Eur. J . Biochem. 67, 23-29 (1976)
Geometry of the Protein S4 from Escherichia coli Ribosomes H. Hasko PARADIES and Alfred F R A N Z Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, Berlin und Fachbereich Biologic, Fachrichtung Biochemie der Pflanzen, Freie Universitat Berlin (Received November 19, 1975/April 24, 1976)
The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low-angle X-ray scattering. The molecular weight of 24000 determined by low-angle X-ray scattering is within 3 % of that found by sedimentation equilibrium analysis and 8 of that determined by amino acid sequence work. The radius of gyration of 3.36 nm. the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape. Calculations of different conformations, e.g. random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod-like structure of S4 with a length of 14 nm and a diameter of 1 nm. This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.
Physiochemical characterizations of ribosomal proteins, especially those which specifically bind to 23-S, 16-S or 5-S RNA, are of importance for studies on protein-RNA interactions and on the topography of the ribosoinal particle. Although the chemical and immunological properties of the isolated proteins [l - 51 have been studied intensively, very little is known about the shape of the individual proteins, e.g. if they are globular or highly asymmetric. Only the shapes of the proteins L7 and L12 have recently been determined [6]. Protein S4, of which the primary sequence has been determined [7], is one of the most interesting proteins in the Escherichiu coli 30-S ribosomal subunits. It binds specifically to the 16-S RNA [8], and details of the interaction between proteins S4 and 16-S RNA have been studied [9- 131. Furthermore, mutants with drastically altered S4 proteins, whose binding to 16-S RNA can be impaired, have been investigated [14211. Information on the shape of protein S4 would help to understand its role in protein-RNA interactions. In the present study attempts were made to determine the shape of protein S4 in solution, e . g . reconstitution buffer [22]. The gross conformation of the S4 molecule in solution was determined by different techniques, of which low-angle X-ray scattering proved to be the most useful. Moreover, several hydrodynamic methods revealed that S4 has a markedly extended shape.
MATERIALS AND METHODS Isolation of Protein S4 Isolation of protein S4 from E. coli ribosomes was performed according to Hindenach et a/. 1231. The freshly lyophilized protein S4 (10 mg by weight, corresponding to 7.4 mg as determined by the method of Lowry et al. [24] was dissolved in 5 ml of the different buffers, A, B, C, and D. Buffer A contained 0.01 M Tris-HC1, pH 7.0, 0.1 M KCl and 0.005 M MgCl2; buffer B contained 0.01 M CH3COONa(K), pH 5.5, 0.1 M NaCl (KCl), 0.005 M MgClz; buffer contained 0.005 M potassium cacodylate, pH 7.5, 0.1 M KC1 and 0.005 M MgC12. BufTer D contained 0.01 M KzHP04, pH 7.5,O.3 M KCI, 0.05 M MgC13. Gel Filtration
A calibrated Sephadex G-100 column (1.5 x 100cm) with a void volume of 70 ml and an elution volume for dinitrophenyl-alanine of 185 ml was applied. The Stokes' radius of protein S4 was determined from a logarithmic plot of Ro versus elution volume [erf-' (l-KD)], after calibration of the column with standard proteins of known Stokes' radius, by determining the distribution coefficient, KD, according to Ackers [25]. Viscosity Determinations Two Ostwald-type viscosimeters with an average shear gradient of 140 s-' were used for the viscosity
24
Ribosomal Protein S4
measurements. No kinetic energy corrections were made because it was believed to be negligible. Temperature fluctuations were & 0.02 "C (Beckman thermometer). Sedimentation Velocity Ultracentrifugation
All runs were performed in a Beckman model E ultracentrifuge equipped with an RCT temperature control and a photoelectric scanner. The sedimentation boundary was followed either with schlieren optics in double-sector cells with quartz windows or with ultraviolet optics at 274 nm for dilute solutions of protein S4. Sedimentations coefficients were obtained from log r vs t diagrams and converted for standard conditions (SZO,~)in the usual manner 1261.
90 nm. The scattered intensities were placed on an absolute scale by comparing them with the scattering from a calibrated lupolene sheet [30]. Slit desmearing was performed usig a modified computerr program from Lake [31] and Taylor and Smith [32]. The calculations were performed on a C D 3600 at the Institut fur Theoretische Physik, Rechenzentrum, Freie Universitat Berlin. Scattering curves were recorded for different concentrations, buffers and temperatures. The apparent radii of gyration were determined by plotting the logarithm of the scattered intensities against (20)' in the 0.9-0.6-Z0 region where one obtains a straight line, and the slope of this line is proportional to the apparent radius of gyration (Rg).The values of the apparent R, were plotted against S4 concentration and extrapolated to infinite dilution.
Sedimentation Equilibrium
A double-sector, aluminum-filled epon centerpiece, 12-mm high, with sapphire windows, was used. The cell thickness was measured with a Nikon comparator. Photographs, using Kodak type 11-G spectroscopic plates, were taken at the start of each experiment to inspect for leaks of the cells. At equilibrium, schlieren pictures were taken in order to locate the bottom and the meniscus position of the cell. Rayleigh interferometric optics [27] were used for protein concentration determinations. An evaluation of molecular weight averages of proteins S4 (fin, and was made according to Yphantis [28] using the measured value for the partial specific volume or protein S4 of 0.725 ml g-'.
a,,, a,)
Other Methods
Values of (hn/6c),, and ( 6 [ / 6 ~(where )~ p refers to the chemical potential of all diffusible components) for protein S4 in buffers A, B, C and D were determined by measurements of the samples that had been exhaustively dialyzed against the buffers. The methods are similar to those described by Eisenberg and Felsenfeld [33]. The measured value of (6n/hcp)p was 0.0403 I/equiv. S4 at 20 "C, that of (6[/6c),, was 0.725; cp denotes the concentration in equivalents of monomer S4 per liter, and c the concentration in grams of S4 per milliliter.
RESULTS Determination of the Diffusion Coefficient
Diffusion experiments were performed in the analytical ultracentrifuge using an AN-G rotor with capillary cells at 22 "C. The diffusion experiments were carried out at 5200 rev./min. The apparent values of the translational diffusion constant (Dapp) at various S4 concentrations were evaluated from the area and height of the boundary of the schlieren peaks by means of standard procedures [29]. Lou>-AngleX-Ray Scattering of Protein S4 in Solution
Low-angle X-ray scattering experiments of protein S4 in the different buffers, A, B, C, and D, were performed in a Kratky camera, equipped with an electronically programmed step-scanning device (MiillerSeiffert) using moonochromatized (quartz monochromator) CuK, radiation. Slit opticcs were used with a 90-pm entrance slit, 270-p-receiving slit and a sample-to-detector distance of 220 mm. These settings resulted in a theoretical resolution of approximately
The best buffer solutions for the study of hydrodynamic parameters for protein S4 were buffers B, D, and a 2 % acetic acid solution, pH 2.5, containing 0.1 M KC1. These solvents have the advantage that protein S4 does not aggregate or precipitate in a wide range of ionic strengths of monovalent or divalent cations. At high protein concentrations (c > 10 mg/ ml) and pH values above 9 protein S4 (pKi = 10.4) tends to form higher aggregates and precipitates. The peculiar solution properties of protein S4 in relation to ionic strength and pH limits the evaluation of buffer constituents considerably. Molecular Weights
Molecular weights of the protein S4 and of the other proteins of the 30-S subunit have been obtained by Craven et al. [34] and Dzionara et al. [35]. We determined the molecular weight of protein S4 under non-denaturing conditions using different buffers, including the reconstitution buffer according to Nomura [22], and obtained a value of 24500 & 1500.
H. H. Parddies and A . Franz
Furthermore, no self-association of S4 under these conditions were detected. Shape
of Protein S4 from Hydrodynamic Data
The shape of protein S4 in solution is estimated from the ratio of frictional coefficients, f’, from the Simha, v, and from the Mandelkern-Scheraga parameter, [36]. A first estimate o f 8 6 is obtained from the elution behavior of protein S4 on a Sephadex G-100 column. The elution volume of protein S4 deviated appreciably from the value expected for a globular protein of a molecular weight of 24500 (see Table 1). The Stokes’ radius of protein S4, obtained from a plot of log ROversus elution volume from a calibrated Sephadex G-100 column, was R = 3.17 nm; this is defined as the radius of a sphere that is hydrodynamically equivalent to the true hydrated molecule. If one takes the frictional coefficient of S4 and the value expected for a sphere with the volume of S4 the Stokes’ radius of this sphere would be
with R,= average molecular weight, N = AvogadrG’s number, and 1’ = the partial specific volume. 6, represents the extent of solvation of the molecule S4 taken as zero for calculating the minimum possible frictional coefficient [37]. Fo refers to water with the assumption that the buffer or water has no particularly strong affinity to the monomeric molecule. From the data obtained we get a minimum value of the frictional ratio, R/Ro = f / h = 1.69, for S4. Sedimentation Velocity Independent values offfo were obtained from the sedimentation and diffusion constants at different [H’ ] concentrations, ionic strengths and various S4 concentrations. For a sphere with a Stokes’ radius of 1.98 nm the sedimentation coefficient, so, can be evaluated :
R(l
-
a@)
= 3.36. = 3.36 S 6 n N . ylo .% where e is the density and yo the viscosity of the buffer at 20 “C. The sedimentation coefficient, corrected for standard conditions, is ~ 2 0 = , ~1.98 S for protein S4 between pH 2.5-7.5. It follows that the minimum frictional ratio
so =
25 Table 1. Physical properties of protein S4,from E. coli riho.rome.7 Parameter
Unit
SZ0.W
DSo,, [? 1
I’
61
JlJi Molecular weight: M ,from sedimentation equilibrium M ,from low-angle X-ray scattering M,from sedimentation equilibrium M , from so and Do M ,from so and [ q ] Stokes’ radius
P
Value
1.95 0.06 (3.70 0.25) x 1012.5 f 2.7 0.725 0.35 1.69
+
25000
’
500
24800 f 200 25000 25 000 25 000
+ 500
3.17 & 0.05 2.46~ loCb
protein concentrations from 5.5 to 12.0 mg/ml. N o aggregation of S4 at ionic strengths 0.08 to 0.2 M was observed. Furthermore, working at different pH values in acetic acid (2 ‘%;),acetate buffer (pH 5.0- 6.5) and phosphate buffer (pH 7.0-7.8) we obtained a single peak with a sedimentation coefficient of S Z O , ~ = 1.95 S. At an ionic strength of 0.12 M, e.g. in 2 % acetic acid and at pH 5.5 in 0.01 M sodium acetate buffer containing 0.1 M KC1, a diffusion constant of D 0Z O ,=~ 3.5 x lo7 cm2 . s-’ was obtained (Table 1). From these diffusion data a frictional ratio, Do/D = j h / f = 1.90, for protein S4 was obtained. Viscosities studies were performed at ionic strengths of 0.08 to 0.15 M. The intrinsic viscosity of asymmetric particles depends very much on their axial ratios (a/h) which vary approximately with (a/b)‘ [38] at fairly dilute solutions. Measurements at different temperatures yielded the same value for the intrinsic viscosity, namely, 12.5 & 2.7 ml/g, and a Simha parameter of v = 17.3, which is considerably larger than that of suspended spheres. According to the assumptions of Scheraga and Mandelkern [36] we obtained a p value of 2.46 . lo6 for protein S4 (see Table 1) that is hydrodynamically equivalent to an ellipsoid of revolution which does not necessarily have the same volume as the real molecule. Only an extended ellipsoid of revolution is consistent with the value obtained. However, a value of 2.15 x lo6 does not exceed the same value for oblate ellipsoids of any ,ixial ratio [38]. Low-Angle X-Ray Scattering
Sedimentation velocity runs at different ionic strengths, ranging from 0.08 to 0.15 M, were performed at S4
Scattering curves of solutions of protein S4 were recorded, using different buffer systems and the reconstitution buffer according to Nomura [22].
Ribosomal Protein S4
26
2.50 0
0.5
1.0
1.5
2.0
2.5
Special care was taken to ineasure all materials in the same cell and under identical technical conditions. For filled particles, having an elongated shape, the radius of gyration of the cross section, R,, can be determined from the slope of the linear portion of a plot of ln(hI/c) versus h2, since ln(hZ/c) = ln(hZo/c) (A2 R:/2). The radius of gyration of the whole scattering particle for sufficiently low scattering angles was obtained from the slope of a plot of ln(l/c) = In(lo/c) - (h2R2/3)with h = 4 njl. sin 0, the magnitude of the reciprocal lattice vector, and A = 0.154 nm (CuK, radiation) [39]. Data obtained at the lowest angles for four concentrations of protein S4 are shown in a Guinier plot in Fig. 1. We were concerned that the protein S4 might bear sufficient charge at pH 5.5, as well as at pH 7.5, so that interparticle interference effects would be troublesome. Since the inner portion of Fig.1 shows no downward curvature we conclude that the ionic strength of the buffers are sufficiently high and that no aggregation of protein S4 has occurred to suppress interparticle interference effects. A similar plot of log (Izljc) versus lz2 is shown in Fig.2, whereas in Fig. 3 the concentration dependence of the radius of gyration of protein S4 is shown. There is also no severe concentration dependence of protein S4 on the radius of gyration of the cross section, R,, up to 15 mg/ml and ionic strengths 0.1 -0.2 M. Extrapolation to infinite dilution yields the molecular weight M,, which is directly proportional to ( I / C ) , = O ,with M , = 24800, R, = 3.36 nm and R, = 0.41 nm (Fig. 1 and 2). Additional parameters could be determined
3.0
lo4 h2 (ntf2) Fig. 1. Guinier plot of protciiz S4 ut l o l i w t un,qles f i v f o u r thffcrcwt concentrations, c of'5.0, 7.0, 10.0, untl 15.0 n7g nil. '. The same graphs were obtained for all buffers
0.89 0.88 0.87 0.86
-
2 0.85 c
c
15.0 0.0 7.0 5 .0
m
0.84 0.83 0.82 0.80
iI
-
through the use of the invariant term Q
= Sfhdh,
as
0
described by Porod [40,41]. Since insensity measurements must be terminated when the excess intensity becomes too small to measure with precision the integral c'dn be evaluated if the outer part of the scattering curve exhibits a Porod region in which f varies with K 3 .If there is some degree of internal order in the scattering particle the scattered intensity is written
Fig. 2. Plot of determinutions qf the radius oj'gyrution of' the cross srction ut j&tr protein S4 concentrutions, c of 15.0, 10.0, 1.0,tin& 5.0 mg . mi-'.The same graphs were obtained for all buffers
1
4.0
u I _ 2 4 6 8 1
I
~
1
1 0
OO
c (mgirnl)
Fig. 3 Contrntration dependentr of the iudius of gjration of protein S4
in buffers
A , B, C uizd
D
_
_
H. H Paradies and A . Franz
21
Table 2. Molecular puramcters of'protein S4 derived fkom Iow-anglr X-ru.v scattering Parameter
Unit
Value
Radius of gyration, R,
3.36
Radius of gyration of the cross section, R ,
0.41
Molecular weight
24800
Volume
121
Surface
565
Degree of hydration
0.2s
Area of cross section
2.30
ulh from (RL
15.0
14.8 Assuming prolate ellipsoid of revolution. Assuming prolate ellipsoid of revolution with 4 R, = b I/ (2 + a2):5 and V = nh3 . n. 3 a
~
1c .c
1 .@
0.1 P (nn";
Fig. 4. E.xpcrimcntaI (-) cud theor-uticai .scuticsing r u r w s ( ~ f prowin S4 ,/kr a radius of gyration of R, = 3.36 nrii. ellip.witl,\ of' rcidution w i t h lengths of2a = 17 und 18 nm i --),and r(1d.c with heighrs of H = 14.5 and 15.5 n m und ellipticul ends with half'uxes of 0.55 and 0.30 nm (-.-. ) ~
f = k l K 3 + k2. In this case h3fis plotted
against h3, and kl is evaluated by extrapolating the linear region found at high angles back to h3 = 0. The area of the outer tail of the hfcurve is then evaluated analytically, taking f = k l K 3 [42]. The value obtained for this invariant term for protein S4 is 0 = 0.786. The volume per S4 molecule, V , calculated from the relation V = 4n210/0is 121 nm3. The average area of the cross-sectional surface, So, is given by So = 4n(hZ)0/2, and found to be 2.30 nm2. The surface per unit volume, ,F, according t o S = 4tllll/Q with fl1 = the volume fraction of solvent, was found to be 0.0475 nm-', so that the total surface area per molecule S4, SV, is 565 nm2 [42]. The molecular parameters derived from low-angle X-ray scattering of protein S 4 in solution are listed in Table 2.
DISCUSSION The sedimentation, diffusion and viscosity studies of protein S 4 presented here provide evidence for an extended, rather than a globular particle. From Table 1 it is seen that the frictional coefficients, fife, obtained with different methods, agree quite well with each other and are consistent with the values obtained by Rhode et al. [43]. Calculation of the ratio of long t o short axis (a/h)of an equivalent extended ellipsoid of revolution, assuming no hydration (61 = 0), leads to an average value of the axial ratio of 14. However, the knowledge of the diffusion constant, Do, the anhydrous molecular weight from sequence work and the partial specific
~
volume permits one to draw certain conclusions about the degree ofhydration of S4 according to R . T/N . Do = (6nqflf;) [3 M,(G + S1VIo)/4 n N ] l i 3with d1 = the hydration of the particle. Since,flji is also known, the maximum hydration obtained in this manner is 0.35 g HzO/g of S4, which is comparable with the value of 0.25 g HzO/g of S4 obtained from low-angle X-ray scattering. The scattering curve of protein S4 is compared with scattering curves for different models, r . g . for prolate ellipsoids with different axial ratios and for rods of different lengths, with R , = = 3.36 nm where a = long half axis and h = short half axis, but different length I = 2 a (Fig.4). The best correspondence with the experimental curve is obtained for an ellipsoid of three half axes, N = 6.25 nm, h = 0.525 nm and c = 0.25 nm, or for a cylindrical rod of height H = 14.5 nm and elliptical ends with half axes of 0.55 and 0.30 nm. There is no reason for a clear preference of one of the hydrodynamic scattering analogues over the simple prolate ellipsoid of revolution. Moreover, we examined the possibility that the S 4 protein molecule exists as an open coil in solution using the model of the worm-like chain with finite chain length. The Debye scattering function for a random coil shows for h values near the Guinier region a Z z h-2 dependence. This implies that a polypeptide chain with finite persistent length will undergo a transition with increasing
v
m
28
scattering angle from coil behavior ( I = K 2 )to rodlike behavior ( I = K ' ) . Inspections of plots of h2Z versus I7 for solutions of protein S4 show no horizontal plateau regions corresponding to the I z K 2 region; only linearly increasing functions with Z FZ h - l , evidently only rod-like domains, are to be seen, Moreover, applying Luzzati's theory [44] for the interpretation of curves of low-angle X-ray scattering for rod-like particles in conditions of collimating approximation of an infinitely high slit-point receiver, the radius of gyration of the cross section, 0.41 nni, was directly obtained. This implies that, if sufficiently prolonged regions of S4 threads can be approximated by a cylinder, R , would characterize the cross dimensions of the S4 thread. This agrees well with a rod diameter of 1.15 nm, corresponding to a length of 15.0 nm, and supports the conclusions concerning the shape of S4, also. Almost the same results are obtained from electron microscopic studies of antibody-labeled subunits by Lake et al. [45] and Tischendorf et al. [46]. Hydrodynamic studies of S4 and low-angle X-ray scattering show that S4 does not behave as a typical spherical protein, since frictional ratio, intrinsic viscosity and radius of gyration are much higher than for globular proteins. Calculations of the radii of gyration for different conformations show that only a rod-like structure rather than a flexible coil for S4 is possible. Moreover, the intrinsic viscosity of 12.5 mg/ ml is somewhat smaller than the value to be expected for a solvent-immobilized Gaussian coil of 210 amino acid residues. This value would be 23.5 ml/g and would correspond to a radius of gyration of 4.62 nm. Neither value is comparable with the experimentally determined ones. Furthermore, circular dichroism studies of S4 in reconstitution buffer, and in the buffers used in this study, show a certain degree of order, displaying both cc-helical (35 %) and P-pleated sheet structures (30 %) (Paradies, unpublished results). It is rather unlikely that only hydration of such an ordered molecule accounts for the high frictional ratio and intrinsic viscosity; otherwise this would require solvations of the order of 10 g H2 0 /g protein. We are grateful to Dr H. G. Wittmann for critical discussions in the course of this work and to Miss Else Nielsen for help in the preparation and typing of the English text.
REFERENCES 1. Wittmann, H. G. & Stoffler, G. (1972) Protein Biosynthesis (Bosch, L., ed.) vol. 27, p. 285, North-Holland, Amsterdam. 2. Kurland, C. G. (1972) Annu. Rev. Biochem. 41, 377-408. 3. Garrett, R. A. & Wittmann, H. G. (1973) Protein Biosynthesis in Reproductive Tissue (Diczfalusy, ed.), Karolinska Symposium om Research Methods in Reproductive Endocrinology, 6th Symposium, p. 75.
Ribosomal Protein S4 4. Wittmann, H. G. (1974) The Ribosomes, pp. 93- 114, Cold Spring Harbor Laboratory. 5. Wittmann, H . G . & Wittmann-Liebold, B. (1974) The Ribosomes, pp. 115- 140, Cold Spring Harbor Laboratory. 6. Wong, K . P. & Paradies, H. H. (1974) Biochem. Biophys. Res. Commun. 61, 178-184. 7. Reinbolt, J. & Schiltz, F. (1973) FEBS Lett. 36, 250-252. 8. Traub, P., Mizushima, S., Lowry, C. U. & Nomura, M. (1971) Method. Enzymol. 20, 391 - 407. 9. Schaup, H. W., Green, W. & Kurland, C. G. (1971) Mol. Gcw. Genet. 112, 1 - 8 . 10. Schaup, H. W., Sogin, M., Woese, C. & Kurland. C. G . (1971) Mol. Gcn. Genet. 114, 1-8. 11. Nanninga, N., Garrett, R. A,, Stomer, G. & Klotz, G. (1972) Mol. G m . Genet. 119, 175-184. 12. Zimmermann, R . A., Muto, A., Fellner, P., Ehresmann, C. & Bralant, C. (2972) Proc. Nutl Acad. Sci. U . S . A . 69, 12821286. 13. Daya-Grosjean, L., Geisser, M., Stoffler, G. & Garrett, R. A. (1973) FEBSLett. 37, 17-20. 14. Donner, D. & Kurland, C. G. (1972) Mol. Gen. Genet. 115, 49- 53. 15. Deusser, E., Stoffler, G., Wittmann, H. G . &Apirion, D.(1970) Mol. Gen. Genet. 109, 298 302. 16. Kreider, J . & Brownstein, B. L. (1971) J . Mol. Bid. 61, 135145. 17. Zimmermann, R. A., Garvin, R. T. & Gorini, L. (1971) Proc. Nutl Acad. Sci. U.S.A. 68, 2263-2261. 18. Funatzu, G., Puls, W., Schiltz, E., Reinboldt, J. & Wittmann, H. G . (1972) Mol. Gen. Genet. 115, 131-139. 19. Schaup, H. W. & Kurland, C. G. (1972) Mol. Gen. Genet. 114, 350- 357. 20. Hasenbank, R., Guthrie, C., Stoffler, G., Wittmann, H. G., Rosen, L. & Apirion, D. (1973) Mol. Gen. Gene?. 127, 1 - 18. 21. Olsson, M., Isaksson, L. & Kurland, C. G. (1974) Mol. Gen. Genet. 135, 191 -202. 22. Mizushima, S. & Nomura, M. (1970) Nature (Lond.) 226, 1214- 1218. 23. Hindenach, I., Stoffler, G . & Wittmann, H. G. (1971) Eur. J . Biochem. 23, 7 - 11. 24. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 25. Ackers, G. (1964) Biochemistry 3, 723-730. 26. Schachman, H. K. (1959) Ultracentrifugation in Biochemistry, pp. 82-83, Academic Press, New York. 27. Richards, E. G. & Schachman, H. K . (1959) J . Phys. Chem. 63, 1578-1591. 28. Yphantis, D. A. (1964) Biochemistry, 3, 297-317. 29. Chervenka, C. H. (1970) A Manual of Methods for the Analytical Ulti.acentr~fuge,p. 50, Beckinan Instruments, Inc., Palo Alto, Calif., U.S.A. 30. Kratky, O., Pilz, I. & Schmitz, P. J. (1966) J . Colloid Interface Sci. 21, 24-34. 31. Lake, J. A. (1967) Acta Crystallogr. 23, 191-194. 32. Taylor, T. R. & Schmidt, P. W. (1969) J . Appl. Crystallogr. 2, 143- 144. 33. Eisenberg, H. & Felsenfeld, G. (1967) J . Mol. Biol. 30, 17-37. 34. Craven, G. R., Voynow, P., Hardy, S. J. S. & Kurland, C. G. (1969) Biochemistry, 8,2906-2915. 35. Dzionara, M., Kaltschmidt, E. & Wittmann, H. G. (1970) Proc. Nut1 Acad. Sci. U . S . A . 67, 1276- 1282. 36. Mandelkern, L. & Scheraga, H. A. (1953) 1. An?. Chrm. Soc. 75, 179 - 184. 37. Tanford, Ch. (1961) Plzvsicul Chemistry of Macromoleculczs, pp. 357-397, Wiley, New York. 38. Yang, J. T. (1961) Adv. Protein Chem. 16. 323-400. 39. Guinier. A . & Fournet, G. (1955) SnwIl-A,~,y/c,SCa / / c r / / i y ( I / ' X-Rt/j,.\. Wiley. Ncw, l ' o r k . 40. Porod, G. (1951) KolloidZ. 124, 83- 114. -
H. H. Paradies and A. Frdnz 41. Porod, G. (1951) KolloidZ. 125, 51-57. 42. Hosemann, R., Schonfeld, A. & Wilke, W. (1970) in Ahwices in Structure Research by D$Truction Meihods (Brill, R . & Mason, F., eds)vol. 3, pp. 101 - 172, PergamonPress, Oxford 43. Rohde, M. F., O’Brien, S., Cooper, S. & Aune, K . C. (1975) Biochemistry, 14, 1079- 1087.
29 44. Luzzati, V. (1960) Acia Crystallogr. 13, 939-942. 45. Lake, J. A , , Pendergast, M., Kahan, L. & Nomura, M . (1974) Proc. Nail Acad. Sci. U.S.A. 71, 4688-4692. 46. Tischendorf, J. W., Zeichardt, H. & Stoffler, G. (1974) Mol. G m . Genet. 134, 187-208.
H. H. Paradies, Fachrichtung Biochemie der Pflanzen, Institul fur Pflanzenphysiologie und Zellbiologie, Fachbereich Biologie der Freien Universitat Berlin, Konigin-Luke-StraBe 12/16 a, D-1000 Berlin (West) 33-Dahlem A. Franz, Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, IhnestraBe 63/73, D-1000 Berlin (West) 33-Dahlem
Geometry of the Protein S4 from Escherichia coli Ribosomes H. Hasko PARADIES and Alfred F R A N Z Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, Berlin und Fachbereich Biologic, Fachrichtung Biochemie der Pflanzen, Freie Universitat Berlin (Received November 19, 1975/April 24, 1976)
The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low-angle X-ray scattering. The molecular weight of 24000 determined by low-angle X-ray scattering is within 3 % of that found by sedimentation equilibrium analysis and 8 of that determined by amino acid sequence work. The radius of gyration of 3.36 nm. the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape. Calculations of different conformations, e.g. random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod-like structure of S4 with a length of 14 nm and a diameter of 1 nm. This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.
Physiochemical characterizations of ribosomal proteins, especially those which specifically bind to 23-S, 16-S or 5-S RNA, are of importance for studies on protein-RNA interactions and on the topography of the ribosoinal particle. Although the chemical and immunological properties of the isolated proteins [l - 51 have been studied intensively, very little is known about the shape of the individual proteins, e.g. if they are globular or highly asymmetric. Only the shapes of the proteins L7 and L12 have recently been determined [6]. Protein S4, of which the primary sequence has been determined [7], is one of the most interesting proteins in the Escherichiu coli 30-S ribosomal subunits. It binds specifically to the 16-S RNA [8], and details of the interaction between proteins S4 and 16-S RNA have been studied [9- 131. Furthermore, mutants with drastically altered S4 proteins, whose binding to 16-S RNA can be impaired, have been investigated [14211. Information on the shape of protein S4 would help to understand its role in protein-RNA interactions. In the present study attempts were made to determine the shape of protein S4 in solution, e . g . reconstitution buffer [22]. The gross conformation of the S4 molecule in solution was determined by different techniques, of which low-angle X-ray scattering proved to be the most useful. Moreover, several hydrodynamic methods revealed that S4 has a markedly extended shape.
MATERIALS AND METHODS Isolation of Protein S4 Isolation of protein S4 from E. coli ribosomes was performed according to Hindenach et a/. 1231. The freshly lyophilized protein S4 (10 mg by weight, corresponding to 7.4 mg as determined by the method of Lowry et al. [24] was dissolved in 5 ml of the different buffers, A, B, C, and D. Buffer A contained 0.01 M Tris-HC1, pH 7.0, 0.1 M KCl and 0.005 M MgCl2; buffer B contained 0.01 M CH3COONa(K), pH 5.5, 0.1 M NaCl (KCl), 0.005 M MgClz; buffer contained 0.005 M potassium cacodylate, pH 7.5, 0.1 M KC1 and 0.005 M MgC12. BufTer D contained 0.01 M KzHP04, pH 7.5,O.3 M KCI, 0.05 M MgC13. Gel Filtration
A calibrated Sephadex G-100 column (1.5 x 100cm) with a void volume of 70 ml and an elution volume for dinitrophenyl-alanine of 185 ml was applied. The Stokes' radius of protein S4 was determined from a logarithmic plot of Ro versus elution volume [erf-' (l-KD)], after calibration of the column with standard proteins of known Stokes' radius, by determining the distribution coefficient, KD, according to Ackers [25]. Viscosity Determinations Two Ostwald-type viscosimeters with an average shear gradient of 140 s-' were used for the viscosity
24
Ribosomal Protein S4
measurements. No kinetic energy corrections were made because it was believed to be negligible. Temperature fluctuations were & 0.02 "C (Beckman thermometer). Sedimentation Velocity Ultracentrifugation
All runs were performed in a Beckman model E ultracentrifuge equipped with an RCT temperature control and a photoelectric scanner. The sedimentation boundary was followed either with schlieren optics in double-sector cells with quartz windows or with ultraviolet optics at 274 nm for dilute solutions of protein S4. Sedimentations coefficients were obtained from log r vs t diagrams and converted for standard conditions (SZO,~)in the usual manner 1261.
90 nm. The scattered intensities were placed on an absolute scale by comparing them with the scattering from a calibrated lupolene sheet [30]. Slit desmearing was performed usig a modified computerr program from Lake [31] and Taylor and Smith [32]. The calculations were performed on a C D 3600 at the Institut fur Theoretische Physik, Rechenzentrum, Freie Universitat Berlin. Scattering curves were recorded for different concentrations, buffers and temperatures. The apparent radii of gyration were determined by plotting the logarithm of the scattered intensities against (20)' in the 0.9-0.6-Z0 region where one obtains a straight line, and the slope of this line is proportional to the apparent radius of gyration (Rg).The values of the apparent R, were plotted against S4 concentration and extrapolated to infinite dilution.
Sedimentation Equilibrium
A double-sector, aluminum-filled epon centerpiece, 12-mm high, with sapphire windows, was used. The cell thickness was measured with a Nikon comparator. Photographs, using Kodak type 11-G spectroscopic plates, were taken at the start of each experiment to inspect for leaks of the cells. At equilibrium, schlieren pictures were taken in order to locate the bottom and the meniscus position of the cell. Rayleigh interferometric optics [27] were used for protein concentration determinations. An evaluation of molecular weight averages of proteins S4 (fin, and was made according to Yphantis [28] using the measured value for the partial specific volume or protein S4 of 0.725 ml g-'.
a,,, a,)
Other Methods
Values of (hn/6c),, and ( 6 [ / 6 ~(where )~ p refers to the chemical potential of all diffusible components) for protein S4 in buffers A, B, C and D were determined by measurements of the samples that had been exhaustively dialyzed against the buffers. The methods are similar to those described by Eisenberg and Felsenfeld [33]. The measured value of (6n/hcp)p was 0.0403 I/equiv. S4 at 20 "C, that of (6[/6c),, was 0.725; cp denotes the concentration in equivalents of monomer S4 per liter, and c the concentration in grams of S4 per milliliter.
RESULTS Determination of the Diffusion Coefficient
Diffusion experiments were performed in the analytical ultracentrifuge using an AN-G rotor with capillary cells at 22 "C. The diffusion experiments were carried out at 5200 rev./min. The apparent values of the translational diffusion constant (Dapp) at various S4 concentrations were evaluated from the area and height of the boundary of the schlieren peaks by means of standard procedures [29]. Lou>-AngleX-Ray Scattering of Protein S4 in Solution
Low-angle X-ray scattering experiments of protein S4 in the different buffers, A, B, C, and D, were performed in a Kratky camera, equipped with an electronically programmed step-scanning device (MiillerSeiffert) using moonochromatized (quartz monochromator) CuK, radiation. Slit opticcs were used with a 90-pm entrance slit, 270-p-receiving slit and a sample-to-detector distance of 220 mm. These settings resulted in a theoretical resolution of approximately
The best buffer solutions for the study of hydrodynamic parameters for protein S4 were buffers B, D, and a 2 % acetic acid solution, pH 2.5, containing 0.1 M KC1. These solvents have the advantage that protein S4 does not aggregate or precipitate in a wide range of ionic strengths of monovalent or divalent cations. At high protein concentrations (c > 10 mg/ ml) and pH values above 9 protein S4 (pKi = 10.4) tends to form higher aggregates and precipitates. The peculiar solution properties of protein S4 in relation to ionic strength and pH limits the evaluation of buffer constituents considerably. Molecular Weights
Molecular weights of the protein S4 and of the other proteins of the 30-S subunit have been obtained by Craven et al. [34] and Dzionara et al. [35]. We determined the molecular weight of protein S4 under non-denaturing conditions using different buffers, including the reconstitution buffer according to Nomura [22], and obtained a value of 24500 & 1500.
H. H. Parddies and A . Franz
Furthermore, no self-association of S4 under these conditions were detected. Shape
of Protein S4 from Hydrodynamic Data
The shape of protein S4 in solution is estimated from the ratio of frictional coefficients, f’, from the Simha, v, and from the Mandelkern-Scheraga parameter, [36]. A first estimate o f 8 6 is obtained from the elution behavior of protein S4 on a Sephadex G-100 column. The elution volume of protein S4 deviated appreciably from the value expected for a globular protein of a molecular weight of 24500 (see Table 1). The Stokes’ radius of protein S4, obtained from a plot of log ROversus elution volume from a calibrated Sephadex G-100 column, was R = 3.17 nm; this is defined as the radius of a sphere that is hydrodynamically equivalent to the true hydrated molecule. If one takes the frictional coefficient of S4 and the value expected for a sphere with the volume of S4 the Stokes’ radius of this sphere would be
with R,= average molecular weight, N = AvogadrG’s number, and 1’ = the partial specific volume. 6, represents the extent of solvation of the molecule S4 taken as zero for calculating the minimum possible frictional coefficient [37]. Fo refers to water with the assumption that the buffer or water has no particularly strong affinity to the monomeric molecule. From the data obtained we get a minimum value of the frictional ratio, R/Ro = f / h = 1.69, for S4. Sedimentation Velocity Independent values offfo were obtained from the sedimentation and diffusion constants at different [H’ ] concentrations, ionic strengths and various S4 concentrations. For a sphere with a Stokes’ radius of 1.98 nm the sedimentation coefficient, so, can be evaluated :
R(l
-
a@)
= 3.36. = 3.36 S 6 n N . ylo .% where e is the density and yo the viscosity of the buffer at 20 “C. The sedimentation coefficient, corrected for standard conditions, is ~ 2 0 = , ~1.98 S for protein S4 between pH 2.5-7.5. It follows that the minimum frictional ratio
so =
25 Table 1. Physical properties of protein S4,from E. coli riho.rome.7 Parameter
Unit
SZ0.W
DSo,, [? 1
I’
61
JlJi Molecular weight: M ,from sedimentation equilibrium M ,from low-angle X-ray scattering M,from sedimentation equilibrium M , from so and Do M ,from so and [ q ] Stokes’ radius
P
Value
1.95 0.06 (3.70 0.25) x 1012.5 f 2.7 0.725 0.35 1.69
+
25000
’
500
24800 f 200 25000 25 000 25 000
+ 500
3.17 & 0.05 2.46~ loCb
protein concentrations from 5.5 to 12.0 mg/ml. N o aggregation of S4 at ionic strengths 0.08 to 0.2 M was observed. Furthermore, working at different pH values in acetic acid (2 ‘%;),acetate buffer (pH 5.0- 6.5) and phosphate buffer (pH 7.0-7.8) we obtained a single peak with a sedimentation coefficient of S Z O , ~ = 1.95 S. At an ionic strength of 0.12 M, e.g. in 2 % acetic acid and at pH 5.5 in 0.01 M sodium acetate buffer containing 0.1 M KC1, a diffusion constant of D 0Z O ,=~ 3.5 x lo7 cm2 . s-’ was obtained (Table 1). From these diffusion data a frictional ratio, Do/D = j h / f = 1.90, for protein S4 was obtained. Viscosities studies were performed at ionic strengths of 0.08 to 0.15 M. The intrinsic viscosity of asymmetric particles depends very much on their axial ratios (a/h) which vary approximately with (a/b)‘ [38] at fairly dilute solutions. Measurements at different temperatures yielded the same value for the intrinsic viscosity, namely, 12.5 & 2.7 ml/g, and a Simha parameter of v = 17.3, which is considerably larger than that of suspended spheres. According to the assumptions of Scheraga and Mandelkern [36] we obtained a p value of 2.46 . lo6 for protein S4 (see Table 1) that is hydrodynamically equivalent to an ellipsoid of revolution which does not necessarily have the same volume as the real molecule. Only an extended ellipsoid of revolution is consistent with the value obtained. However, a value of 2.15 x lo6 does not exceed the same value for oblate ellipsoids of any ,ixial ratio [38]. Low-Angle X-Ray Scattering
Sedimentation velocity runs at different ionic strengths, ranging from 0.08 to 0.15 M, were performed at S4
Scattering curves of solutions of protein S4 were recorded, using different buffer systems and the reconstitution buffer according to Nomura [22].
Ribosomal Protein S4
26
2.50 0
0.5
1.0
1.5
2.0
2.5
Special care was taken to ineasure all materials in the same cell and under identical technical conditions. For filled particles, having an elongated shape, the radius of gyration of the cross section, R,, can be determined from the slope of the linear portion of a plot of ln(hI/c) versus h2, since ln(hZ/c) = ln(hZo/c) (A2 R:/2). The radius of gyration of the whole scattering particle for sufficiently low scattering angles was obtained from the slope of a plot of ln(l/c) = In(lo/c) - (h2R2/3)with h = 4 njl. sin 0, the magnitude of the reciprocal lattice vector, and A = 0.154 nm (CuK, radiation) [39]. Data obtained at the lowest angles for four concentrations of protein S4 are shown in a Guinier plot in Fig. 1. We were concerned that the protein S4 might bear sufficient charge at pH 5.5, as well as at pH 7.5, so that interparticle interference effects would be troublesome. Since the inner portion of Fig.1 shows no downward curvature we conclude that the ionic strength of the buffers are sufficiently high and that no aggregation of protein S4 has occurred to suppress interparticle interference effects. A similar plot of log (Izljc) versus lz2 is shown in Fig.2, whereas in Fig. 3 the concentration dependence of the radius of gyration of protein S4 is shown. There is also no severe concentration dependence of protein S4 on the radius of gyration of the cross section, R,, up to 15 mg/ml and ionic strengths 0.1 -0.2 M. Extrapolation to infinite dilution yields the molecular weight M,, which is directly proportional to ( I / C ) , = O ,with M , = 24800, R, = 3.36 nm and R, = 0.41 nm (Fig. 1 and 2). Additional parameters could be determined
3.0
lo4 h2 (ntf2) Fig. 1. Guinier plot of protciiz S4 ut l o l i w t un,qles f i v f o u r thffcrcwt concentrations, c of'5.0, 7.0, 10.0, untl 15.0 n7g nil. '. The same graphs were obtained for all buffers
0.89 0.88 0.87 0.86
-
2 0.85 c
c
15.0 0.0 7.0 5 .0
m
0.84 0.83 0.82 0.80
iI
-
through the use of the invariant term Q
= Sfhdh,
as
0
described by Porod [40,41]. Since insensity measurements must be terminated when the excess intensity becomes too small to measure with precision the integral c'dn be evaluated if the outer part of the scattering curve exhibits a Porod region in which f varies with K 3 .If there is some degree of internal order in the scattering particle the scattered intensity is written
Fig. 2. Plot of determinutions qf the radius oj'gyrution of' the cross srction ut j&tr protein S4 concentrutions, c of 15.0, 10.0, 1.0,tin& 5.0 mg . mi-'.The same graphs were obtained for all buffers
1
4.0
u I _ 2 4 6 8 1
I
~
1
1 0
OO
c (mgirnl)
Fig. 3 Contrntration dependentr of the iudius of gjration of protein S4
in buffers
A , B, C uizd
D
_
_
H. H Paradies and A . Franz
21
Table 2. Molecular puramcters of'protein S4 derived fkom Iow-anglr X-ru.v scattering Parameter
Unit
Value
Radius of gyration, R,
3.36
Radius of gyration of the cross section, R ,
0.41
Molecular weight
24800
Volume
121
Surface
565
Degree of hydration
0.2s
Area of cross section
2.30
ulh from (RL
15.0
14.8 Assuming prolate ellipsoid of revolution. Assuming prolate ellipsoid of revolution with 4 R, = b I/ (2 + a2):5 and V = nh3 . n. 3 a
~
1c .c
1 .@
0.1 P (nn";
Fig. 4. E.xpcrimcntaI (-) cud theor-uticai .scuticsing r u r w s ( ~ f prowin S4 ,/kr a radius of gyration of R, = 3.36 nrii. ellip.witl,\ of' rcidution w i t h lengths of2a = 17 und 18 nm i --),and r(1d.c with heighrs of H = 14.5 and 15.5 n m und ellipticul ends with half'uxes of 0.55 and 0.30 nm (-.-. ) ~
f = k l K 3 + k2. In this case h3fis plotted
against h3, and kl is evaluated by extrapolating the linear region found at high angles back to h3 = 0. The area of the outer tail of the hfcurve is then evaluated analytically, taking f = k l K 3 [42]. The value obtained for this invariant term for protein S4 is 0 = 0.786. The volume per S4 molecule, V , calculated from the relation V = 4n210/0is 121 nm3. The average area of the cross-sectional surface, So, is given by So = 4n(hZ)0/2, and found to be 2.30 nm2. The surface per unit volume, ,F, according t o S = 4tllll/Q with fl1 = the volume fraction of solvent, was found to be 0.0475 nm-', so that the total surface area per molecule S4, SV, is 565 nm2 [42]. The molecular parameters derived from low-angle X-ray scattering of protein S 4 in solution are listed in Table 2.
DISCUSSION The sedimentation, diffusion and viscosity studies of protein S 4 presented here provide evidence for an extended, rather than a globular particle. From Table 1 it is seen that the frictional coefficients, fife, obtained with different methods, agree quite well with each other and are consistent with the values obtained by Rhode et al. [43]. Calculation of the ratio of long t o short axis (a/h)of an equivalent extended ellipsoid of revolution, assuming no hydration (61 = 0), leads to an average value of the axial ratio of 14. However, the knowledge of the diffusion constant, Do, the anhydrous molecular weight from sequence work and the partial specific
~
volume permits one to draw certain conclusions about the degree ofhydration of S4 according to R . T/N . Do = (6nqflf;) [3 M,(G + S1VIo)/4 n N ] l i 3with d1 = the hydration of the particle. Since,flji is also known, the maximum hydration obtained in this manner is 0.35 g HzO/g of S4, which is comparable with the value of 0.25 g HzO/g of S4 obtained from low-angle X-ray scattering. The scattering curve of protein S4 is compared with scattering curves for different models, r . g . for prolate ellipsoids with different axial ratios and for rods of different lengths, with R , = = 3.36 nm where a = long half axis and h = short half axis, but different length I = 2 a (Fig.4). The best correspondence with the experimental curve is obtained for an ellipsoid of three half axes, N = 6.25 nm, h = 0.525 nm and c = 0.25 nm, or for a cylindrical rod of height H = 14.5 nm and elliptical ends with half axes of 0.55 and 0.30 nm. There is no reason for a clear preference of one of the hydrodynamic scattering analogues over the simple prolate ellipsoid of revolution. Moreover, we examined the possibility that the S 4 protein molecule exists as an open coil in solution using the model of the worm-like chain with finite chain length. The Debye scattering function for a random coil shows for h values near the Guinier region a Z z h-2 dependence. This implies that a polypeptide chain with finite persistent length will undergo a transition with increasing
v
m
28
scattering angle from coil behavior ( I = K 2 )to rodlike behavior ( I = K ' ) . Inspections of plots of h2Z versus I7 for solutions of protein S4 show no horizontal plateau regions corresponding to the I z K 2 region; only linearly increasing functions with Z FZ h - l , evidently only rod-like domains, are to be seen, Moreover, applying Luzzati's theory [44] for the interpretation of curves of low-angle X-ray scattering for rod-like particles in conditions of collimating approximation of an infinitely high slit-point receiver, the radius of gyration of the cross section, 0.41 nni, was directly obtained. This implies that, if sufficiently prolonged regions of S4 threads can be approximated by a cylinder, R , would characterize the cross dimensions of the S4 thread. This agrees well with a rod diameter of 1.15 nm, corresponding to a length of 15.0 nm, and supports the conclusions concerning the shape of S4, also. Almost the same results are obtained from electron microscopic studies of antibody-labeled subunits by Lake et al. [45] and Tischendorf et al. [46]. Hydrodynamic studies of S4 and low-angle X-ray scattering show that S4 does not behave as a typical spherical protein, since frictional ratio, intrinsic viscosity and radius of gyration are much higher than for globular proteins. Calculations of the radii of gyration for different conformations show that only a rod-like structure rather than a flexible coil for S4 is possible. Moreover, the intrinsic viscosity of 12.5 mg/ ml is somewhat smaller than the value to be expected for a solvent-immobilized Gaussian coil of 210 amino acid residues. This value would be 23.5 ml/g and would correspond to a radius of gyration of 4.62 nm. Neither value is comparable with the experimentally determined ones. Furthermore, circular dichroism studies of S4 in reconstitution buffer, and in the buffers used in this study, show a certain degree of order, displaying both cc-helical (35 %) and P-pleated sheet structures (30 %) (Paradies, unpublished results). It is rather unlikely that only hydration of such an ordered molecule accounts for the high frictional ratio and intrinsic viscosity; otherwise this would require solvations of the order of 10 g H2 0 /g protein. We are grateful to Dr H. G. Wittmann for critical discussions in the course of this work and to Miss Else Nielsen for help in the preparation and typing of the English text.
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29 44. Luzzati, V. (1960) Acia Crystallogr. 13, 939-942. 45. Lake, J. A , , Pendergast, M., Kahan, L. & Nomura, M . (1974) Proc. Nail Acad. Sci. U.S.A. 71, 4688-4692. 46. Tischendorf, J. W., Zeichardt, H. & Stoffler, G. (1974) Mol. G m . Genet. 134, 187-208.
H. H. Paradies, Fachrichtung Biochemie der Pflanzen, Institul fur Pflanzenphysiologie und Zellbiologie, Fachbereich Biologie der Freien Universitat Berlin, Konigin-Luke-StraBe 12/16 a, D-1000 Berlin (West) 33-Dahlem A. Franz, Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, IhnestraBe 63/73, D-1000 Berlin (West) 33-Dahlem
