VOL. 16, 1167-1181 (1977)
BIOPOLY MERS
A Study of Paramyosin Aggregation Using Transient Electric Birefringence Techniques* SONJA KRAUSE and DONALD E. DeLANEY, Department of Chemistry, Rensselaer Polytechnic Institute, Troy, N e w York 12181
Synopsis The aggregation of several forms of the molluscan muscle protein paramyosin a t low concentration and at low ionic strength was studied in the pH range 6-10 using transient electric birefringence techniques. In the lower part of this pH range, the aggregates exhibit negative birefringence, hut this changes to positive birefringence when the pH increases. Analysis of the field-free birefringence decay transients of the paramyosin solutions showed that all aggregates coexisted with paramyosin monomer and allowed a determination of the rotational diffusion constant of the aggregates present a t each pH. T h e size and shape of‘these aggregates were estimated from their rotational diffusion constants and were compared with the known characteristics of larger aggregates such as paracrystals. The positively hiref‘ringent aggregates appear to he staggered dimers a t certain values of pH; a t other pH values. these aggregates appear to he higher aggregates, probably formed by lateral aggregation of monomer or dimer onto one of these staggered dimers. The staggered dimers are formed by overlap of 200-600 8, along each cylindrical paramyosin molecule, in agreement with the 530-8, overlap distance found in paramyosin paracrystals by Cohen et al. (1971) J . Mol. R i d . 56,223-237. Some speculations are presented about the nature of the negatively hirefringent species.
INTRODUCTION Certain molluscan muscles have the ability to contract for long periods of time without expending any appreciable energy. This type of contraction is often called the “catch contraction” and appears to be associated with the presence of large amounts of the protein paramyosin in the muscles. Some workers’ feel, nevertheless, that paramyosin has no role in the catch contraction, while others2,“have proposed a more active mechanism for paramyosin in “catch.” In particular, Johnson has proposed that “catch” involves the aggregation of the large filaments in molluscan catch muscles, mediated by interactions between paramyosin molecules on the outside surfaces of these large filaments (manuscript in preparation). This makes it of particular interest to study the aggregation of paramyosin molecules in uitro as well as in uiuo. Paramyosin is a rod-shaped protein molecule with a two chain a-helical * Taken, in part, from a thesis by D. E. DeLaney, submitted to Rensselaer Polytechnic Institute in partial fulfillment of the requirements for the Ph.D., 1975. Portions of this work were presented a t Meetings of the Biophysical Society in Minneapolis, 1973, and in Philadelphia, 1974, a t the International Congress of Biophysics in Copenhagen, 1975, and at the International Symposium on Molecular Electro-Optics, Asilornar, 1975. 1167 (0 1977 by
John Wiley & Sons, Inc.
1168
KRAUSE AND DELANEY
coiled coil ~ t r u c t u r e . ~The . ~ two chains, which appear to be identical6 probably both run in the same direction giving rise to a molecular polarity. The length of the molecule determined by light scattering7 and electron microscopy8 is in the range of 1200-1300 A with some differences between species. The molecular weight lies in the range of 190,000-220,000 also with some interspecies differences8 Paramyosin may be extracted from molluscan muscle by different procedures which lead to molecules with slightly different properties. The presumed native form of the molecule9 is extracted from the muscle either under acid conditions or in the presence of EDTA to prevent partial digestion by a hitherto unidentified metalloenzyme and in the presence of dithiothreitol to prevent oxidation of the free sulfhydryl groups in the molecule. This form of paramyosin is usually called a-R-paramyosin. If paramyosin is extracted from molluscan muscle, e.g., from the adductor muscle of Mercenaria mercenaria, using ethanol in the absence of EDTA and dithiothreitol,2J0 P-paramyosin is obtained. Stafford and Yphantisll found, using SDS gel electrophoresis, that 0-paramyosin appears to have a molecular weight about 5% less than that of a-R-paramyosin. We12 found, using transient electric birefringence techniques, that a-R-paramyosin molecules are about 6% longer, 1220 f 40 A, than P-paramyosin molecules, 1150 f 20 A, if measurements are performed at pH 3.2. The molecules of 0-paramyosin because of their method of preparation, probably have some of their sulfhydryl groups oxidized. In our study of monomeric paramyosin,12we found that the length of the a-R-paramyosin molecule, as calculated from the field-free birefringence decay transient, decreased to that of P-paramyosin as the pH was increased from 3.2 to 3.8. The decrease was not gradual but abrupt, and occurred suddenly between pH 3.21 and 3.29, It was postulated that the extra piece or pieces that were present in the a-R-paramyosin molecule become hinged or flexible in that pH range. We also found a large difference in the dipole moments of a-R- and P-paramyosin a t pH 3.2 which disappeared a t higher pH. This implied that the larger dipole moment of the a-R-paramyosin at low pH was connected with one or more extra positive charges on the extra piece of the molecule; because of the very low pH a t which these charges appeared to lose their effect, possible aspartic acid residues (which titrate at very low pH) were also postulated in the extra piece. When these are titrated, the negatively charged carboxyl groups should cancel the effects of the positive charges. Possible mechanisms for the increase in flexibility of the extra piece on a-paramyosin as the pH changes from 3.21 to 3.29 were discussed but not proven. The extra piece appeared to remain flexible up to pH 11,that is, throughout the physiological pH region around 7. This may prove significant in the explanation of the “catch” mechanism in paramyosin-containing muscles even though our data were obtained a t millimolar ionic strengths, several orders of magnitude below physiological ionic strength. In the present work, we have used the transient electric birefringence
STUDY OF PARAMYOSIN AGGREGATION
1169
technique to study the aggregates of a-R- and of P-paramyosin at millimolar ionic strength in the pH range in which these aggregates are soluble. One preliminary study of a-R-paramyosin paracrystals was also obtained. The transient electric birefringence technique is described in some recent book^'"^'^ and reviews.'"17 Most aspects of the technique of interest in this work were also discussed in a previous paper.12
EXPERIMENTAL Materials The chowder clams, Mercenaria mercenaria, used as a source of paramyosin in these experiments were obtained fresh from a local fish market. The various forms of paramyosin were all extracted from the white portion of the adductor muscles. P-Paramyosin was extracted from the muscle tissue by the ethanol extraction method of Bailey'O as modified by Johnson et al.2 described previously.12 The a-R-paramyosin was isolated using the ethanol extraction method with the addition of lOmM potassium EDTA and 0.5mM dithiothreitol to all of the steps, following Stafford,l8also described previously.12 In preparation for electric birefringence measurements, paramyosin stock solutions were diluted and dialyzed a t 4°C against 1mM buffer solutions at the desired pH for 24-28 hr using five changes of buffer solution. Buffers used in the experiments discussed in this paper included N,N-bis(2-hydroxyethy1)piperazinesulfonic acid, pKa = 7.15; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pKa = 7.55; N,N-bis(2-hydroxyethy1)glycine, pKa = 8.35; and 3-(cyclohexylamino)propanesulfonicacid, pKa = 10.40. All pKa's are a t 20"C, the temperature of most of our birefringence measurements. After dialysis, the concentration of paramyosin in each solution was determined spectrophotometrically from the tyrosinetryptophan absorbance a t 277 nm as described previously.12
Birefringence Apparatus The birefringence apparatus, as described previously,12had a He-Ne laser as a light source and contained a quarter-wave plate so that the sign of any birefringence could be determined. Glan-Thompson prisms were used as polarizer and analyzer, light signals were picked up and amplified by an EM1 9558 B photomultiplier, and a Tektronix 547 Dual Trace Oscilloscope was used to obtain simultaneous voltage pulse and birefringence signals which were photographed on Polaroid transparencies. For this work, the voltage pulses were single rectangular pulses produced either by a medium-range (0-1000 V DC) transistorized pulser constructed in this laboratory12 or by a high-voltage unit (0-4400 V DC) constructed from a stacked combination of two commercially available Cober 605 P High-Power Pulse Generators. The voltage pulses were applied to the
1170
KRAUSE AND DELANEY RunXl30-5
8 -Poromyosm i
Conc. Img/mN Temp. (CJ
i
Shorr ielozotmn rime componenr =
(radians)
.u'u[
?;':
l7,;psec
,005
o
20
,
,
1
,y
60 ao 100 T I M E (pet)
40
7.9 0.68
= 2O0
1 2 0 140
Fig. 1. Field-free birefringence decay curve of a sample of P-paramyosin with positive birefringence in terms of loglo(optical retardation, 6) us time; TL is the longer, and lS is the shorter relaxation time obtained from the data.
solution in a 1-cm spectrophotometer cell by means of platinum electrodes which were 2.30 f 0.05 mm apart.
Calculations In this paper, we discuss only the field-free decay of the birefringence of the paramyosin solutions after the electric field is turned off. In a monodisperse solution of very anisometric macromolecules, this birefringence decay is exp~nential'~: AnlAno = e - t / T
(1)
where An is the magnitude of the birefringence of time t , An0 is the birefringence when t = 0, and T is the birefringence relaxation time for the molecule. For a cylindrically symmetric molecule, T is related to the rotational diffusion constant of the symmetry axis d:
e = l/sT
(2)
For polydisperse systems containing i discrete, noninteracting components the decay of the birefringence takes the formlg
where Ano,;is the contribution of the ith species to the birefringence a t t = 0 and ~i is the relaxation time of that species. Equation (3) shows that the observed birefringence decay curve can be broken down into its components if the 71' are sufficiently different from each other. Figure 1shows how this was done for one of our samples with positive birefringence, a typical sample in the sense that two components with different relaxation
STUDY OF PARAMYOSIN AGGREGATION
1171
/
,/
(SIGNAL
FROM LAROE
SPECIES
TIME
Fig. 2. Birefringence signal from a solution in which a small positively birefringent species coexists with a large negatively birefringent species. The breakdown of the actual signal (solid curve) into its components (dashed curves) is shown.
times appeared to be present. The optical retardation d was calculated from photographs of the birefringence signal in the usual manner.12 At those values of pH a t which the paramyosin aggregates had negative birefringence, the birefringence signals looked very much like Figure 2. Figure 2 also shows how the actual birefringence signal was broken down into its components parts, a positive signal from a small species and a negative signal from a large species.20 Birefringence relaxation times were than obtained for the negative species; for the positive species, only the initial slope of the relaxation could be determined in order to estimate its relaxation time. Rotational diffusion constants can be related to the size and shape of the rotating species by means of equations developed by PerrinZ1if they are ellipsoids of revolution, by Bud0 et a1.22 If they are general ellipsoids, and by Broersma2:3if they are cylindrical. Equations for other shapes exist, but were not used in this work.
RESULTS The precision of tabulated values of the relaxation times is stated in terms of average deviation from the mean of a t least three measurements, i.e., three voltage pulses, on each sample except for the negatively birefringent samples. Results from a single voltage pulse only are shown in Table I. Table I contains the results obtained from the analysis of the relaxation times of a-R- and of P-paramyosin in the pH 6-7 region. Although all of these runs showed the same type of signal, the relaxation times measured
KRAUSE AND DELANEY
1172
TABLE I Electric Birefringence Properties of Paramyosin, pH 6.5-7.0 toc
PH
Concentration (mg/ml)
20 20 20 30
6.92 6.82 7.01 7.00
25
6.46
'
Negative Signal T (msec)
Positive Signal Ti (psec)
a-R-paramy osin 0.51 0.47 0.45 0.45
1.01 0.53 0.90 0.99
10.5 13.9 26.3
(3-paramyosin 0.58
1.67
-
were not always the same, varying from 0.53 to 1.67 psec for the negative signal. In some cases, the relaxation of the positive signal was lost in noise; in other cases, estimates of its relaxation time varied from 10 to 45 psec. It was not possible, with the data at hand, to establish whether the species produced were dependent on the conditions of pH and ionic strength. Nevertheless, in measurements made on a-R-paramyosin, the large negatively birefringent aggregate showed some thermal stability. The pH range in which the mixed signals were observed was found to be dependent upon the form studied. a-R-paramyosin existed in this aggregated form over the greatest range, pH 6.0-7.3. (In some cases small negatively birefringent signals were noted at pH values as high as 7.6.) In contrast, P-paramyosin showed the presence of a large negatively birefringent species only in the narrower pH range of pH 6-6.5. A t higher pH, only positive birefringence was observed. , from the The relaxation time of the positive signal is listed as ~ i taken initial slope of the plot of log6 us time. The probable error in this value is therefore very large. Negative signal relaxation times are all of the order of magnitude 1 msec. ) times of a-RTable I1 shows the long ( T L ) and short ( T ~ relaxation paramyosin and P-paramyosin in the pH region where positively birefringent aggregates occur and also the relaxation time calculated from the initial slope of the log6-us-time plot (Ti). In some cases, it was not possible to disentangle two relaxation times from the data even though more than one such relaxation time appeared to be present. In such cases, only 7;is shown in Table 11. Data a t very high pH, where monomeric paramyosin exists alone in the solution, have been presented previously. l 2 Since there is some concentration dependence of the relaxation time of a-R-paramyosin above 0.50 mg/ml a t pH 3.2,12the data for this molecule shown in Figure 3 are only for concentrations at and below 0.50 mg/ml. Figure 3 shows the long and the short relaxation times, when both were observed, of a-R-paramyosin as a function of pH, including pH ranges discussed previously.12 In the pH range 4-6, the a-R-paramyosin precipitates from solution as a gel, and from pH 6-7, the aggregates exhibited
STUDY OF PARAMYOSIN AGGREGATION
1173
TABLE I1 pH Dependence of the Properties of Positively Birefringent Aggregated Paramyosin a t 20°C PH 7.35 8.10 8.40 8.67 8.80 8.82 8.87 9.00 9.10 9.20 9.58 9.61 9.70 9.75 9.75 9.80 9.88 6.63 6.72 6.90 6.90 7.13 7.24 7.31 7.48 7.54 7.61 7.79 7.92 8.17 8.55 8.72 8.97 9.00 9.47
Concentration (mg/ml)
TL (psec)
7s
(wet)
Ti (psec)
a-R-Paramyosin (0.5mM Dithiothreitol in All Samples) 0.46 24.3 f 0.43 63.3 f 2.1 16.4 t 0.6 28.9 f 0.50 31.5 f 0.50 81.7 f 3.8 31.8 t 18.4 + 0.9 17.6 i 1.1 25.8 f 0.69 79.8 f 4.6 1.30 75.4 f 6.8 14.8 + 1.7 26.6 t 0.64 17.8 i 0.9 31.3 r 65.3 i 2.2 0.46 33.4 f 0.48 81.3 f 7 ; l 32.2 f 17.3 f 0.2 0.42 78.9 i 5.6 35.8 f 17.9 i 0.2 0.42 68.2 i 6.6 15.8 i 2.4 26.3 f 1.02 68.7 2 1.9 16.6 t 0.2 31.1 i 0.83 24.3 t 0.63 29.9 t 25.3 t 0.64 39.1 f 5.2 14.7 i 0.6 0.40 26.7 f 0.50 25.7 i 0.70 0.89 0.56 0.63 0.72 0.64 0.63 0.56 0.74 0.90 0.92 0.68 0.80 0.81 0.83 0.81 0.58 0.88
(3-Paramyosin 68.5 f 4.8 67.3 f 0.5 67.8 t 2.5 74.9 f 3.1 67.9 f 5.7 82.4 t 8.3 73.6 t 5.6 79.0 f 4.5 93.9 t 6.2 89.9 i 10.3 78.9 f 3.1 77.2 5 3.5 87.9 i 2.7 62.3 f 6.7 47.7 t 0.7 -
17.5 i 0.4 17.3 f 1.0 19.5 t 1.6 16.6 t 1.6 19.5 f 1.3 17.2 f 0.1 20.0 f 0.7 19.7 f 1.3 19.9 i 0.6 19.3 t 0.5 17.4 f 1.0 19.6 f 0.4 19.2 i 1.4 18.5 f 0.3 15.3 i 0.4 -
36.5 38.6 36.1 32.9 44.7 48.0 33.6 45.4 48.7 52.3 53.0 34.0 46.6 48.6 41.8 29.9 26.6 22.5
t f
i
t f 2
i i i
i f f
i i i i i f
3.1 0.5 3.1 2.2 1.9 1.5 2.0 1.7 0.2 1.2 3.3 1.4 1.1 1.0 1.2 0.4 0.7 1.5 1.5 4.2 2.0 0.9 1.8 0.5 2.5 1.4 1.6 1.3 0.8 1.0 2.5 1.4 1.1 0.7 0.6
negative birefringence. Figure 4 shows similar data for P-paramyosin; this molecule was studied at higher concentration because of its low dependence of relaxation time on concentration at pH 3.2.12 The differences in the pH dependence of the relaxation times of a-R-paramyosin and P-paramyosin can best be seen in a superposition of the two sets of data, as in Figure 5. It seemed reasonable to suppose that the negatively birefringent species seen in the pH 6-7 region might arise from small paramyosin crystallites,
KRAUSE AND DELANEY
1174
90 - (3-R-faromyosin
I001
90
~
I
-
I
I
I
I
I
I
I
I
I
p - Paromyosin Ternperolure -20°C
80 70-
z-
60-
( p s e c ) 50I
I
I
1
30-
G
I BJ I' ;1 '4A P
&.I; I
10
I
I
I
E L IN.;
r
20-
I
I' I!
I
40 -
I
1
1
1
I
I
'SIX
Z I , I
I
I
I
especially since paracrystals precipitate from such solutions at higher ionic strength.24 However, since monomeric paramyosin has positive birefringence, a negatively birefringent crystallite would probably tend to have the molecules oriented perpendicular to the major axis of the crystallite. Since this seemed improbable, some paracrystals of a-R-paramyosin were prepared a t pH 6, suspended in distilled water, centrifuged to remove large aggregates, and exposed to a 5-msec pulse of 3500 V/cm in the birefringence cell. A large, positively birefringent signal with an pxtremely long field-free
STUDY OF PARAMYOSIN AGGREGATION
90
t
d - R - Porornvos,n :p-Porom od,n = ----
2
i
Comb,ne/Monorner Values = - Temperature - 2 0 ° C
I
10
1175
I
I
I
I
,
I
,
I
I
I
3
4
5
6
7
8
9
10
II
12
PH Fig. 5. Superposition of the best f i t of relaxation times us pH data for wR- and ¶ m yosin.
decay (up to 1sec) was observed. If the negatively birefringent species are crystallite nuclei, therefore, they are very different from the paracrystals that can be precipitated from solution a t slightly higher ionic strength.
DISCUSSION In general, paramyosin appears to exhibit three types of aggregation a t low ionic strength. 1)Both a-R- and P-paramyosin form a gel in the pH 4-6 region. 2) A combination of a slowly relaxing, negatively birefringent aggregate and a positively birefringent species with a fast relaxation time is seen over the pH 6-7.2 range for a-R-paramyosin and in the region of pH 6-6.5 for 0-paramyosin. 3) Smaller, positively birefringent aggregates in equilibrium with monomer are observed in the range of pH 7.2-10 for aR-paramyosin and pH 6.5-10 for the form of the molecule. No birefringence studies were done in the pH 4-6 region where both the a-R and forms of paramyosin form a gel. However, it is of interest to note that this pH region contains the isolectric point, pH 5.8.25
Aggregated Species with Mixed Positive and Negative Birefringence The aggregated state that gives rise to a mixed positive and negative birefringence signal appears over a wider range of pH for a-R-paramyosin than for the p form. However, analysis of the birefringence signals from the different forms of paramyosin indicates that the species present are similar. As mentioned above, the monomeric paramyosin molecule probably has
1176
KRAUSE AND DELANEY
its permanent dipole moment directed close to or directly along its major axis, since it gives rise to a positively birefringent signal. Thus, the presence of a negative birefringence would indicate that the direction of the dipoles responsible for the orientation of the aggregated species is perpendicular to the major axis of the aggregate. The very long relaxation times observed for these aggregates also indicate that they are quite large. Combining the above observations, it appears that the negatively birefringent signals could arise from a large lateral aggregate of paramyosin molecules, i.e., with the major axes of the molecules perpendicular to the major axis of the aggregate. If it is assumed that the aggregate is in the form of an oblate spheroid (i.e., disk shaped) it is possible to estimate its size from the relaxation time and the Perrin equation for the rotational diffusion constant of an oblate spheroid.21 A series of calculations were done using this equation with the rotational diffusion constants calculated from the experimentally determined relaxation times. To calculate unique values for the largest dimension of the oblate spheroid, i.e., the diameter, it was necessary to choose specific values for the thickness. When it was assumed that the thickness of the aggregate corresponded to the estimated monomer, whose length was taken as 1200 A for this purpose, the experimentally determined relaxation times corresponded to diameters from 2000-3000 A. Using the general ellipsoid calculations of Bud0 et a1.,22many ellipsoids whose longest relaxation time is of the order of 1 msec can be calculated, for example, an ellipsoid with one axis approximately monomer length, 1200 A, a second axis 3600 A, and a third axis about 600 A, a rectangular parallelopipedlike aggregate. One must note two things about such speculations: 1) the aggregates in solution are not really ellipsoids, and 2) aggregates of this type would probably exhibit two birefringence relaxation times, although these might be hard to disentangle from the data. A t any rate, the negatively birefringent species may be a laterally aggregated species whose thickness is equal to the length of a monomer molecule, but this speculation needs much further investigation, for example, direct electron microscopic observation of the aggregates. It is interesting to note that the pH region in which the combination of postively and negatively birefringent species appears (pH 6-7) also corresponds to the pH region in which paramyosin precipitates as paracrystals a t higher ionic strength.24 The a-R-paramyosin, for which the presence of the mixed positively and negatively birefringent species persists over the pH range of 6-7.2, produces much larger and thicker paracrystals than P-paramyosin, for which the presence of the mixed species was observed over the more limited range of pH 6-6.5. In spite of the positive birefringence observed for the paracrystals themselves, the negatively birefringent aggregates may nevertheless be representative of their nuclei. The relaxation times of the positively birefringent species which was mixed with the negatively birefringent species ranged from 10 to 45 gsec. This indicates that the observed signals probably originate from monomer or small aggregates of the molecule. Some of these relaxation times were
STUDY OF PARAMYOSIN AGGREGATION
1177
much lower than those observed for the monomer of any of the forms of paramyosin, but this may be experimental error.
Positively Birefringent Aggregates of Paramyosin In the region of pH 6.5-7.2, where solutions of a-R-paramyosin show the presence of mixed positively birefringent and negatively birefringent species, the 0 form of the molecule apparently exists as small positively birefringent aggregates in equilibrium with monomer. When the pH of the a-R-paramyosin is increased to 7.4, its negative birefringence also disappears and the resulting positive birefringence has a relaxation time of 24.3 psec, probably monomer plus a very small amount of aggregate. A t the same pH, the relaxation times of the 0-paramyosin species indicate small aggregates with relaxation times of 65 f 5 psec in equilibrium with monomer. As the pH is raised further, both a-R- and 0-paramyosin show increases in the relaxation time. At pH values above 8.0, it would appear that the relaxation time us pH curves for a-R- and 0-paramyosin are similar, except that the curve for the former is shifted to a position about one pH unit higher than that of the latter (Fig. 5). Each form of the molecule has a maximum relaxation time, at pH 7.6-8.0 for 0-paramyosin and pH 8.8-9.2 for a-R-paramyosin. A t higher pH values, the relaxation times of both forms of paramyosin fall off rapidly until only monomeric forms are present a t pH 10. Although a-R- and 0-paramyosin show striking differences in the pH dependence of aggregation, it appears that the positively birefringent aggregates formed by these two forms of the molecule are very similar. From Figure 5 and Table I1 it can be seen that the maximum relaxation times of both the a-R- and 0-paramyosin are essentially the same with values of TL = 80 f 5 psec. With few exceptions, other positively birefringent aggregates of both forms have TL values not less than 65 f 5 psec. Using these observations, one may speculate that both forms of the molecule form similar positively birefringent aggregates in a roughly stepwise manner. Several calculations were done to obtain an estimate of the size of the aggregated species from their relaxation times and the monomer lengths determined earlier,12 1220 f 40 A for a-R-paramyosin and 1150 f 20 A for P-paramyosin. The relaxation time of an end to end dimer was calculated using the Broersma”j equation. The value obtained, 130-160 psec, depending on species, appears to preclude the presence of any such end to end aggregates in the solutions of a-R or 0-paramyosin. From this observation it was concluded that the paramyosin monomers must aggregate in the form of overlapped dimers, possibly with some lateral aggregation. Since these aggregates could no longer be considered cylindrical, the Perrin equation for a prolate ellipsoid,21which was considered a better representation of the shape of the aggregates, was used. Plots of relaxation time us length were made from the Perrin equation using several different values for the diameter. The diameters used were in the range of those formed by ag-
KRAUSE AND DELANEY
1178
TABLE I11 Calculated Relaxation Times for Prolate Ellipsoids with a 1910 * 80 A Major Axis Diameter (Minor Axis)
(8) 20
35 40 60 70
100
Relaxation Time (wec ) 51 1 62t 64 t 721 74 t 84k
7 8 8 8 9 10
gregates of close packed rods with individual diameters the same as that of the molecule, 20 8. It was found that the length of the laterally aggregated species, from dimer to symmetrically packed lgmer, that would produce a relaxation time of 80 f 5 psec varied over the relatively small range of 1900-2100 A. These values predict that the molecules overlap each other for 200-600 8, of their length. The molecular overlaps calculated above include the value obtained by Cohen et a1.8 from electron micrographs of stained paracrystals of M . mercenaria paramyosin. From the banding patterns in these crystals they determined that the paramyosin molecules overlap each other by 530 A and that no end to end bonding is present. Thus the length of a dimer of aR-paramyosin would be expected to be 1910 f 80 A. Table I11 shows the birefringence relaxation times calculated for prolate ellipsoids21with short axes from 20 to 100 A, if their length is set a t 1910 f 80 A. The overlapped dimer is not cylindrically symmetrical, but its relaxation time should be in the range of those calculated for prolate ellipsoids with diameters from 20 to 40 A. (The overlapped dimer, a t its center of mass, has one 20- and one 40-Asemiaxis). Table I11 shows that the expected relaxation time of the overlapped dimer varies from 57 f 7 to 62 f 8 psec, within the range of the 65 f 5 psec observed in most of the smallest aggregated species. A P-paramyosin dimer would have a length of 1750 f 50 A, with a relaxation time of 51 f 4 psec. The maximum relaxation time of either species then corresponds to a more laterally aggregated species, at least a heptamer, with a thickness of 60-70 A. (A close-packed heptamer would have a diameter, at its center, of 60 A.) Thus, from these observations, one may hypothesize that the aggregation begins with the formation of an overlapped dimer to which molecules add on in a lateral direction so that the thickness of the aggregate increases while its length remains constant. The relaxation times of the smaller species present in equilibrium with the observed aggregates correspond, within the error of the curve analysis technique, to values expected for monomer. The median of the relaxation times observed for the samples of P-paramyosin is somewhat greater than the a-R-paramyosin value, but due to the large experimental error this difference cannot be considered significant. The most striking difference between the relaxation time us pH curves
STUDY OF PARAMYOSIN AGGREGATION
1179
Fig. 6. Superposition o f t h e best fit of the relaxation time us pH curve for ij-paramyosin on the titration curve of Johnson and Kahn (Ref. 26).
of the two forms of paramyosin (Fig. 5) is the shift of the maximum relaxation time in the a-R-paramyosin curve to a value about one pH unit higher than that observed for the P form. Since the size of the aggregates formed by the two kinds of paramyosin appear to be similar, the basis of this difference probably lies in the number andlor nature of the residues that titrate in the pH 7-10 region. It is thus of interest to compare the relaxation time us pH behavior with the titration curve obtained for the protein. Figure 6 is a plot of the titration curve for P-paramyosin obtained by Johnson and Kahn26superposed upon the relaxation times observed for aggregates of this molecule over the same range of pH. The titration data obtained by Riddiford and Scheraga25were not used in this comparison because their solutions, which were prepared from lyophilized samples of p-paramyosin, showed the presence of aggregation in 0.6M KC1, pH 7. A t these same conditions, fresh samples of 0-paramyosin generally are in monomeric form. The Johnson and Kahn data,26however, all were obtained using samples of freshly prepared P-paramyosin. From the superposed curves, several observations can be made. The region in which P-paramyosin forms a gel, pH 4-6, ends a t the center of a large buffering zone which Johnson and Kahn attributed to the titration of glutamic acid residues. However, most of the aggregation observed by transient electric birefringence in the present experiments appears in a region of the titration curve where there is no obvious titration taking place, at least a t high ionic strength. The region where the mixed positively and negatively birefringent species are seen, pH 6-6.5, does fall a t the end of the buffering zone mentioned above. However, in the region where only positively birefringent aggregates are present, there is no apparent titration
1180
KRAUSE AND DELANEY
taking place. Nevertheless, it is possible that the observed aggregation is related to the terminal -NH2 groups (pKa = 7.5) or the cystine residues (pKa = 8.3) that normally titrate in this pH region. Because of their small number in paramyosin, they might not be detected in a titration, but they might nevertheless be important to the aggregation. The aggregates dissociate into monomer in a region of the titration curve (pH 9-10) where there is a large buffering zone which can be attributed to the titration of the tyrosine (pKa = 9.6) and lysine (pKa = 10.4) residues. Unfortunately, no precise comparison of these data with the birefringence results can be made since the titration curves were obtained a t an ionic strength of 0.3M KC1 while all of the birefringence measurements were made on solutions in 1mM buffer. Since it has been shown that the pKa of the tyrosine groups in paramyosin depend upon the ionic strength,27it is probable that such shifts would take place for other groups as well. A titration curve at low ionic strength for each form of paramyosin will be obtained in the future. The titration curve of the a-R form of paramyosin has not yet been obtained, and, because of the differences in the aggregation behavior, the relaxation time and specific Kerr constant data cannot be compared with the titration curve for P-paramyosin. However, it seems that the pH range in which the dissociation of the aggregates occurs, although shifted to a somewhat higher pH in the case of a-R-paramyosin, still lies in the region of pH where one would expect tyrosine, lysine, and arginine to titrate. The shift in these values may possibly arise from the fact that these residues are involved in inter- or intramolecular interactions that are not present in the P form of the molecule. Some investigators have speculated, using spectrophotometric data, that the tyrosine residues of P-paramyosin are involved in the molecular a g g r e g a t i ~ n . ~Although, ~ . ~ ~ the data presented here would seem to support this thesis, Cowgill’s theory of the tyrosine fluorescence of P-paramyosin28provides evidence that these hydrophobic groups do not enter into any intermolecular interactions. However, the data obtained for the monomer of a-R- and P-paramyosin a t low pH indicate that both forms of the molecule contain a significant number of basic groups which probably remain charged in the aggregation region. Since the aggregates of both the a-R and P forms of paramyosin dissociate to the monomeric form in the region of the pKa of the lysine residues, one might suspect the participation of this group in the aggregation. The shift of the aggregation curve of a-R-paramyosin to higher pH values relative to Pparamyosin may arise from a difference in the number and environment of the lysine residues in the two forms of the molecule. A study of the titration curve of the a-R form of paramyosin over a range of ionic strength will be very helpful in further interpretation of the data obtained from electric birefringence. Our thanks to the donors of the Petroleum Research Fund of the American Chemical Society who supported this work at its start and to the National Institutes of Health for the support
STUDY OF PARAMYOSIN AGGREGATION
1181
of one of us (S.K.) recently by means of a Research Career Award. Thanks also to Dr. J. Garber, who built our original electric birefringence apparatus and who performed the first preliminary experiments on aggregated paramyosin, and to Mr. S. Jacobson, President of Cober Electronics, f i r lending us the pulsers for this work.
References 1. Lowey, J., Millman, B. M. & Hansen, J. (1964) Proc. Roy. Soc. B 160,525-536. 2. Johnson, W. H., Kahn, J. & Szent-Gyorgyi, A. G. (1959) Science 130,160-161. 3. Ruegg, J . C. (1961) Proc. Roy. Soc. R 154,209-223,224-249. 4. Cohen, C. & Holmes, K. C. (1963) J . Mol. Biol. 6,423-432. 5. Elliot, A. (1968) Symposium on Fibrous Proteins-Australia, Plenum, New York. 6. Weisel, J. W. (1974) Biophysical Society Abstracts, 18th Annual Meeting, Minneapolis, p. 76a. 7. Lowey, S., Kucera, J . & Holzer, A. (1963) J . Mol. Biol. 7,234-244. 8. Cohen, C., Szent-Gyorgyi, A. G. & Kendrick-Jones, J . (1971) J . Mol. R i d . 56, 223237. 9. Cowgill, R. W. (1974) Biochemistry 13,2467-2474. 10. Bailey, K. (1957) Pubb. S ~ WZool. . Napoli 29,96-108. 11. Stafford, W. F. & Yphantis, D. A. (1972) Riochem. Biophys. Res. Commun. 49,848854. 12. DeLaney, D. E. & Krause, S. (1976) Macromolecules 9,455-463. 13. Fredericq, E. & Houssier, C. (1973) Electric Dichroism and Electric Birefringence, Clarendon, Oxford. 14. Molecular Electric-Optics, O’Konski, C. T., Ed. (1976), Dekker, New York,Vols. I and 11. 15. O’Konski, C. T. (1968) Encyclopedia Polymer Sci. Technol. 8,551-590. 16. Yoshioka, K. & Watanabe, H. (1969) in PhysicalPrinciples and Techniques ofprotein Chemistry, Part A, Leach, S. J.. Ed., Academic, New York, Part A, pp. 335-367. 17. Stoylov, S. P. (1971) Aduan. Colloid Interface Sci. 3,45-110. 18. Stafford, W. F. (1973) Ph.D. dissertation, University of Connecticut, Storrs, Conn. 19. Benoit, H. (1952) Ann. Phys. (Paris) 6,561-609. 20. Matusmoto, M., Watanabe, H. & Yoshioka, K. (1972) Biopolymers 11,1711-1722. 21. Perrin, F. (1934) J . Phys. Radium 5,497-511. 22. Budo, A,, Fischer, E. & Miyamoto, S. (1939) Phys. 2.40,337-345. 23. Broersma, S. (1960) J . Chem. Phys. 32,1626-1631. 24. Merrick, J. P. & Johnson, W. H. (1977) Biochemistry (to be published). 25. Riddiford, L. M. & Scheraga, H. A. (1962) Biochemistry I, 95-107. 26. Johnson, W. H. & Kahn, J. S. (1959) Science 129,1190-1191. 27. Lowey, S.(1965) J . R i d . Chem. 240,2421-2427. 28. Cowgill, R. W. (1968) Biochim. Biophys. Acta 168,417-430.
Received June 18,1976 Accepted November 2,1976
BIOPOLY MERS
A Study of Paramyosin Aggregation Using Transient Electric Birefringence Techniques* SONJA KRAUSE and DONALD E. DeLANEY, Department of Chemistry, Rensselaer Polytechnic Institute, Troy, N e w York 12181
Synopsis The aggregation of several forms of the molluscan muscle protein paramyosin a t low concentration and at low ionic strength was studied in the pH range 6-10 using transient electric birefringence techniques. In the lower part of this pH range, the aggregates exhibit negative birefringence, hut this changes to positive birefringence when the pH increases. Analysis of the field-free birefringence decay transients of the paramyosin solutions showed that all aggregates coexisted with paramyosin monomer and allowed a determination of the rotational diffusion constant of the aggregates present a t each pH. T h e size and shape of‘these aggregates were estimated from their rotational diffusion constants and were compared with the known characteristics of larger aggregates such as paracrystals. The positively hiref‘ringent aggregates appear to he staggered dimers a t certain values of pH; a t other pH values. these aggregates appear to he higher aggregates, probably formed by lateral aggregation of monomer or dimer onto one of these staggered dimers. The staggered dimers are formed by overlap of 200-600 8, along each cylindrical paramyosin molecule, in agreement with the 530-8, overlap distance found in paramyosin paracrystals by Cohen et al. (1971) J . Mol. R i d . 56,223-237. Some speculations are presented about the nature of the negatively hirefringent species.
INTRODUCTION Certain molluscan muscles have the ability to contract for long periods of time without expending any appreciable energy. This type of contraction is often called the “catch contraction” and appears to be associated with the presence of large amounts of the protein paramyosin in the muscles. Some workers’ feel, nevertheless, that paramyosin has no role in the catch contraction, while others2,“have proposed a more active mechanism for paramyosin in “catch.” In particular, Johnson has proposed that “catch” involves the aggregation of the large filaments in molluscan catch muscles, mediated by interactions between paramyosin molecules on the outside surfaces of these large filaments (manuscript in preparation). This makes it of particular interest to study the aggregation of paramyosin molecules in uitro as well as in uiuo. Paramyosin is a rod-shaped protein molecule with a two chain a-helical * Taken, in part, from a thesis by D. E. DeLaney, submitted to Rensselaer Polytechnic Institute in partial fulfillment of the requirements for the Ph.D., 1975. Portions of this work were presented a t Meetings of the Biophysical Society in Minneapolis, 1973, and in Philadelphia, 1974, a t the International Congress of Biophysics in Copenhagen, 1975, and at the International Symposium on Molecular Electro-Optics, Asilornar, 1975. 1167 (0 1977 by
John Wiley & Sons, Inc.
1168
KRAUSE AND DELANEY
coiled coil ~ t r u c t u r e . ~The . ~ two chains, which appear to be identical6 probably both run in the same direction giving rise to a molecular polarity. The length of the molecule determined by light scattering7 and electron microscopy8 is in the range of 1200-1300 A with some differences between species. The molecular weight lies in the range of 190,000-220,000 also with some interspecies differences8 Paramyosin may be extracted from molluscan muscle by different procedures which lead to molecules with slightly different properties. The presumed native form of the molecule9 is extracted from the muscle either under acid conditions or in the presence of EDTA to prevent partial digestion by a hitherto unidentified metalloenzyme and in the presence of dithiothreitol to prevent oxidation of the free sulfhydryl groups in the molecule. This form of paramyosin is usually called a-R-paramyosin. If paramyosin is extracted from molluscan muscle, e.g., from the adductor muscle of Mercenaria mercenaria, using ethanol in the absence of EDTA and dithiothreitol,2J0 P-paramyosin is obtained. Stafford and Yphantisll found, using SDS gel electrophoresis, that 0-paramyosin appears to have a molecular weight about 5% less than that of a-R-paramyosin. We12 found, using transient electric birefringence techniques, that a-R-paramyosin molecules are about 6% longer, 1220 f 40 A, than P-paramyosin molecules, 1150 f 20 A, if measurements are performed at pH 3.2. The molecules of 0-paramyosin because of their method of preparation, probably have some of their sulfhydryl groups oxidized. In our study of monomeric paramyosin,12we found that the length of the a-R-paramyosin molecule, as calculated from the field-free birefringence decay transient, decreased to that of P-paramyosin as the pH was increased from 3.2 to 3.8. The decrease was not gradual but abrupt, and occurred suddenly between pH 3.21 and 3.29, It was postulated that the extra piece or pieces that were present in the a-R-paramyosin molecule become hinged or flexible in that pH range. We also found a large difference in the dipole moments of a-R- and P-paramyosin a t pH 3.2 which disappeared a t higher pH. This implied that the larger dipole moment of the a-R-paramyosin at low pH was connected with one or more extra positive charges on the extra piece of the molecule; because of the very low pH a t which these charges appeared to lose their effect, possible aspartic acid residues (which titrate at very low pH) were also postulated in the extra piece. When these are titrated, the negatively charged carboxyl groups should cancel the effects of the positive charges. Possible mechanisms for the increase in flexibility of the extra piece on a-paramyosin as the pH changes from 3.21 to 3.29 were discussed but not proven. The extra piece appeared to remain flexible up to pH 11,that is, throughout the physiological pH region around 7. This may prove significant in the explanation of the “catch” mechanism in paramyosin-containing muscles even though our data were obtained a t millimolar ionic strengths, several orders of magnitude below physiological ionic strength. In the present work, we have used the transient electric birefringence
STUDY OF PARAMYOSIN AGGREGATION
1169
technique to study the aggregates of a-R- and of P-paramyosin at millimolar ionic strength in the pH range in which these aggregates are soluble. One preliminary study of a-R-paramyosin paracrystals was also obtained. The transient electric birefringence technique is described in some recent book^'"^'^ and reviews.'"17 Most aspects of the technique of interest in this work were also discussed in a previous paper.12
EXPERIMENTAL Materials The chowder clams, Mercenaria mercenaria, used as a source of paramyosin in these experiments were obtained fresh from a local fish market. The various forms of paramyosin were all extracted from the white portion of the adductor muscles. P-Paramyosin was extracted from the muscle tissue by the ethanol extraction method of Bailey'O as modified by Johnson et al.2 described previously.12 The a-R-paramyosin was isolated using the ethanol extraction method with the addition of lOmM potassium EDTA and 0.5mM dithiothreitol to all of the steps, following Stafford,l8also described previously.12 In preparation for electric birefringence measurements, paramyosin stock solutions were diluted and dialyzed a t 4°C against 1mM buffer solutions at the desired pH for 24-28 hr using five changes of buffer solution. Buffers used in the experiments discussed in this paper included N,N-bis(2-hydroxyethy1)piperazinesulfonic acid, pKa = 7.15; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pKa = 7.55; N,N-bis(2-hydroxyethy1)glycine, pKa = 8.35; and 3-(cyclohexylamino)propanesulfonicacid, pKa = 10.40. All pKa's are a t 20"C, the temperature of most of our birefringence measurements. After dialysis, the concentration of paramyosin in each solution was determined spectrophotometrically from the tyrosinetryptophan absorbance a t 277 nm as described previously.12
Birefringence Apparatus The birefringence apparatus, as described previously,12had a He-Ne laser as a light source and contained a quarter-wave plate so that the sign of any birefringence could be determined. Glan-Thompson prisms were used as polarizer and analyzer, light signals were picked up and amplified by an EM1 9558 B photomultiplier, and a Tektronix 547 Dual Trace Oscilloscope was used to obtain simultaneous voltage pulse and birefringence signals which were photographed on Polaroid transparencies. For this work, the voltage pulses were single rectangular pulses produced either by a medium-range (0-1000 V DC) transistorized pulser constructed in this laboratory12 or by a high-voltage unit (0-4400 V DC) constructed from a stacked combination of two commercially available Cober 605 P High-Power Pulse Generators. The voltage pulses were applied to the
1170
KRAUSE AND DELANEY RunXl30-5
8 -Poromyosm i
Conc. Img/mN Temp. (CJ
i
Shorr ielozotmn rime componenr =
(radians)
.u'u[
?;':
l7,;psec
,005
o
20
,
,
1
,y
60 ao 100 T I M E (pet)
40
7.9 0.68
= 2O0
1 2 0 140
Fig. 1. Field-free birefringence decay curve of a sample of P-paramyosin with positive birefringence in terms of loglo(optical retardation, 6) us time; TL is the longer, and lS is the shorter relaxation time obtained from the data.
solution in a 1-cm spectrophotometer cell by means of platinum electrodes which were 2.30 f 0.05 mm apart.
Calculations In this paper, we discuss only the field-free decay of the birefringence of the paramyosin solutions after the electric field is turned off. In a monodisperse solution of very anisometric macromolecules, this birefringence decay is exp~nential'~: AnlAno = e - t / T
(1)
where An is the magnitude of the birefringence of time t , An0 is the birefringence when t = 0, and T is the birefringence relaxation time for the molecule. For a cylindrically symmetric molecule, T is related to the rotational diffusion constant of the symmetry axis d:
e = l/sT
(2)
For polydisperse systems containing i discrete, noninteracting components the decay of the birefringence takes the formlg
where Ano,;is the contribution of the ith species to the birefringence a t t = 0 and ~i is the relaxation time of that species. Equation (3) shows that the observed birefringence decay curve can be broken down into its components if the 71' are sufficiently different from each other. Figure 1shows how this was done for one of our samples with positive birefringence, a typical sample in the sense that two components with different relaxation
STUDY OF PARAMYOSIN AGGREGATION
1171
/
,/
(SIGNAL
FROM LAROE
SPECIES
TIME
Fig. 2. Birefringence signal from a solution in which a small positively birefringent species coexists with a large negatively birefringent species. The breakdown of the actual signal (solid curve) into its components (dashed curves) is shown.
times appeared to be present. The optical retardation d was calculated from photographs of the birefringence signal in the usual manner.12 At those values of pH a t which the paramyosin aggregates had negative birefringence, the birefringence signals looked very much like Figure 2. Figure 2 also shows how the actual birefringence signal was broken down into its components parts, a positive signal from a small species and a negative signal from a large species.20 Birefringence relaxation times were than obtained for the negative species; for the positive species, only the initial slope of the relaxation could be determined in order to estimate its relaxation time. Rotational diffusion constants can be related to the size and shape of the rotating species by means of equations developed by PerrinZ1if they are ellipsoids of revolution, by Bud0 et a1.22 If they are general ellipsoids, and by Broersma2:3if they are cylindrical. Equations for other shapes exist, but were not used in this work.
RESULTS The precision of tabulated values of the relaxation times is stated in terms of average deviation from the mean of a t least three measurements, i.e., three voltage pulses, on each sample except for the negatively birefringent samples. Results from a single voltage pulse only are shown in Table I. Table I contains the results obtained from the analysis of the relaxation times of a-R- and of P-paramyosin in the pH 6-7 region. Although all of these runs showed the same type of signal, the relaxation times measured
KRAUSE AND DELANEY
1172
TABLE I Electric Birefringence Properties of Paramyosin, pH 6.5-7.0 toc
PH
Concentration (mg/ml)
20 20 20 30
6.92 6.82 7.01 7.00
25
6.46
'
Negative Signal T (msec)
Positive Signal Ti (psec)
a-R-paramy osin 0.51 0.47 0.45 0.45
1.01 0.53 0.90 0.99
10.5 13.9 26.3
(3-paramyosin 0.58
1.67
-
were not always the same, varying from 0.53 to 1.67 psec for the negative signal. In some cases, the relaxation of the positive signal was lost in noise; in other cases, estimates of its relaxation time varied from 10 to 45 psec. It was not possible, with the data at hand, to establish whether the species produced were dependent on the conditions of pH and ionic strength. Nevertheless, in measurements made on a-R-paramyosin, the large negatively birefringent aggregate showed some thermal stability. The pH range in which the mixed signals were observed was found to be dependent upon the form studied. a-R-paramyosin existed in this aggregated form over the greatest range, pH 6.0-7.3. (In some cases small negatively birefringent signals were noted at pH values as high as 7.6.) In contrast, P-paramyosin showed the presence of a large negatively birefringent species only in the narrower pH range of pH 6-6.5. A t higher pH, only positive birefringence was observed. , from the The relaxation time of the positive signal is listed as ~ i taken initial slope of the plot of log6 us time. The probable error in this value is therefore very large. Negative signal relaxation times are all of the order of magnitude 1 msec. ) times of a-RTable I1 shows the long ( T L ) and short ( T ~ relaxation paramyosin and P-paramyosin in the pH region where positively birefringent aggregates occur and also the relaxation time calculated from the initial slope of the log6-us-time plot (Ti). In some cases, it was not possible to disentangle two relaxation times from the data even though more than one such relaxation time appeared to be present. In such cases, only 7;is shown in Table 11. Data a t very high pH, where monomeric paramyosin exists alone in the solution, have been presented previously. l 2 Since there is some concentration dependence of the relaxation time of a-R-paramyosin above 0.50 mg/ml a t pH 3.2,12the data for this molecule shown in Figure 3 are only for concentrations at and below 0.50 mg/ml. Figure 3 shows the long and the short relaxation times, when both were observed, of a-R-paramyosin as a function of pH, including pH ranges discussed previously.12 In the pH range 4-6, the a-R-paramyosin precipitates from solution as a gel, and from pH 6-7, the aggregates exhibited
STUDY OF PARAMYOSIN AGGREGATION
1173
TABLE I1 pH Dependence of the Properties of Positively Birefringent Aggregated Paramyosin a t 20°C PH 7.35 8.10 8.40 8.67 8.80 8.82 8.87 9.00 9.10 9.20 9.58 9.61 9.70 9.75 9.75 9.80 9.88 6.63 6.72 6.90 6.90 7.13 7.24 7.31 7.48 7.54 7.61 7.79 7.92 8.17 8.55 8.72 8.97 9.00 9.47
Concentration (mg/ml)
TL (psec)
7s
(wet)
Ti (psec)
a-R-Paramyosin (0.5mM Dithiothreitol in All Samples) 0.46 24.3 f 0.43 63.3 f 2.1 16.4 t 0.6 28.9 f 0.50 31.5 f 0.50 81.7 f 3.8 31.8 t 18.4 + 0.9 17.6 i 1.1 25.8 f 0.69 79.8 f 4.6 1.30 75.4 f 6.8 14.8 + 1.7 26.6 t 0.64 17.8 i 0.9 31.3 r 65.3 i 2.2 0.46 33.4 f 0.48 81.3 f 7 ; l 32.2 f 17.3 f 0.2 0.42 78.9 i 5.6 35.8 f 17.9 i 0.2 0.42 68.2 i 6.6 15.8 i 2.4 26.3 f 1.02 68.7 2 1.9 16.6 t 0.2 31.1 i 0.83 24.3 t 0.63 29.9 t 25.3 t 0.64 39.1 f 5.2 14.7 i 0.6 0.40 26.7 f 0.50 25.7 i 0.70 0.89 0.56 0.63 0.72 0.64 0.63 0.56 0.74 0.90 0.92 0.68 0.80 0.81 0.83 0.81 0.58 0.88
(3-Paramyosin 68.5 f 4.8 67.3 f 0.5 67.8 t 2.5 74.9 f 3.1 67.9 f 5.7 82.4 t 8.3 73.6 t 5.6 79.0 f 4.5 93.9 t 6.2 89.9 i 10.3 78.9 f 3.1 77.2 5 3.5 87.9 i 2.7 62.3 f 6.7 47.7 t 0.7 -
17.5 i 0.4 17.3 f 1.0 19.5 t 1.6 16.6 t 1.6 19.5 f 1.3 17.2 f 0.1 20.0 f 0.7 19.7 f 1.3 19.9 i 0.6 19.3 t 0.5 17.4 f 1.0 19.6 f 0.4 19.2 i 1.4 18.5 f 0.3 15.3 i 0.4 -
36.5 38.6 36.1 32.9 44.7 48.0 33.6 45.4 48.7 52.3 53.0 34.0 46.6 48.6 41.8 29.9 26.6 22.5
t f
i
t f 2
i i i
i f f
i i i i i f
3.1 0.5 3.1 2.2 1.9 1.5 2.0 1.7 0.2 1.2 3.3 1.4 1.1 1.0 1.2 0.4 0.7 1.5 1.5 4.2 2.0 0.9 1.8 0.5 2.5 1.4 1.6 1.3 0.8 1.0 2.5 1.4 1.1 0.7 0.6
negative birefringence. Figure 4 shows similar data for P-paramyosin; this molecule was studied at higher concentration because of its low dependence of relaxation time on concentration at pH 3.2.12 The differences in the pH dependence of the relaxation times of a-R-paramyosin and P-paramyosin can best be seen in a superposition of the two sets of data, as in Figure 5. It seemed reasonable to suppose that the negatively birefringent species seen in the pH 6-7 region might arise from small paramyosin crystallites,
KRAUSE AND DELANEY
1174
90 - (3-R-faromyosin
I001
90
~
I
-
I
I
I
I
I
I
I
I
I
p - Paromyosin Ternperolure -20°C
80 70-
z-
60-
( p s e c ) 50I
I
I
1
30-
G
I BJ I' ;1 '4A P
&.I; I
10
I
I
I
E L IN.;
r
20-
I
I' I!
I
40 -
I
1
1
1
I
I
'SIX
Z I , I
I
I
I
especially since paracrystals precipitate from such solutions at higher ionic strength.24 However, since monomeric paramyosin has positive birefringence, a negatively birefringent crystallite would probably tend to have the molecules oriented perpendicular to the major axis of the crystallite. Since this seemed improbable, some paracrystals of a-R-paramyosin were prepared a t pH 6, suspended in distilled water, centrifuged to remove large aggregates, and exposed to a 5-msec pulse of 3500 V/cm in the birefringence cell. A large, positively birefringent signal with an pxtremely long field-free
STUDY OF PARAMYOSIN AGGREGATION
90
t
d - R - Porornvos,n :p-Porom od,n = ----
2
i
Comb,ne/Monorner Values = - Temperature - 2 0 ° C
I
10
1175
I
I
I
I
,
I
,
I
I
I
3
4
5
6
7
8
9
10
II
12
PH Fig. 5. Superposition of the best f i t of relaxation times us pH data for wR- and ¶ m yosin.
decay (up to 1sec) was observed. If the negatively birefringent species are crystallite nuclei, therefore, they are very different from the paracrystals that can be precipitated from solution a t slightly higher ionic strength.
DISCUSSION In general, paramyosin appears to exhibit three types of aggregation a t low ionic strength. 1)Both a-R- and P-paramyosin form a gel in the pH 4-6 region. 2) A combination of a slowly relaxing, negatively birefringent aggregate and a positively birefringent species with a fast relaxation time is seen over the pH 6-7.2 range for a-R-paramyosin and in the region of pH 6-6.5 for 0-paramyosin. 3) Smaller, positively birefringent aggregates in equilibrium with monomer are observed in the range of pH 7.2-10 for aR-paramyosin and pH 6.5-10 for the form of the molecule. No birefringence studies were done in the pH 4-6 region where both the a-R and forms of paramyosin form a gel. However, it is of interest to note that this pH region contains the isolectric point, pH 5.8.25
Aggregated Species with Mixed Positive and Negative Birefringence The aggregated state that gives rise to a mixed positive and negative birefringence signal appears over a wider range of pH for a-R-paramyosin than for the p form. However, analysis of the birefringence signals from the different forms of paramyosin indicates that the species present are similar. As mentioned above, the monomeric paramyosin molecule probably has
1176
KRAUSE AND DELANEY
its permanent dipole moment directed close to or directly along its major axis, since it gives rise to a positively birefringent signal. Thus, the presence of a negative birefringence would indicate that the direction of the dipoles responsible for the orientation of the aggregated species is perpendicular to the major axis of the aggregate. The very long relaxation times observed for these aggregates also indicate that they are quite large. Combining the above observations, it appears that the negatively birefringent signals could arise from a large lateral aggregate of paramyosin molecules, i.e., with the major axes of the molecules perpendicular to the major axis of the aggregate. If it is assumed that the aggregate is in the form of an oblate spheroid (i.e., disk shaped) it is possible to estimate its size from the relaxation time and the Perrin equation for the rotational diffusion constant of an oblate spheroid.21 A series of calculations were done using this equation with the rotational diffusion constants calculated from the experimentally determined relaxation times. To calculate unique values for the largest dimension of the oblate spheroid, i.e., the diameter, it was necessary to choose specific values for the thickness. When it was assumed that the thickness of the aggregate corresponded to the estimated monomer, whose length was taken as 1200 A for this purpose, the experimentally determined relaxation times corresponded to diameters from 2000-3000 A. Using the general ellipsoid calculations of Bud0 et a1.,22many ellipsoids whose longest relaxation time is of the order of 1 msec can be calculated, for example, an ellipsoid with one axis approximately monomer length, 1200 A, a second axis 3600 A, and a third axis about 600 A, a rectangular parallelopipedlike aggregate. One must note two things about such speculations: 1) the aggregates in solution are not really ellipsoids, and 2) aggregates of this type would probably exhibit two birefringence relaxation times, although these might be hard to disentangle from the data. A t any rate, the negatively birefringent species may be a laterally aggregated species whose thickness is equal to the length of a monomer molecule, but this speculation needs much further investigation, for example, direct electron microscopic observation of the aggregates. It is interesting to note that the pH region in which the combination of postively and negatively birefringent species appears (pH 6-7) also corresponds to the pH region in which paramyosin precipitates as paracrystals a t higher ionic strength.24 The a-R-paramyosin, for which the presence of the mixed positively and negatively birefringent species persists over the pH range of 6-7.2, produces much larger and thicker paracrystals than P-paramyosin, for which the presence of the mixed species was observed over the more limited range of pH 6-6.5. In spite of the positive birefringence observed for the paracrystals themselves, the negatively birefringent aggregates may nevertheless be representative of their nuclei. The relaxation times of the positively birefringent species which was mixed with the negatively birefringent species ranged from 10 to 45 gsec. This indicates that the observed signals probably originate from monomer or small aggregates of the molecule. Some of these relaxation times were
STUDY OF PARAMYOSIN AGGREGATION
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much lower than those observed for the monomer of any of the forms of paramyosin, but this may be experimental error.
Positively Birefringent Aggregates of Paramyosin In the region of pH 6.5-7.2, where solutions of a-R-paramyosin show the presence of mixed positively birefringent and negatively birefringent species, the 0 form of the molecule apparently exists as small positively birefringent aggregates in equilibrium with monomer. When the pH of the a-R-paramyosin is increased to 7.4, its negative birefringence also disappears and the resulting positive birefringence has a relaxation time of 24.3 psec, probably monomer plus a very small amount of aggregate. A t the same pH, the relaxation times of the 0-paramyosin species indicate small aggregates with relaxation times of 65 f 5 psec in equilibrium with monomer. As the pH is raised further, both a-R- and 0-paramyosin show increases in the relaxation time. At pH values above 8.0, it would appear that the relaxation time us pH curves for a-R- and 0-paramyosin are similar, except that the curve for the former is shifted to a position about one pH unit higher than that of the latter (Fig. 5). Each form of the molecule has a maximum relaxation time, at pH 7.6-8.0 for 0-paramyosin and pH 8.8-9.2 for a-R-paramyosin. A t higher pH values, the relaxation times of both forms of paramyosin fall off rapidly until only monomeric forms are present a t pH 10. Although a-R- and 0-paramyosin show striking differences in the pH dependence of aggregation, it appears that the positively birefringent aggregates formed by these two forms of the molecule are very similar. From Figure 5 and Table I1 it can be seen that the maximum relaxation times of both the a-R- and 0-paramyosin are essentially the same with values of TL = 80 f 5 psec. With few exceptions, other positively birefringent aggregates of both forms have TL values not less than 65 f 5 psec. Using these observations, one may speculate that both forms of the molecule form similar positively birefringent aggregates in a roughly stepwise manner. Several calculations were done to obtain an estimate of the size of the aggregated species from their relaxation times and the monomer lengths determined earlier,12 1220 f 40 A for a-R-paramyosin and 1150 f 20 A for P-paramyosin. The relaxation time of an end to end dimer was calculated using the Broersma”j equation. The value obtained, 130-160 psec, depending on species, appears to preclude the presence of any such end to end aggregates in the solutions of a-R or 0-paramyosin. From this observation it was concluded that the paramyosin monomers must aggregate in the form of overlapped dimers, possibly with some lateral aggregation. Since these aggregates could no longer be considered cylindrical, the Perrin equation for a prolate ellipsoid,21which was considered a better representation of the shape of the aggregates, was used. Plots of relaxation time us length were made from the Perrin equation using several different values for the diameter. The diameters used were in the range of those formed by ag-
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TABLE I11 Calculated Relaxation Times for Prolate Ellipsoids with a 1910 * 80 A Major Axis Diameter (Minor Axis)
(8) 20
35 40 60 70
100
Relaxation Time (wec ) 51 1 62t 64 t 721 74 t 84k
7 8 8 8 9 10
gregates of close packed rods with individual diameters the same as that of the molecule, 20 8. It was found that the length of the laterally aggregated species, from dimer to symmetrically packed lgmer, that would produce a relaxation time of 80 f 5 psec varied over the relatively small range of 1900-2100 A. These values predict that the molecules overlap each other for 200-600 8, of their length. The molecular overlaps calculated above include the value obtained by Cohen et a1.8 from electron micrographs of stained paracrystals of M . mercenaria paramyosin. From the banding patterns in these crystals they determined that the paramyosin molecules overlap each other by 530 A and that no end to end bonding is present. Thus the length of a dimer of aR-paramyosin would be expected to be 1910 f 80 A. Table I11 shows the birefringence relaxation times calculated for prolate ellipsoids21with short axes from 20 to 100 A, if their length is set a t 1910 f 80 A. The overlapped dimer is not cylindrically symmetrical, but its relaxation time should be in the range of those calculated for prolate ellipsoids with diameters from 20 to 40 A. (The overlapped dimer, a t its center of mass, has one 20- and one 40-Asemiaxis). Table I11 shows that the expected relaxation time of the overlapped dimer varies from 57 f 7 to 62 f 8 psec, within the range of the 65 f 5 psec observed in most of the smallest aggregated species. A P-paramyosin dimer would have a length of 1750 f 50 A, with a relaxation time of 51 f 4 psec. The maximum relaxation time of either species then corresponds to a more laterally aggregated species, at least a heptamer, with a thickness of 60-70 A. (A close-packed heptamer would have a diameter, at its center, of 60 A.) Thus, from these observations, one may hypothesize that the aggregation begins with the formation of an overlapped dimer to which molecules add on in a lateral direction so that the thickness of the aggregate increases while its length remains constant. The relaxation times of the smaller species present in equilibrium with the observed aggregates correspond, within the error of the curve analysis technique, to values expected for monomer. The median of the relaxation times observed for the samples of P-paramyosin is somewhat greater than the a-R-paramyosin value, but due to the large experimental error this difference cannot be considered significant. The most striking difference between the relaxation time us pH curves
STUDY OF PARAMYOSIN AGGREGATION
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Fig. 6. Superposition o f t h e best fit of the relaxation time us pH curve for ij-paramyosin on the titration curve of Johnson and Kahn (Ref. 26).
of the two forms of paramyosin (Fig. 5) is the shift of the maximum relaxation time in the a-R-paramyosin curve to a value about one pH unit higher than that observed for the P form. Since the size of the aggregates formed by the two kinds of paramyosin appear to be similar, the basis of this difference probably lies in the number andlor nature of the residues that titrate in the pH 7-10 region. It is thus of interest to compare the relaxation time us pH behavior with the titration curve obtained for the protein. Figure 6 is a plot of the titration curve for P-paramyosin obtained by Johnson and Kahn26superposed upon the relaxation times observed for aggregates of this molecule over the same range of pH. The titration data obtained by Riddiford and Scheraga25were not used in this comparison because their solutions, which were prepared from lyophilized samples of p-paramyosin, showed the presence of aggregation in 0.6M KC1, pH 7. A t these same conditions, fresh samples of 0-paramyosin generally are in monomeric form. The Johnson and Kahn data,26however, all were obtained using samples of freshly prepared P-paramyosin. From the superposed curves, several observations can be made. The region in which P-paramyosin forms a gel, pH 4-6, ends a t the center of a large buffering zone which Johnson and Kahn attributed to the titration of glutamic acid residues. However, most of the aggregation observed by transient electric birefringence in the present experiments appears in a region of the titration curve where there is no obvious titration taking place, at least a t high ionic strength. The region where the mixed positively and negatively birefringent species are seen, pH 6-6.5, does fall a t the end of the buffering zone mentioned above. However, in the region where only positively birefringent aggregates are present, there is no apparent titration
1180
KRAUSE AND DELANEY
taking place. Nevertheless, it is possible that the observed aggregation is related to the terminal -NH2 groups (pKa = 7.5) or the cystine residues (pKa = 8.3) that normally titrate in this pH region. Because of their small number in paramyosin, they might not be detected in a titration, but they might nevertheless be important to the aggregation. The aggregates dissociate into monomer in a region of the titration curve (pH 9-10) where there is a large buffering zone which can be attributed to the titration of the tyrosine (pKa = 9.6) and lysine (pKa = 10.4) residues. Unfortunately, no precise comparison of these data with the birefringence results can be made since the titration curves were obtained a t an ionic strength of 0.3M KC1 while all of the birefringence measurements were made on solutions in 1mM buffer. Since it has been shown that the pKa of the tyrosine groups in paramyosin depend upon the ionic strength,27it is probable that such shifts would take place for other groups as well. A titration curve at low ionic strength for each form of paramyosin will be obtained in the future. The titration curve of the a-R form of paramyosin has not yet been obtained, and, because of the differences in the aggregation behavior, the relaxation time and specific Kerr constant data cannot be compared with the titration curve for P-paramyosin. However, it seems that the pH range in which the dissociation of the aggregates occurs, although shifted to a somewhat higher pH in the case of a-R-paramyosin, still lies in the region of pH where one would expect tyrosine, lysine, and arginine to titrate. The shift in these values may possibly arise from the fact that these residues are involved in inter- or intramolecular interactions that are not present in the P form of the molecule. Some investigators have speculated, using spectrophotometric data, that the tyrosine residues of P-paramyosin are involved in the molecular a g g r e g a t i ~ n . ~Although, ~ . ~ ~ the data presented here would seem to support this thesis, Cowgill’s theory of the tyrosine fluorescence of P-paramyosin28provides evidence that these hydrophobic groups do not enter into any intermolecular interactions. However, the data obtained for the monomer of a-R- and P-paramyosin a t low pH indicate that both forms of the molecule contain a significant number of basic groups which probably remain charged in the aggregation region. Since the aggregates of both the a-R and P forms of paramyosin dissociate to the monomeric form in the region of the pKa of the lysine residues, one might suspect the participation of this group in the aggregation. The shift of the aggregation curve of a-R-paramyosin to higher pH values relative to Pparamyosin may arise from a difference in the number and environment of the lysine residues in the two forms of the molecule. A study of the titration curve of the a-R form of paramyosin over a range of ionic strength will be very helpful in further interpretation of the data obtained from electric birefringence. Our thanks to the donors of the Petroleum Research Fund of the American Chemical Society who supported this work at its start and to the National Institutes of Health for the support
STUDY OF PARAMYOSIN AGGREGATION
1181
of one of us (S.K.) recently by means of a Research Career Award. Thanks also to Dr. J. Garber, who built our original electric birefringence apparatus and who performed the first preliminary experiments on aggregated paramyosin, and to Mr. S. Jacobson, President of Cober Electronics, f i r lending us the pulsers for this work.
References 1. Lowey, J., Millman, B. M. & Hansen, J. (1964) Proc. Roy. Soc. B 160,525-536. 2. Johnson, W. H., Kahn, J. & Szent-Gyorgyi, A. G. (1959) Science 130,160-161. 3. Ruegg, J . C. (1961) Proc. Roy. Soc. R 154,209-223,224-249. 4. Cohen, C. & Holmes, K. C. (1963) J . Mol. Biol. 6,423-432. 5. Elliot, A. (1968) Symposium on Fibrous Proteins-Australia, Plenum, New York. 6. Weisel, J. W. (1974) Biophysical Society Abstracts, 18th Annual Meeting, Minneapolis, p. 76a. 7. Lowey, S., Kucera, J . & Holzer, A. (1963) J . Mol. Biol. 7,234-244. 8. Cohen, C., Szent-Gyorgyi, A. G. & Kendrick-Jones, J . (1971) J . Mol. R i d . 56, 223237. 9. Cowgill, R. W. (1974) Biochemistry 13,2467-2474. 10. Bailey, K. (1957) Pubb. S ~ WZool. . Napoli 29,96-108. 11. Stafford, W. F. & Yphantis, D. A. (1972) Riochem. Biophys. Res. Commun. 49,848854. 12. DeLaney, D. E. & Krause, S. (1976) Macromolecules 9,455-463. 13. Fredericq, E. & Houssier, C. (1973) Electric Dichroism and Electric Birefringence, Clarendon, Oxford. 14. Molecular Electric-Optics, O’Konski, C. T., Ed. (1976), Dekker, New York,Vols. I and 11. 15. O’Konski, C. T. (1968) Encyclopedia Polymer Sci. Technol. 8,551-590. 16. Yoshioka, K. & Watanabe, H. (1969) in PhysicalPrinciples and Techniques ofprotein Chemistry, Part A, Leach, S. J.. Ed., Academic, New York, Part A, pp. 335-367. 17. Stoylov, S. P. (1971) Aduan. Colloid Interface Sci. 3,45-110. 18. Stafford, W. F. (1973) Ph.D. dissertation, University of Connecticut, Storrs, Conn. 19. Benoit, H. (1952) Ann. Phys. (Paris) 6,561-609. 20. Matusmoto, M., Watanabe, H. & Yoshioka, K. (1972) Biopolymers 11,1711-1722. 21. Perrin, F. (1934) J . Phys. Radium 5,497-511. 22. Budo, A,, Fischer, E. & Miyamoto, S. (1939) Phys. 2.40,337-345. 23. Broersma, S. (1960) J . Chem. Phys. 32,1626-1631. 24. Merrick, J. P. & Johnson, W. H. (1977) Biochemistry (to be published). 25. Riddiford, L. M. & Scheraga, H. A. (1962) Biochemistry I, 95-107. 26. Johnson, W. H. & Kahn, J. S. (1959) Science 129,1190-1191. 27. Lowey, S.(1965) J . R i d . Chem. 240,2421-2427. 28. Cowgill, R. W. (1968) Biochim. Biophys. Acta 168,417-430.
Received June 18,1976 Accepted November 2,1976
