- Email: [email protected]
Molecular interactions between ribosomal proteins: A study of the S6-S18 interaction
ARCHIVES
OF BIOCHEMISTRY
Vol. 187, No. 2, April
Molecular
AND
BIOPHYSICS
30, pp. 399-405,
Interactions
1978
between S6-S18
V. PRAKASH Marrs
McLean
Department
Received
of Biochemistry, November
Ribosomal Interaction’ AND Baylor
KIRK College
1, 1977; revised
Proteins:
A Study
of the
C. AUNE of Medicine, January
Houston,
Texas
77030
16, 1978
The proteins S6 and S18 from the 30 S ribosomal subunit of Escherichia coli were isolated to a purity of greater than 95%, characterized in solution, and investigated by sedimentation equilibrium for possible intermolecular interactions in a dilute salt reconstitution buffer. It was observed that neither protein S6 nor S18 has a tendency to selfassociate in the concentration range studied. An analysis of solution mixtures containing proteins S6 and S18 revealed a species of molecular weight greater than either of the proteins. Proteins S6 and SIB were found to interact with an equilibrium constant of association of 6.6 + 4.2 X IO4 M-’ at 3’C with a Gibbs free energy of interaction, AG” = -6.1 kcal/mol. These data are part of those collected to help in building a map of the energetics in the 30 S ribosomal subunit, which provides for the stabilization of the structure.
The structure of the ribosome of Escherichia coli with regard to the spatial relationship of the various individual proteins of the 30 and 50 S subunits has been the focus of many studies. The identification of the neighborhood of the proteins and their interactions can be studied by isolating the protein pairs formed by bifimctional reagents and characterizing them by various methods. This laboratory has examined several interactions by direct energy of interaction analysis (l-4). In the present work, the proteins of interest from the 30 S ribosome are isolated and characterized; the characterized proteins with known concentrations are mixed and analyzed in the analytical ultracentrifuge by the method of sedimentation equilibrium (1, 3, 5, 6). Proteins S6 and S18 from the 30 S component have been of interest as they have been chemically crosslinked among themselves and to some of the presumptive neighboring proteins. Morgan and Brimacombe (7) place S6 and S18 as neighbors from their fragmentation studies with ri-
bonucleases. Clegg and Hayes (8) have obtained S6 and S18 in pair by treatment of 30 S subunits with dimethylsuberimidate. Huang et al. (9) have suggested from singlet energy transfer measurements using fluorescent probes that S6 and S18 are proximal. Sommer and Traut (10) have also isolated a chemically crosslinked complex of S6 and S18. They obtained an apparent molecular weight of 27,500 for the complex as compared to their reported monomer molecular weights of 16,000 and 11,000 for S6 and S18, respectively. Expert-Bezancon et al. (11) have also crosslinked S6 and S18 by treating the 30 S subunit with bisimidoesters. They report a value of 28,009 for the molecular weight of the S6-S18 complex. Further S6 or S7 and S18 have been shown to interact with S21 (12-14). Also, from the recent topographical models of Traut et al. (15), Nomura and Held (16), and Cornick and Kretainger (17), proteins S6 and S18 are shown to be near neighbors in the 30 S ribosomal subunit. Cornick and Kretsinger (17) place S6, S18, and S12 only as in reasonable proximity. At present, no study of a direct physical S6-S18 interaction is available. Knowledge of these intrasubunit interactions is useful in developing an under-
i This work was supported in part by the National Institute of Health (GM 22244) and the Robert A. Welch Foundation (Q-592). K.A. is the recipient of an NIH Research Career Development Award (KO4 GMOOO711. 399
IMO3-9861/78/1872-03SS$O2.00/0 Cop.pight 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
400
PRAKASH
standing of the neighborhood of each of the proteins in the 30 S subunit and also for revealing the centers of cooperativity and energy of interaction within the subunit. These data will help to construct a topographical model of the proteins of the 30 S subunit of Escherichia coli from an energetics point of view, possessing information regarding stabilization and cooperativity. The present study discusses the isolation and characterization of S6 and S18 and their interaction, using mainly the technique of sedimentation equilibrium. MATERIALS
AND
METHODS
The 70 S ribosomes were isolated from E. coli strain B cells and the 30 and 50 S subunits were separated on a sucrose gradient under low Mg*+ conditions, as has been described by Rohde et al. (1). From the 30 S subunits thus obtained, fractions containing S6 and S18 were obtained by chromatography on phosphocellulose using the procedure of Hardy et al. (18) and Rohde et al. (1). The fractions were tentatively identified based upon their mobility in urea gel electrophoresis and comparison to their prior elution positions (1, 18). Isolation ofS6. The fraction containing protein S6, obtained from phosphocellulose chromatography, was loaded onto a Sepharose 6B column equilibrated with 6 M GuHCl.* The protein S6 was the major component eluting from the column. The protein fractions were pooled and further purified by rechromatography on a Sephadex G-100 column equilibrated with 6 M urea buffer (0.05 M sodium phosphate, 0.012 M methylamine hydrochloride, and 7 x 10m4 M 2-mercaptoethanol, pH 5.6) from which pure S6 was obtained. The homogeneity of the sample was judged by urea gel electrophoresis (Fig. 1) and by a modified SDS gel electrophoresis (19), the details of which are described by Rohde et al. (1). The amido black-stained gels were scanned in an ISCO UA-5 absorbance monitor at 546 nm. Isolation of S18. The fraction containing protein S18 obtained from the phosphocellulose column coeluted with another protein, S9. Initial separation of the two proteins was achieved on a Sephadex G-100 column equilibrated with 6 M urea buffer at pH 5.6. The partially purified protein was further processed by repeated gel filtration on the G-100 column in urea buffer, and a homogenous protein thus obtained was appraised by the procedures mentioned above for S6. The tentative assignments of S6 and S18 to those products were confirmed by comparing the amino acid ’ Abbreviations used: GuHCl, guanidine ride; SDS, sodium dodecyl sulfate; TMK M Tris, 0.02 M MgC12, 0.1 M KCI, and 0.01 toethanol, pH 7.36; DNP, dinotrophenyl.
hydrochlobuffer, 0.03 M 2-mercap-
AND
AUNE
0
I
2 3 4 Mobdily (cm)
5
6
FIG. 1. Scans at 546 nm of S6 and S18 gels from urea gel electrophoresis. analysis of these proteins with that of the published compositions of Craven et al. (20) and Kaltachmidt et al. (21). The comparisons were quantitated by determining the correlation coefficient. In this method, the mole fraction of amino acid residue i in protein k, XJ~ was compared to the mole fraction of amino acid residue i in protein 1, &. The variations were quantitated by computing a correlation coefficient defined
by
Rki=c (xti.&,/[~x?,c a]‘.
(1)
It is seen that a perfect correlation will yield a value of 1 and a totally imperfect correlation would yield a value of 0. Stokes’ radii. Stokes’ radii of the proteins were determined by gel chromatography in a Sephadex G100 column employing low salt TMK buffer (0.03 M Tris, 0.02 M MgClx, 0.1 M KCl, and 0.01 M 2-mercaptoethanol, pH 7.36). The column was equilibrated with the above buffer and calibrated with the standard proteins ovalbumin, bovine carbonic anhydrase, trypsin inhibitor, myoglobin, and cytochrome c. The Stokes’ radius, R,, was evaluated by the procedure of Ackers (22) using the equation
R. = B + A erfc-’ (a),
PI
where o is the partition coefficient and A and B are constants. The frictional coefficient, f/fmti, of the protein can be calculated using the value of R, from the equation fjfmin =
RJRmin,
[31
where Rmin is calculated from the partial specific volume and molecular weight of the protein, assuming it to be a globular nonhydrated sphere. The quantity f/f,,,i”, was also calculated using the molecular weight and sedimentation coefficient by the equation f/fmm where specific
= M( 1 - UP I/( 6ml NsRmin
M is the molecular volume,
p is
),
r41
weight, Sr is the partial the density of the solution, n is
RIBOSOMAL
PROTEIN
the viscosity of the solution, N is Avogadro’s number, and s is the sedimentation coefficient. Refolding. The isolated proteins were refolded in 0.1 M KC1 TMK, instead of the usual 0.35 M KC1 TMK, since the solubility of S18 was higher in the former solvent. Refolded proteins were obtained by dissolving -0.7 mg of the protein in 20 ~1 of 6 M urea, to which was added 0.8 ml of the TMK buffer. This solution was incubated at 37°C for 1 h and dialyzed for at least 48 h at 3°C against the TMK buffer. Molecular weight: (i) Gel filtration. The molecular weights of the proteins were obtained from gel filtration by calibrating a Sephadex G-100 column in 6 M urea buffer (pH 5.66) using the standard protein trypsin inhibitor, P-lactoglobulin, lysozyme, a-chymotrypsin, and insulin (peptides) as standards. The partition coefficient, o, was determined by the equation, o=(V-
vo)/(VT-
vn),
[51
where V represents the elution volume of protein, Vo, the elution volume of totally excluded blue dextran, and VT, the elution volume of DNP-glycine. All the volumes were determined gravimetrically for better precision. A plot of erf-’ (0) vesus Ma.% was constructed, from which the molecular weight of the unknown protein was interpolated. The relevance of the exponent of M, 0.555, has been discussed by Fish
et al. (23). (iiJ Sedimentation
equilibrium. Molecular weights were determined by sedimentation equilibrium employing the high-speed technique of Yphantis (5), and the calculations were performed using a Hewlett Packard 9810A programmable calculator. The details of the procedure have been discussed by Rohde et al. (I), Kar and Aune (6), and Aune and Rohde (3). The .SZQ,~ values were calculated using the Hewlett Packard 9810A programmable calculator, which received the digital output from the phototube of the scanner optics of the ultracentrifuge through an integrating digital voltmeter. The details of this procedure are discussed by Inners et al. (24). (ii4 SDS-polyacrylamide gel electrophoresis. The ultimate molecular weight of proteins can also be approximated by utilizing the method of SDS electrophoresis as described by Weber and Osborn (19). Markers of bovine serum albumin, pepsinogen, chymotrypsinogen, /?-lactoglobulin, lysozyme, and a-chymotrypsin chains in 12.5% gels with 1.25% crosslinking were employed. RESULTS
AND
DISCUSSION
In order to investigate any association phenomena of the mixtures of the two proteins S6 and S18, the individual proteins themselves have to be characterized for the monomer molecular weight, the degree of self-association, and other parameters. This is done by utilizing the technique of sedi-
S6-S18
401
INTERACTION
mentation equilibrium. In the above method the concentration distribution of a single species or a mixture of two proteins is given by
C(r) = 2 Ci( U)[eXp{Mi(
1 - &Pip)
where C is the concentration of the ith component at the meniscus, Mi is its molecular weight, Uiis the partial specific volume of component i, p is the solution density, w is the angular velocity, r is the radial position, r, is the radial position at meniscus, R is the gas constant, and T is the absolute temperature. After the proteins were tentatively assigned based on mobility in urea gel and SDS gel electrophoresis and chromatography elution position, they were isolated to the highest degree of purity and final identification was made. This was performed by carrying out an amino acid analysis and computing correlation coefficients relative to previously assigned proteins (20). Values of 0.989 and 0.973 were obtained for S6 and S18, respectively, with no other protein assignment being remotely suitable. To determine the concentration of the protein solution, the absorptivity is required. The absorptivity of S6 has already been determined by Rohde et al. (1) as 0.99 ml mg-’ cm-’ in 0.35 M KC1 TMK buffer at 280 nm. The absorptivity of S18 was determined to be 1.00 ml mg-’ cm-’ in 15%acetic acid as solvent. Lysozyme, used as the calibrating protein, yielded a refractive increment value of 1.803 X 1O-5liter-’ g-’ in 15% acetic acid at 280 nm. The absorptivities were assumed to be the same in both 15% acetic acid and TMK buffer for the purposes of determination of concentration of proteins. The molecular weights of the proteins were determined by the methods already discussed under Materials and Methods. The partition coefficients obtained from gel filtration data for the standard proteins are: trypsin inhibitor (21,000), 0.246; P-lactoglobulin, (18,40(l), 0.368; lysozyme (14,308), 0.597; a-chymotrypsin (ll,OOO), 0.675; and insulin (6000), 0.851. The partition coefficients of S6 and S18 obtained using the
402
PRAKASH
above data for standard proteins were 0.61 and 0.73, respectively. Table I presents the molecular weights of S6 and S18 as determined by the above methods. The reported molecular weight of S6 from E. coli B in 0.35 M KC1 TMK buffer is 16,106 f 500 (1). The value obtained here for S6 from E. coli strain B from a separate source and preparation is 16,400 + 500 by sedimentation equilibrium. This is in close agreement with the previously reported value. The value of 17,500 + 1500 in SDS gel electrophoresis is also close to the above reported value. But a value of 19,000 f 2000 by urea gel filtration was slightly on the high side. Further, S6 from E. coli strain K has been sequenced by Hits et al. (25) and the molecular weight calculated from the sequence data gives a value of 15,187. The protein S18 from E. coli strain K has also been sequenced by Yaguchi (26), who reported the molecular weight to be 8951. However, the results of SDS gel electrophoresis and urea gel filtration in the present study indicate higher molecular weights than the molecular weight calculated from the sequence data. The result from sedimentation equilibrium gives a value of 8900 f 500, which is in excellent agreement with the molecular weight calculated from the sequence data of Yaguchi (26). From the above molecular weight data for both S6 and S18 it is clear that sedimentation equilibrium gives the lowest value of all the methods described and is very close to the molecular weight calculated from the sequence. However, proteins S6 and S18 provide anomalous molecular weights in SDS gel electrophoresis and urea gel filtration. The higher molecular weight in SDS gel electrophoresis could arise from the fact that the SDS-protein binding ratio TABLE
AND
AUNE
may not be the same as that of the standard SDS-protein complex ratio, which has been shown to be 1.4 g of SDS per gram of protein (27). Further, the detergent-bound protein molecule may be in a more extended state than would be the other proteins in this solvent, resulting in a lowering of the mobility. The SDS gel electrophoresis technique is mainly a transport method as compared to other methods used here and should not be expected to yield an exact molecular weight, particularly when the protein is small. Similarly, the anomalous high molecular weight in gel filtration under denaturing solvent conditions could be due to the unusual shape of the protein in urea solution. Since in the present study the technique of sedimentation equilibrium is used to study the interaction between the proteins, the molecular weight values of 16,400 for S6 and 8900 for S18 obtained from sedimentation equilibrium were used for all the calculations. There was no significant change in the molecular weights of either S6 or S18 in sedimentation equilibrium studies over a temperature range of &5”C. The frictional properties of the proteins have been determined both by sedimentation velocity and gel filtration experiments. Table II presents some of the data obtained from the above studies. The ~20,~ value of 1.66 S for S6 is in good agreement with the value reported by Rohde et al. (1). The SZO,W value of S18 could not be obtained due to its low molecular weight and high diffusion coefficient. However, f/fminfor S18 has been computed from gel filtration data to be 1.40. Protein S18 appears to be more extended in its conformation than S6, taking into account f/fmin of each protein. Typical values of f/fminfor globular proteins are of the order of 1.20 to 1.30.
I
MOLECULAR~EIGHTOF S6 AND S18 BY VARIOUS METHODS zz
TABLE
II
FRICTIONALPROPERTIESOF S6 AND S18 IN 0.1 M KC1 TMK BUFFER ProSm.v f/fma 0 R. f/fmmb tein
S6 Sl8
16,400*500(3) 8,900~500(2)
n Numbers minations.
17,5OOf1,500(2) 12,7OOf1,500(2)
in parentheses
19,000xk2,000(2) 14,900~2,000(1)
are the number
of deter-
S6 S18
IA,
1.66ztO.l -
a Computed b Computed
1.45 from from
Eq. [S]. Eq. [3].
0.610 0.730
20.8 19.0
1.25 1.40
RIBOSOMAL
PROTEIN
The Stokes’ radii of the proteins were obtained by calibrating the column, as has been described under Materials and Methods. Standard proteins were used to provide the CJvalues in the range of 0.25 to 0.85. The Stokes’ radius of S6 is higher than that of S18, which is in conformity with the higher molecular weight of S6 as determined by other methods. It should be noted that the frictional properties of S6 obtained here in 0.1 M KC1 TMK are in good agreement with those previously obtained to 0.35 M KC1 TMK (1). The studies pertaining to proteins S6 and S18 were performed in 0.1 M KC1 TMK instead of the 0.35 M KC1 TMK used in the previous 30 S ribosomal protein interaction studies from this laboratory (1, 2, 4). This was because S18 could not be sufficiently solubilized in salt concentrations greater than 0.1 M. The protein solubility is nearly four times higher in 0.1 M KC1 TMK than in 0.35 M KC1 TMK. This is presumably due to the fact that the protein is salted out at the above salt concentration (especially in the presence of ~0.3 M urea, which is incorporated into the solution while refolding). The solubility of S6, although less in 0.1 M KC1 TMK than in 0.35 M KC1 TMK, presented no major problems. The sedimentation equilibrium patterns of S6 and S18 are shown in Figs. 2 and 3, respectively. The plot of fringe displacement (in microns) versus radial position gave a displacement of nearly 800 and 500 pm at the bottom of the cell for S6 and S18,
I
I
I
7.0
7.05
7.10
I
7.15
1
7 20
r lcml
FIG. 2. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: S6, 0.2 mg/ml at 35,600 rpm at 3°C. The solid line is the best fit for a single species.
S6-S18
INTERACTION
FIG. 3. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: S18,0.14 mg/ml at 40,006 rpm at 3°C. The solid line is the best fit for a single species.
FIG. 4. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: 1:3 ratio of S&S18 at 40,000 rpm at 3°C. The dashed line is the best fit for two species and the solid line is the best fit for three species.
respectively. The data obtained were subjected to curve-fitting procedures where pure monomer was assumed to be present in one model, and dimer in equilibrium with monomer in a second model. The solid line indicates the best fit for the experimental curve and represents the single species fit in both figures. Obviously, dimer species were not required in either case to improve the fit. This indicates that both S6 and S18 are monomeric in 0.1 M KC1 TMK under the concentration conditions over the entire range of the cell. The average residuals for S6 and S18 were 10.2 and 9.9 pm. The average residual is defined as R = i [I&l/( iv-S-l)],
PI
404
PRAKASH
AND
TABLE PARAMETERS Pair
S6-S18
Trial
1 2 3
DETERMINED
R"
FROM
a Composition ratio, S18S6; the total concentration for the experiments. b Computed according to Eq. [4] of Ref. (4). c Weighted mean with the root-mean square error
elm) 13.1 11.7 11.2
34.6 25.8 30.1 mg/mI
K(xW4 5.8 8.8 11.9 6.6
and the range
A f f f
M) 4.3 9.3 16.7 4.2’
was -0.0-1.0
mg/mI
on it.
PI a)S6C(
CURVE-FITTING
Percentage* mass as complex
was -0.3-0.4
where N is the number of data points, S is the number of species in the system, and 6 is the absolute value of the residual at each point where the residual is the difference between the experimental data and the caIculated curve (1, 3). The data obtained for the heterogeneous sample, S6-S18, are shown in Fig. 4. It can be seen that the data fit very weIl to a model with a three-species fit, i.e., S6, S18, and S6-S18, instead of just two noninteracting species, S6 and S18. This establishes a complex formation between $6 and S18 presumed to be of the simplest stoichiometry, 1:l. Naturally, this is presumptive for higher-ordered stoichiometry cannot reveal itself if the fit is already at the level of experimental error. More complex models need not be considered in the absence of more data. The average residual errors in the three trials were 13.1,11.7, and 11.2 w for a three-species fit, as indicated in Table III. Since the average residual is reasonably well within the range of plate-reading error, no further fitting with variation in parameters is deemed necessary. Further, the percentage mass as complexes formed in the system is quite significant, as shown in Table III. The data on the interaction study are utilized for the calculation of the equihbrium constant of association, which is given by the equation,
. c( U)@s+Sls)/C(
EQUILIBRIUM
Residual
0.59 2.86 3.10
K = [MsdMsls/M(s6+sle,l
III
SEDIMENTATION
R computed
0.50 3.00 3.00
AUNE
ah8
where Mi is the molecular weight of component i and Ci( a) is the concentration of component i at the meniscus. The procedures utilized in obtaining the meniscus
concentrations of the species present in the system have been described by Aune and Rohde (3). The data collected for the mixture yielded an average equilibrium constant of association of 6.6 + 4.2 X lo* M-’ and a Gibbs free energy of interaction, AG” = -6.1 kcaI/mol at 3°C in TMK buffer. The values of AG” obtained are about 1.2 and 1.3 kcaI/mol less negative than the reported values for the S3-S5 and S5-SlO interactions, respectively, and about 1.0 and 1.3 kcaI/mol more negative than the reported values for the S3-S4 and S4-S5 interactions, respectively (1, 2, 4). It has been shown elsewhere that S6 or S7 and S18 may interact with S21 (12-14). In view of this, the present study of the S6-S18 interaction would help in the anaiysis of the putative ternary complex S6-SlB-S21 which is under progress in this laboratory. In the present state of ambiguities and discrepancies of the positions of various 30 S subunit proteins, the results of the various energetics of interaction between the different proteins would assist in developing a working model of the thermodynamicahy stable ribosomal30 S subunit. ACKNOWLEDGMENTS We would Iike to express our thanks to Mrs. Linda TaIbert for her expert technical assistance, Mr. Steve Tindah for providing the fractions from the phosphocellulose column, Dr. Harris Busch for the use of the zonal rotor used in the preparation of 30s subunits, and Stanley Moore for the assistance in obtaining ammo acid analysis. REFERENCES 1. ROHDE,
M.
F.,
O’BRIEN,
S., COOPER,
S.,
AND
RIBOSOMAL
PROTEIN
AUNE, K. C. (1975) Biochemistry 14, 1079. 2. ROHDE, M. F., AND AUNE, K. C. (1975) Biochemistry 14,4344. 3. AUNE, K. C., AND ROHDE, M. F. (1977) Anal. B&hem. 79, 110. 4. AUNE, K. C. (1977) Arch. B&hem. Biophys. 180, 172. 5. YPHANTIS, D. A. (1964) Biochemistry 3,297. 6. KAR, E. G., AND AUNE, K. C. (1974) Anal. Bio-
them. 62.1. 7. MORGAN,
J., AND BRIMACOMBE,
B&hem. 8. CLEGG,
R. (1972)
Eur. J.
29,542.
C., AND
HAYES,
D. (1974)
Eur. J. Bio-
them. 42,21. 9. HUANG, K. H., FAIRCLOUGH, R. H., AND CANTOR, C. R. (1975) J. Mol. Biol. 97,443. 10. SOMMER, A., AND TRAUT, R. R. (1976) J. Mol. Biol. 106, 995. 11. EXPERT-BEZANCON, A., BARRITAULT, D., MILET, M., GUERIN, M.-F., AND HAYES, D. H. (1977) J. Mol. Biol. 112, 603. 12. CHANG, F. N., AND FLAKS, J. G. (1972) J. Mol.
Biol. 68, 177. 13. KURLAND, C. G., GREEN, M., SCHAUP, H. W., DONNER, D., LUTTER, L., AND BIRGE, E. A. (1972) FEBS Symp. 23, 75. 14. LUTTER, L. C., ZEICHARDT, H., KURLAND, C. G., AND STOFFLER, G. (1972) Mol. Gen. Genet. 119, 357.
S6-S18
405
INTERACTION
15. TRAUT, R. R., HEIMARK, R. L., SUN, T. T., HERSHEYE, J. W. B., AND BOLLEN, A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, P., eds.), p. 271, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 16. NOMUF~A, M., AND HELD, W. A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, P., eds.), p. 193, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 17. CORNICK, G., C., AND KRETSINGER, R. H. (1977)
B&him.
Biophys. Acta 474,398.
18. HARDY, S. J. S., KURLAND, P., AND MORA, G. (1969) 19. WEBER, K., AND OSBORN,
C. G., AND VOYNOW,
Biochemistry M.
(1969)
8,2897. Biol.
J.
Chem. 244,4406. 20. CRAVEN, G. R., Voy~ow, P., HARDY, S. J. S., AND KURLAND, C. G. (1969) Biochemistry 8, 2906. 21. KALTSCHMIDT, E., DZIONARA, M., AND WITTMANN, H. G. (1970) Mol. Gen. Genet. 109,292. 22. ACKERS, G. K. (1967) J. Biol. Chem. 242,3237. 23. FISH, W. W., MANN, K. G., AND TANFORD, C. (1969) J. Biol. Chem. 244,4989. 24. INNERS, L. D., TINDALL, S., AND AUNE, K. C. (1978) Anal. Biochem., in press. 25. HITZ, H., SCHAFER, D., AND WITTMANN-LIEBOLD, B. (1975) FEBS Lett. 56, 259. 26. YAGUCHI, M. (1975) FEBS Lett. 59,217. 27. REYNOLDS, J. A., AND TANFORD, C. (1970) Proc. Nat. Acad. Sci. USA 66, 1002.
OF BIOCHEMISTRY
Vol. 187, No. 2, April
Molecular
AND
BIOPHYSICS
30, pp. 399-405,
Interactions
1978
between S6-S18
V. PRAKASH Marrs
McLean
Department
Received
of Biochemistry, November
Ribosomal Interaction’ AND Baylor
KIRK College
1, 1977; revised
Proteins:
A Study
of the
C. AUNE of Medicine, January
Houston,
Texas
77030
16, 1978
The proteins S6 and S18 from the 30 S ribosomal subunit of Escherichia coli were isolated to a purity of greater than 95%, characterized in solution, and investigated by sedimentation equilibrium for possible intermolecular interactions in a dilute salt reconstitution buffer. It was observed that neither protein S6 nor S18 has a tendency to selfassociate in the concentration range studied. An analysis of solution mixtures containing proteins S6 and S18 revealed a species of molecular weight greater than either of the proteins. Proteins S6 and SIB were found to interact with an equilibrium constant of association of 6.6 + 4.2 X IO4 M-’ at 3’C with a Gibbs free energy of interaction, AG” = -6.1 kcal/mol. These data are part of those collected to help in building a map of the energetics in the 30 S ribosomal subunit, which provides for the stabilization of the structure.
The structure of the ribosome of Escherichia coli with regard to the spatial relationship of the various individual proteins of the 30 and 50 S subunits has been the focus of many studies. The identification of the neighborhood of the proteins and their interactions can be studied by isolating the protein pairs formed by bifimctional reagents and characterizing them by various methods. This laboratory has examined several interactions by direct energy of interaction analysis (l-4). In the present work, the proteins of interest from the 30 S ribosome are isolated and characterized; the characterized proteins with known concentrations are mixed and analyzed in the analytical ultracentrifuge by the method of sedimentation equilibrium (1, 3, 5, 6). Proteins S6 and S18 from the 30 S component have been of interest as they have been chemically crosslinked among themselves and to some of the presumptive neighboring proteins. Morgan and Brimacombe (7) place S6 and S18 as neighbors from their fragmentation studies with ri-
bonucleases. Clegg and Hayes (8) have obtained S6 and S18 in pair by treatment of 30 S subunits with dimethylsuberimidate. Huang et al. (9) have suggested from singlet energy transfer measurements using fluorescent probes that S6 and S18 are proximal. Sommer and Traut (10) have also isolated a chemically crosslinked complex of S6 and S18. They obtained an apparent molecular weight of 27,500 for the complex as compared to their reported monomer molecular weights of 16,000 and 11,000 for S6 and S18, respectively. Expert-Bezancon et al. (11) have also crosslinked S6 and S18 by treating the 30 S subunit with bisimidoesters. They report a value of 28,009 for the molecular weight of the S6-S18 complex. Further S6 or S7 and S18 have been shown to interact with S21 (12-14). Also, from the recent topographical models of Traut et al. (15), Nomura and Held (16), and Cornick and Kretainger (17), proteins S6 and S18 are shown to be near neighbors in the 30 S ribosomal subunit. Cornick and Kretsinger (17) place S6, S18, and S12 only as in reasonable proximity. At present, no study of a direct physical S6-S18 interaction is available. Knowledge of these intrasubunit interactions is useful in developing an under-
i This work was supported in part by the National Institute of Health (GM 22244) and the Robert A. Welch Foundation (Q-592). K.A. is the recipient of an NIH Research Career Development Award (KO4 GMOOO711. 399
IMO3-9861/78/1872-03SS$O2.00/0 Cop.pight 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
400
PRAKASH
standing of the neighborhood of each of the proteins in the 30 S subunit and also for revealing the centers of cooperativity and energy of interaction within the subunit. These data will help to construct a topographical model of the proteins of the 30 S subunit of Escherichia coli from an energetics point of view, possessing information regarding stabilization and cooperativity. The present study discusses the isolation and characterization of S6 and S18 and their interaction, using mainly the technique of sedimentation equilibrium. MATERIALS
AND
METHODS
The 70 S ribosomes were isolated from E. coli strain B cells and the 30 and 50 S subunits were separated on a sucrose gradient under low Mg*+ conditions, as has been described by Rohde et al. (1). From the 30 S subunits thus obtained, fractions containing S6 and S18 were obtained by chromatography on phosphocellulose using the procedure of Hardy et al. (18) and Rohde et al. (1). The fractions were tentatively identified based upon their mobility in urea gel electrophoresis and comparison to their prior elution positions (1, 18). Isolation ofS6. The fraction containing protein S6, obtained from phosphocellulose chromatography, was loaded onto a Sepharose 6B column equilibrated with 6 M GuHCl.* The protein S6 was the major component eluting from the column. The protein fractions were pooled and further purified by rechromatography on a Sephadex G-100 column equilibrated with 6 M urea buffer (0.05 M sodium phosphate, 0.012 M methylamine hydrochloride, and 7 x 10m4 M 2-mercaptoethanol, pH 5.6) from which pure S6 was obtained. The homogeneity of the sample was judged by urea gel electrophoresis (Fig. 1) and by a modified SDS gel electrophoresis (19), the details of which are described by Rohde et al. (1). The amido black-stained gels were scanned in an ISCO UA-5 absorbance monitor at 546 nm. Isolation of S18. The fraction containing protein S18 obtained from the phosphocellulose column coeluted with another protein, S9. Initial separation of the two proteins was achieved on a Sephadex G-100 column equilibrated with 6 M urea buffer at pH 5.6. The partially purified protein was further processed by repeated gel filtration on the G-100 column in urea buffer, and a homogenous protein thus obtained was appraised by the procedures mentioned above for S6. The tentative assignments of S6 and S18 to those products were confirmed by comparing the amino acid ’ Abbreviations used: GuHCl, guanidine ride; SDS, sodium dodecyl sulfate; TMK M Tris, 0.02 M MgC12, 0.1 M KCI, and 0.01 toethanol, pH 7.36; DNP, dinotrophenyl.
hydrochlobuffer, 0.03 M 2-mercap-
AND
AUNE
0
I
2 3 4 Mobdily (cm)
5
6
FIG. 1. Scans at 546 nm of S6 and S18 gels from urea gel electrophoresis. analysis of these proteins with that of the published compositions of Craven et al. (20) and Kaltachmidt et al. (21). The comparisons were quantitated by determining the correlation coefficient. In this method, the mole fraction of amino acid residue i in protein k, XJ~ was compared to the mole fraction of amino acid residue i in protein 1, &. The variations were quantitated by computing a correlation coefficient defined
by
Rki=c (xti.&,/[~x?,c a]‘.
(1)
It is seen that a perfect correlation will yield a value of 1 and a totally imperfect correlation would yield a value of 0. Stokes’ radii. Stokes’ radii of the proteins were determined by gel chromatography in a Sephadex G100 column employing low salt TMK buffer (0.03 M Tris, 0.02 M MgClx, 0.1 M KCl, and 0.01 M 2-mercaptoethanol, pH 7.36). The column was equilibrated with the above buffer and calibrated with the standard proteins ovalbumin, bovine carbonic anhydrase, trypsin inhibitor, myoglobin, and cytochrome c. The Stokes’ radius, R,, was evaluated by the procedure of Ackers (22) using the equation
R. = B + A erfc-’ (a),
PI
where o is the partition coefficient and A and B are constants. The frictional coefficient, f/fmti, of the protein can be calculated using the value of R, from the equation fjfmin =
RJRmin,
[31
where Rmin is calculated from the partial specific volume and molecular weight of the protein, assuming it to be a globular nonhydrated sphere. The quantity f/f,,,i”, was also calculated using the molecular weight and sedimentation coefficient by the equation f/fmm where specific
= M( 1 - UP I/( 6ml NsRmin
M is the molecular volume,
p is
),
r41
weight, Sr is the partial the density of the solution, n is
RIBOSOMAL
PROTEIN
the viscosity of the solution, N is Avogadro’s number, and s is the sedimentation coefficient. Refolding. The isolated proteins were refolded in 0.1 M KC1 TMK, instead of the usual 0.35 M KC1 TMK, since the solubility of S18 was higher in the former solvent. Refolded proteins were obtained by dissolving -0.7 mg of the protein in 20 ~1 of 6 M urea, to which was added 0.8 ml of the TMK buffer. This solution was incubated at 37°C for 1 h and dialyzed for at least 48 h at 3°C against the TMK buffer. Molecular weight: (i) Gel filtration. The molecular weights of the proteins were obtained from gel filtration by calibrating a Sephadex G-100 column in 6 M urea buffer (pH 5.66) using the standard protein trypsin inhibitor, P-lactoglobulin, lysozyme, a-chymotrypsin, and insulin (peptides) as standards. The partition coefficient, o, was determined by the equation, o=(V-
vo)/(VT-
vn),
[51
where V represents the elution volume of protein, Vo, the elution volume of totally excluded blue dextran, and VT, the elution volume of DNP-glycine. All the volumes were determined gravimetrically for better precision. A plot of erf-’ (0) vesus Ma.% was constructed, from which the molecular weight of the unknown protein was interpolated. The relevance of the exponent of M, 0.555, has been discussed by Fish
et al. (23). (iiJ Sedimentation
equilibrium. Molecular weights were determined by sedimentation equilibrium employing the high-speed technique of Yphantis (5), and the calculations were performed using a Hewlett Packard 9810A programmable calculator. The details of the procedure have been discussed by Rohde et al. (I), Kar and Aune (6), and Aune and Rohde (3). The .SZQ,~ values were calculated using the Hewlett Packard 9810A programmable calculator, which received the digital output from the phototube of the scanner optics of the ultracentrifuge through an integrating digital voltmeter. The details of this procedure are discussed by Inners et al. (24). (ii4 SDS-polyacrylamide gel electrophoresis. The ultimate molecular weight of proteins can also be approximated by utilizing the method of SDS electrophoresis as described by Weber and Osborn (19). Markers of bovine serum albumin, pepsinogen, chymotrypsinogen, /?-lactoglobulin, lysozyme, and a-chymotrypsin chains in 12.5% gels with 1.25% crosslinking were employed. RESULTS
AND
DISCUSSION
In order to investigate any association phenomena of the mixtures of the two proteins S6 and S18, the individual proteins themselves have to be characterized for the monomer molecular weight, the degree of self-association, and other parameters. This is done by utilizing the technique of sedi-
S6-S18
401
INTERACTION
mentation equilibrium. In the above method the concentration distribution of a single species or a mixture of two proteins is given by
C(r) = 2 Ci( U)[eXp{Mi(
1 - &Pip)
where C is the concentration of the ith component at the meniscus, Mi is its molecular weight, Uiis the partial specific volume of component i, p is the solution density, w is the angular velocity, r is the radial position, r, is the radial position at meniscus, R is the gas constant, and T is the absolute temperature. After the proteins were tentatively assigned based on mobility in urea gel and SDS gel electrophoresis and chromatography elution position, they were isolated to the highest degree of purity and final identification was made. This was performed by carrying out an amino acid analysis and computing correlation coefficients relative to previously assigned proteins (20). Values of 0.989 and 0.973 were obtained for S6 and S18, respectively, with no other protein assignment being remotely suitable. To determine the concentration of the protein solution, the absorptivity is required. The absorptivity of S6 has already been determined by Rohde et al. (1) as 0.99 ml mg-’ cm-’ in 0.35 M KC1 TMK buffer at 280 nm. The absorptivity of S18 was determined to be 1.00 ml mg-’ cm-’ in 15%acetic acid as solvent. Lysozyme, used as the calibrating protein, yielded a refractive increment value of 1.803 X 1O-5liter-’ g-’ in 15% acetic acid at 280 nm. The absorptivities were assumed to be the same in both 15% acetic acid and TMK buffer for the purposes of determination of concentration of proteins. The molecular weights of the proteins were determined by the methods already discussed under Materials and Methods. The partition coefficients obtained from gel filtration data for the standard proteins are: trypsin inhibitor (21,000), 0.246; P-lactoglobulin, (18,40(l), 0.368; lysozyme (14,308), 0.597; a-chymotrypsin (ll,OOO), 0.675; and insulin (6000), 0.851. The partition coefficients of S6 and S18 obtained using the
402
PRAKASH
above data for standard proteins were 0.61 and 0.73, respectively. Table I presents the molecular weights of S6 and S18 as determined by the above methods. The reported molecular weight of S6 from E. coli B in 0.35 M KC1 TMK buffer is 16,106 f 500 (1). The value obtained here for S6 from E. coli strain B from a separate source and preparation is 16,400 + 500 by sedimentation equilibrium. This is in close agreement with the previously reported value. The value of 17,500 + 1500 in SDS gel electrophoresis is also close to the above reported value. But a value of 19,000 f 2000 by urea gel filtration was slightly on the high side. Further, S6 from E. coli strain K has been sequenced by Hits et al. (25) and the molecular weight calculated from the sequence data gives a value of 15,187. The protein S18 from E. coli strain K has also been sequenced by Yaguchi (26), who reported the molecular weight to be 8951. However, the results of SDS gel electrophoresis and urea gel filtration in the present study indicate higher molecular weights than the molecular weight calculated from the sequence data. The result from sedimentation equilibrium gives a value of 8900 f 500, which is in excellent agreement with the molecular weight calculated from the sequence data of Yaguchi (26). From the above molecular weight data for both S6 and S18 it is clear that sedimentation equilibrium gives the lowest value of all the methods described and is very close to the molecular weight calculated from the sequence. However, proteins S6 and S18 provide anomalous molecular weights in SDS gel electrophoresis and urea gel filtration. The higher molecular weight in SDS gel electrophoresis could arise from the fact that the SDS-protein binding ratio TABLE
AND
AUNE
may not be the same as that of the standard SDS-protein complex ratio, which has been shown to be 1.4 g of SDS per gram of protein (27). Further, the detergent-bound protein molecule may be in a more extended state than would be the other proteins in this solvent, resulting in a lowering of the mobility. The SDS gel electrophoresis technique is mainly a transport method as compared to other methods used here and should not be expected to yield an exact molecular weight, particularly when the protein is small. Similarly, the anomalous high molecular weight in gel filtration under denaturing solvent conditions could be due to the unusual shape of the protein in urea solution. Since in the present study the technique of sedimentation equilibrium is used to study the interaction between the proteins, the molecular weight values of 16,400 for S6 and 8900 for S18 obtained from sedimentation equilibrium were used for all the calculations. There was no significant change in the molecular weights of either S6 or S18 in sedimentation equilibrium studies over a temperature range of &5”C. The frictional properties of the proteins have been determined both by sedimentation velocity and gel filtration experiments. Table II presents some of the data obtained from the above studies. The ~20,~ value of 1.66 S for S6 is in good agreement with the value reported by Rohde et al. (1). The SZO,W value of S18 could not be obtained due to its low molecular weight and high diffusion coefficient. However, f/fminfor S18 has been computed from gel filtration data to be 1.40. Protein S18 appears to be more extended in its conformation than S6, taking into account f/fmin of each protein. Typical values of f/fminfor globular proteins are of the order of 1.20 to 1.30.
I
MOLECULAR~EIGHTOF S6 AND S18 BY VARIOUS METHODS zz
TABLE
II
FRICTIONALPROPERTIESOF S6 AND S18 IN 0.1 M KC1 TMK BUFFER ProSm.v f/fma 0 R. f/fmmb tein
S6 Sl8
16,400*500(3) 8,900~500(2)
n Numbers minations.
17,5OOf1,500(2) 12,7OOf1,500(2)
in parentheses
19,000xk2,000(2) 14,900~2,000(1)
are the number
of deter-
S6 S18
IA,
1.66ztO.l -
a Computed b Computed
1.45 from from
Eq. [S]. Eq. [3].
0.610 0.730
20.8 19.0
1.25 1.40
RIBOSOMAL
PROTEIN
The Stokes’ radii of the proteins were obtained by calibrating the column, as has been described under Materials and Methods. Standard proteins were used to provide the CJvalues in the range of 0.25 to 0.85. The Stokes’ radius of S6 is higher than that of S18, which is in conformity with the higher molecular weight of S6 as determined by other methods. It should be noted that the frictional properties of S6 obtained here in 0.1 M KC1 TMK are in good agreement with those previously obtained to 0.35 M KC1 TMK (1). The studies pertaining to proteins S6 and S18 were performed in 0.1 M KC1 TMK instead of the 0.35 M KC1 TMK used in the previous 30 S ribosomal protein interaction studies from this laboratory (1, 2, 4). This was because S18 could not be sufficiently solubilized in salt concentrations greater than 0.1 M. The protein solubility is nearly four times higher in 0.1 M KC1 TMK than in 0.35 M KC1 TMK. This is presumably due to the fact that the protein is salted out at the above salt concentration (especially in the presence of ~0.3 M urea, which is incorporated into the solution while refolding). The solubility of S6, although less in 0.1 M KC1 TMK than in 0.35 M KC1 TMK, presented no major problems. The sedimentation equilibrium patterns of S6 and S18 are shown in Figs. 2 and 3, respectively. The plot of fringe displacement (in microns) versus radial position gave a displacement of nearly 800 and 500 pm at the bottom of the cell for S6 and S18,
I
I
I
7.0
7.05
7.10
I
7.15
1
7 20
r lcml
FIG. 2. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: S6, 0.2 mg/ml at 35,600 rpm at 3°C. The solid line is the best fit for a single species.
S6-S18
INTERACTION
FIG. 3. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: S18,0.14 mg/ml at 40,006 rpm at 3°C. The solid line is the best fit for a single species.
FIG. 4. Fringe displacement (in microns) versus the radial position. Sedimentation equilibrium experiment conditions: 1:3 ratio of S&S18 at 40,000 rpm at 3°C. The dashed line is the best fit for two species and the solid line is the best fit for three species.
respectively. The data obtained were subjected to curve-fitting procedures where pure monomer was assumed to be present in one model, and dimer in equilibrium with monomer in a second model. The solid line indicates the best fit for the experimental curve and represents the single species fit in both figures. Obviously, dimer species were not required in either case to improve the fit. This indicates that both S6 and S18 are monomeric in 0.1 M KC1 TMK under the concentration conditions over the entire range of the cell. The average residuals for S6 and S18 were 10.2 and 9.9 pm. The average residual is defined as R = i [I&l/( iv-S-l)],
PI
404
PRAKASH
AND
TABLE PARAMETERS Pair
S6-S18
Trial
1 2 3
DETERMINED
R"
FROM
a Composition ratio, S18S6; the total concentration for the experiments. b Computed according to Eq. [4] of Ref. (4). c Weighted mean with the root-mean square error
elm) 13.1 11.7 11.2
34.6 25.8 30.1 mg/mI
K(xW4 5.8 8.8 11.9 6.6
and the range
A f f f
M) 4.3 9.3 16.7 4.2’
was -0.0-1.0
mg/mI
on it.
PI a)S6C(
CURVE-FITTING
Percentage* mass as complex
was -0.3-0.4
where N is the number of data points, S is the number of species in the system, and 6 is the absolute value of the residual at each point where the residual is the difference between the experimental data and the caIculated curve (1, 3). The data obtained for the heterogeneous sample, S6-S18, are shown in Fig. 4. It can be seen that the data fit very weIl to a model with a three-species fit, i.e., S6, S18, and S6-S18, instead of just two noninteracting species, S6 and S18. This establishes a complex formation between $6 and S18 presumed to be of the simplest stoichiometry, 1:l. Naturally, this is presumptive for higher-ordered stoichiometry cannot reveal itself if the fit is already at the level of experimental error. More complex models need not be considered in the absence of more data. The average residual errors in the three trials were 13.1,11.7, and 11.2 w for a three-species fit, as indicated in Table III. Since the average residual is reasonably well within the range of plate-reading error, no further fitting with variation in parameters is deemed necessary. Further, the percentage mass as complexes formed in the system is quite significant, as shown in Table III. The data on the interaction study are utilized for the calculation of the equihbrium constant of association, which is given by the equation,
. c( U)@s+Sls)/C(
EQUILIBRIUM
Residual
0.59 2.86 3.10
K = [MsdMsls/M(s6+sle,l
III
SEDIMENTATION
R computed
0.50 3.00 3.00
AUNE
ah8
where Mi is the molecular weight of component i and Ci( a) is the concentration of component i at the meniscus. The procedures utilized in obtaining the meniscus
concentrations of the species present in the system have been described by Aune and Rohde (3). The data collected for the mixture yielded an average equilibrium constant of association of 6.6 + 4.2 X lo* M-’ and a Gibbs free energy of interaction, AG” = -6.1 kcaI/mol at 3°C in TMK buffer. The values of AG” obtained are about 1.2 and 1.3 kcaI/mol less negative than the reported values for the S3-S5 and S5-SlO interactions, respectively, and about 1.0 and 1.3 kcaI/mol more negative than the reported values for the S3-S4 and S4-S5 interactions, respectively (1, 2, 4). It has been shown elsewhere that S6 or S7 and S18 may interact with S21 (12-14). In view of this, the present study of the S6-S18 interaction would help in the anaiysis of the putative ternary complex S6-SlB-S21 which is under progress in this laboratory. In the present state of ambiguities and discrepancies of the positions of various 30 S subunit proteins, the results of the various energetics of interaction between the different proteins would assist in developing a working model of the thermodynamicahy stable ribosomal30 S subunit. ACKNOWLEDGMENTS We would Iike to express our thanks to Mrs. Linda TaIbert for her expert technical assistance, Mr. Steve Tindah for providing the fractions from the phosphocellulose column, Dr. Harris Busch for the use of the zonal rotor used in the preparation of 30s subunits, and Stanley Moore for the assistance in obtaining ammo acid analysis. REFERENCES 1. ROHDE,
M.
F.,
O’BRIEN,
S., COOPER,
S.,
AND
RIBOSOMAL
PROTEIN
AUNE, K. C. (1975) Biochemistry 14, 1079. 2. ROHDE, M. F., AND AUNE, K. C. (1975) Biochemistry 14,4344. 3. AUNE, K. C., AND ROHDE, M. F. (1977) Anal. B&hem. 79, 110. 4. AUNE, K. C. (1977) Arch. B&hem. Biophys. 180, 172. 5. YPHANTIS, D. A. (1964) Biochemistry 3,297. 6. KAR, E. G., AND AUNE, K. C. (1974) Anal. Bio-
them. 62.1. 7. MORGAN,
J., AND BRIMACOMBE,
B&hem. 8. CLEGG,
R. (1972)
Eur. J.
29,542.
C., AND
HAYES,
D. (1974)
Eur. J. Bio-
them. 42,21. 9. HUANG, K. H., FAIRCLOUGH, R. H., AND CANTOR, C. R. (1975) J. Mol. Biol. 97,443. 10. SOMMER, A., AND TRAUT, R. R. (1976) J. Mol. Biol. 106, 995. 11. EXPERT-BEZANCON, A., BARRITAULT, D., MILET, M., GUERIN, M.-F., AND HAYES, D. H. (1977) J. Mol. Biol. 112, 603. 12. CHANG, F. N., AND FLAKS, J. G. (1972) J. Mol.
Biol. 68, 177. 13. KURLAND, C. G., GREEN, M., SCHAUP, H. W., DONNER, D., LUTTER, L., AND BIRGE, E. A. (1972) FEBS Symp. 23, 75. 14. LUTTER, L. C., ZEICHARDT, H., KURLAND, C. G., AND STOFFLER, G. (1972) Mol. Gen. Genet. 119, 357.
S6-S18
405
INTERACTION
15. TRAUT, R. R., HEIMARK, R. L., SUN, T. T., HERSHEYE, J. W. B., AND BOLLEN, A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, P., eds.), p. 271, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 16. NOMUF~A, M., AND HELD, W. A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, P., eds.), p. 193, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 17. CORNICK, G., C., AND KRETSINGER, R. H. (1977)
B&him.
Biophys. Acta 474,398.
18. HARDY, S. J. S., KURLAND, P., AND MORA, G. (1969) 19. WEBER, K., AND OSBORN,
C. G., AND VOYNOW,
Biochemistry M.
(1969)
8,2897. Biol.
J.
Chem. 244,4406. 20. CRAVEN, G. R., Voy~ow, P., HARDY, S. J. S., AND KURLAND, C. G. (1969) Biochemistry 8, 2906. 21. KALTSCHMIDT, E., DZIONARA, M., AND WITTMANN, H. G. (1970) Mol. Gen. Genet. 109,292. 22. ACKERS, G. K. (1967) J. Biol. Chem. 242,3237. 23. FISH, W. W., MANN, K. G., AND TANFORD, C. (1969) J. Biol. Chem. 244,4989. 24. INNERS, L. D., TINDALL, S., AND AUNE, K. C. (1978) Anal. Biochem., in press. 25. HITZ, H., SCHAFER, D., AND WITTMANN-LIEBOLD, B. (1975) FEBS Lett. 56, 259. 26. YAGUCHI, M. (1975) FEBS Lett. 59,217. 27. REYNOLDS, J. A., AND TANFORD, C. (1970) Proc. Nat. Acad. Sci. USA 66, 1002.