feature of Locker & Leet's (1976) model was t h a t gap filaments were supposed only to extend from one rather than both ends of a thick filament. If, as seems likely, gap filaments are formed from titin strands, the fact t h a t we find these strands at both ends in the same filaments would rule out this idea. The evidence in the literature of a class of elastic filaments in vertebrate skeletal muscle is therefore quite strong, even if it has not been possible to decide their precise location or composition. A major difficulty with much of the data is that it comes from mierographs of thin sections of muscle embedded in plastic, where it is difficult to obtain a resolution of better than about 50 A. Consequently, fine detail that might allow unambiguous identification of particular structures is usually lost. Based on the labelling of myofibrils with antibodies raised to denatured titin and nebulin, Wang (1982a) has suggested that these proteins form elastic filaments linking successive Z-discs independently of thick and thin filaments, but that the elastic filaments are possibly joined to the thick and thin filaments at specific points. In this model, titin would stretch through the A-band and extend into the I-band as far as the N2-1ine. The gap between the N2-1ine and Z-line would be bridged by nebulin and possibly some other protein. I t was suggested that the strong antibody labelling observed at specific points, such as the A - I junction, might result from proteolytieally cleaved elastic filaments having sprung

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back in a rubber-like manner. The present micrographs of titin on thick filaments are not incompatible with these ideas, though there m a y well be alternative explanations. For instance, the titin t h a t is in the A-band m a y be bound to thick filaments and the a n t i b o d y labelling d a t a of W a n g et al. (1979) could be a reflection of differing degrees of antigen accessibility. The titin strands t h a t we see running alongside the cross-bridge regions of separated thick filaments m a y be located in the A-band in vivo, either attached to the surface of the thick filaments or largely independent of them, as suggested by Wang (1982a). However, we cannot rule out the possibility t h a t these strands originally extended into the I - b a n d and have sprung back to the position seen here. These particular strands p r o b a b l y could not have originated from the core of the thick filament, since stain was applied whilst the filaments were being held under roughly physiological ionic conditions. At the m o m e n t then, there are not sufficient d a t a to decide whether any of the various models of elastic filaments is correct, or w h a t the exact relationship of titin to such a system might be. More definite conclusions on the precise location of titin, and therefore on its role, will depend on obtaining electron micrographs of muscle labelled with titin antibodies, preferably raised to the native molecule. I t will also be i m p o r t a n t to demonstrate elasticity in the native molecule, since there is no reason to suppose t h a t the rubber-like consistency of denatured preparations (e.g. see M a r u y a m a et al., 1977) accurately reflects the properties of titin within the myofibril. I f a system of elastic filaments in myofibrils can be proven to exist, it will significantly modify the simple two-filament version of the sarcomere structure t h a t has formed the basis for most modern work on muscle contraction. We are particularly grateful to Dr A. Magid of Duke University for showing us his unpublished work on elastic filaments. We would also like to thank Dr G. Offer for discussion, Dr W. Gratzer for the use of his spectropolarimeter and Messrs D. P~estall and H. F. Brown for help with illustrations. REFERE~TCES Aaron~ B. B. & Gosline, J. M. (1980). Nature (London), 287, 865 867. Craig, l%. & Knight, P. (1983). In Electron Microscopy of Protein8 (Harris, R., ed.), vol. 4, pp. 97 203, Academic Press, London. Craig, R. & Offer, G. (1976). J. Mol. Biol. 102, 325-332. Creeth, J. M. & Knight, C. G. (1965). Biochim. Biophys. Acta, 102, 549-558. Elliott, A. & Offer, G. (1978). J. Mol. Biol. 123,505-519. Eiliott, A., Lowy, J., Parry, D. A. D. & Vibert, P. J. (1968). Nature (London), 218, 656659. Elliott, A., Offer, G. & Burridge, K. (1976). Proc. Roy. Soc. set. B, 193, 43 53. Fujii, K. & Maruyama, K. (1982). Biochem. Biophys. Res. Commun. 104, 633-640. Fujii, K., Kimura, S. & Maruyama, K. (1978). Biochem. Biophys. Res. Commun. 81, 12481253. Greenfield, N. & Fasman, G. D. (t969). Biochemistry, 8, 4108-4116. Hanson: J. & Huxley, H. E. (1953). Nature (London), 172, 530-532. Hanson, J. & Huxley, H. E. (1955). Syrup. Soc. Exp. Biol. 9, 228 264. Huxley, H. E. & Hanson, J. (1957). Biochim. Biophys. Acta, 23,229 249. Kensler, R. W. & Stewart, M. (1983). J. Cell. Biol. 96, 1797-1802.

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Knight, P. J. & Trinick, J. A. (1982). Methods Enzymol. 85B, 9-12. Knight, P. & Triniek, J. (1984). J. Mol. Biol. In the press. Locker, R. H. & Leer, N. G. (1975). J. Ultrastruct. Res. 52, 64-72. Locker, R. H. &Leet, N. G. (1976). J. Ultrastruct. Res. 55, 157-172. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969). J. Mol. Biol. 42, 1-29. Lowry, D. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265 27.5. Luther, P. & Squire, J. (1978). J. Mol. Biol. 125, 313 324. Magid, A. (1983). Biophys. J. 41, 35a. Maruyama, K., Matsubara, S., Natori, R., Nonomura, Y., Kimura, S., Ohashi, K., Murakami, F., Handa, S. & Eguehi, G. (1977). J. Biochem. (Tokyo), 82, 317 337. Maruyama, K., Kimura, S., Toyota, N. & Ohashi, K. (1980). In Fibrous Proteins (Parry, D. A. D. & Creamer, L. K., eds), pp. 33-41, Academic Press, London. Maruyama, K., Kimura, S., Ohashi, K. & Kuwano, Y. (1981). J. Biochem. (Tokyo), 89, 701-709. Maruyama, K., Kimura, S., Toyota, N. & Ohashi, K. (1983). In Muscular Dystrophy: Biomedical Aspects (Ebashi, S. & Oosawa, E., eds), pp. 201-208, Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin. MeNeill, P. A. & Hoyle, G. (1967). Amer. Zoologist, 7, 483-498. Offer, G., Moos, C. & Starr, R. (1973). J. Mol. Biol. 74, 653-676. Pringle, J. W. S. (1977). In Insect Flight Muscle (Tregear, R. T., ed.), pp. 177 196, Elsevier North Holland, Amsterdam. Robins, S. P. & Rueklidge, G. J. (1980). Bioehem. Biophys. Res. Commun. 96, 1240-1247. Sutoh & Harrington (1977). Biochemistry, 16, 2441-2449. Trinick, J. (1981). J. Mol. Biol. 151,309 314. Trinick, J. & Elliott, A. (1979). J. Mol. Biol. 131, 133-135. Triniek, J. & Elliott, A. (1982). J. Microsc. 126, 151-156. Tyler, J. M. & Branton, D. (1980). J. Ultrastruct. Res. 71, 95-102. Waleott, B. & Ridgway, E. B. (1967). Amer. Zoologist, 7, 499-504. Wang, K. (1982a). In Muscle Development molecular and cellular control (Pearson, M. L. & Epstein, H. F., eds), pp. 439-452, Cold Spring Harbor Laboratories, Cold Spring Harbor. Wang, K. (1982b). Methods Enzymol. 85B, 264 274. Wang, K. & Ramirez-Mitehell, R. (1983). Biophys. J. 41, 96a. Wang, K. & Williamson, C. L. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 3254-3258. Wang, K., MeClure, J. & Tu, A. (1979). Proc. :Vat. Acad. Sci., U.S.A. 76: 3698-3702. Edited by H. E. Huxley