Journal of Biochemical and Biophysical Methods, 21 (1990) 247-266

247

Elsevier JBBM 00838

A new method for stoichiometric analysis of proteins in complex mixture - reevaluation of the stoichiometry of E. coli ribosomal proteins Moshe Tal 1, Ishay Weissman 2 and Aaron Silberstein 1 1 Department of Biology and 2 Faculty of Industrial Engineering and Management, Technion- Israel Institute of Technology, Haifa, Israel (Received 16 December 1989) (Accepted 6 May 1990)

Summary

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of $6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.

Key words: Protein mixture; Stoichiometric analysis; Ribosomal protein; E. coil

Correspondence address: M. Tal, Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000 Israel. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

248 Introduction

According to the currently accepted model of E. coli ribosomal proteins (r-proteins), each such protein is present as a single copy per ribosome, except for L7/L12 which appear in two pairs [1]. Hardy based his stoichiometry determinations on a formula which involved the molecular weight of each r-protein, using the polyacrylamide gel electrophoresis and equilibrium sedimentation values, namely the maximal and the minimal ones listed in the Dzionara et al. paper [3]. Now that exact molecular weight values of E. coli r-proteins are available [2], we used them for revision of Hardy's Calculations. Since Hardy's stoichiometric values were due to the two different molecular weight values, the described correction procedure could be expected to yield very similar, if not identical, sets of numbers. In reality, while many pairs of values were indeed almost identical, several major discrepancies were found. In our opinion, revision of Hardy's data is obviously necessary and the above-recalculated results are sufficient grounds for reopening the problem of ribosomal proteins stoichiometry. In the present work we describe a novel procedure of stoichiometric analysis of proteins in a complex mixture, specially suitable for r-proteins. The procedure is relatively simple compared with its predecessors, and is based on different criteria. After a single two-dimensional (2-D) polyacrylamide gel separation of the 70S r-proteins, we determined the amount of Coomassie R bound to each protein, as well as its radioactivity following an incorporation of labeled amino acids [4]. The amount of bound dye was plotted against the number of positive charges in the protein [5], and likewise the amount of radioactivity in each protein was related to the number of residues of the labeled amino acid [6]. The relatively good linear proportionalities observed in both experiments suggest that a linear regression line based on all spots is a manifestation of one copy of r-protein per ribosome, and thus can serve as a calibration curve in stoichiometric analysis. In our method the various proteins are compared not to a single protein serving as standard (such as used by Hardy [1]), but rather to the whole collection of r-proteins. The strong correlation between the two sets of independent values (bound Coomassie on the one hand, and incorporated radioactivity on the other) derived from a single two-dimensional gel inspires confidence in the validity of the general approach and in the conclusions reported below.

Materials and Methods

All chemicals were of analytical grade. Bacteria. The K12 strain A19 rnase 1 - m e t - was used through all our experiments. Growth of bacteria. The bacteria were adapted to Davis and Mingioli [7] minimal medium supplemented with 0.2% glucose by repeated transfers, and incubated after

249 appropriate dilution, so that following overnight growth at 37 ° C with shaking the culture approached the mid-log phase growth in the morning. Three amino acids (Amersham) were used for labeling: L-[4.5-3H]leucine 153 Ci/mmol (5.66 TBq/mmol), L-[4.5-3H]isoleucine 83 Ci/mmol (3.07 TBq/mmol) and L-[3.4(n)3H]valine 42 Ci/mmol (1.55 TBq/mmol). For each labeling experiment the mid-log phase bacteria were inoculated in 100 rnl Davis medium to about 5 Klett units. 0.5 mCi of labeled amino acid were supplemented with 50 /xg/ml of nonradioactive amino acid to ensure constant supply of radioactive material throughout the growth period. The bacteria were grown in a 500 ml Erlenmeyer flask at 37°C under vigorous shaking. To ensure maximal labeling of the r-proteins, the bacteria were harvested at about 4/5 of the logarithmic growth curve, which accounted for about 4{ doublings. In the [3H]valine labeling experiment (in addition to 50 /~g/ml L-valine) 50 /~g/rnl L-isoleucine was added [8]. At the end of the labeling period about 55% of the radioactive amino acids were consumed. • Preparation of ribosomes. The harvested bacteria were suspended in 2 ml of standard buffer: 0.01 M Tris-HCl, pH 7.8, 0.01 M MgClz, 0.06 M NH4C1, 0.006 M 2-mercaptoethanol - sonicated 3 times for 10 s in ice in an MSE Sonicator. The extract was diluted to 3.5 ml in the same buffer and centrifuged on a SorvaU at 12000 x g for 20 rain. The clear crude extract was layered on 1.5 ml of 20% glycerol cushion in standard buffer and the ribosomes were sedimented by centrifuging in an SW 50 rotor at 40000 rpm at 5°C for 3 h on a Spinco ultracentrifuge. The supernatant was decanted and the walls of the tube were wiped carefully several times with wet tissue paper to ensure complete removal of any nonsedimentable proteins from the extract. The sedimentation profile of the ribosomes is shown in Fig. 1. Preparation of proteins. The r-proteins were prepared by acetic acid extraction according to Hardy et al. [9]. The ribosomes were dissolved (about 200 A / m l ) in TSM buffer (0.01 M Tris-HC1, pH 7.6, 0.06 M KC1, 0.3 mM MgC12, 6 mM 2-mercaptoethanol). One-tenth volume of 1 M MgC12 and 2 vol. of acetic acid were added. The suspension was kept on ice and shaken occasionally. After 45 rnin it was centrifuged at 12000 × g for 10 rain (a second extraction of the pellet with 67% acetic acid did not reveal more proteins). The supernatant with the extracted proteins was removed and set up for dialysis against 100 vol. of 6 M freshly deionized urea containing the upper electrophoresis buffer and 6 mM 2mercaptoethanol, with about six changes over a period of 18 h in 4°C. It should be noted that acetone [10] precipitation of the acetic acid extracted r-proteins ended up with a very sit,nificant loss of L7/L12, the amounts of all other proteins were not affected. The use of freshly prepared r-proteins is highly recommended. Gel electrophoresis. One-dimensional (l-D) electrophoresis [11] was carried out in 4 × 110 mm cylindrical 4% polyacrylamide gels at 0.5 mA per tube. The upper buffer contained per liter: 20.9 g Bistris and 45 ml glacial acetic acid, the pH was adjusted to 4.0. The lower buffer contained per liter: 175.7 g potassium aceate and 49 ml glacial acetic acid, the pH was adjusted to 5.0. Electrophoresis in the second dimension was carried out in 150 x 220 mm 18% polyacrylamide gel containing 3 mm spacers. 0.2 M glycine-acetic acid buffer, pH 4.1, was circulated between the

250

Fig. 1. Ribosome sedimentation profile. Solution of ribosomes adjusted as 1.0 24260 nm units in standard buffer (left panel) and in dissociation buffer: 0.01 M Tris-HCl, pH 7.4, 0.2 M NHaC1, 0.001 M MgCi2 (fight panel). The solutions were subjected to ultracentrifugation in a Beckman Model E ultracentrifuge, equipped with electronic speed control and a photoelectric scanner for ultraviolet optics. The S values are given. The profiles show that our ribosomal preparation is mainly 70S particles together with a small amount of 'native' 30S and 50S subunits. Upon dissociation of the 70S the overall ratio of 30S to 50S subunits is normal, 1 : 2.

tanks. The process took place at 8 ° C at 160 V for 48 h, [12]. Fig. 2 shows the separated 70S r-proteins. Staining and destaining. Staining of the gel was carried out with 0.0375~ ( w / v ) Coomassie Brilliant Blue R-250, Serva Heidelberg (Coomassie R) solution in 14~ ( w / v ) trichloroacetic acid (TCA), freshly prepared from 1~ ( w / v ) ethanolic stock solution of Coomassie R. The volume ratio between dye solution and gel was kept constant - 600 ml dye solution per 100 ml gel. Staining took place at 37 ° C for 24 h in a closed plastic container under very mild shaking. Under these conditions the proteins were very effectively fixed in the gel (unpublished data). Destaining was carried out with 7 ~ ( v / v ) acetic acid and 59~ ( v / v ) methanol at r o o m temperature over a period of several days. Solvent extraction of Coomassie R. For processing the gel, each protein zone was removed by means of a 1 cm diameter brass cork borer. The volume of this piece was 0.3 ml, as the thickness of the gel increased to 4 m m during destaining. In the case of close-lying zone spots, the first spot was punched out, the vacant space filled

251

Fig. 2. 2-D-polyacrylamide gel electrophoresis of r-proteins. 200 pg of ribosomal proteins were subjected to electrophoresis as described under Materials and Methods. In spite of the apparent overlapping of L6 and $5 in the piture, these spots were well separated in the gel.

with a slice from a protein-free area, and the adjoining zone then removed in turn, together with part of the filler. Thus, all slices had the same volume and mass of polyacrylamide gel, thereby ensuring identical extraction and counting conditions

252

(with respect to possible quenching), for all samples. The Coomassie R was extracted from the 0.3 ml gel slice by adding 0.7 ml dimethyl sulfoxide (DMSO), at 37°C for 48 h under mild shaking, sufficient conditions for equilibrium to be reached. The concentration of the extracted dye was then determined from its absorbance at 595 nm in a Zeiss spectrophotometer Model M4 QIII. The background value was also determined for every 2-D gel, and subtracted from all readings [4]. Radioactivity measurement. The gel slices were digested with 1 ml H202 at 76 °C for 24 h. 5 ml of scintillation fluid (21 toluene, 1.21 Triton X-100, 80 mg POPOP and 6.4 g PPO) were added to the digested gel slices, and their radioactivity was determined in a Beckman LS 9800 Liquid Scintillation Counter and expressed in dpm. The background value was subtracted from all readings, as well. Protein concentration. Protein detegmination by Coomassie G binding assay was performed according to Read and Northcote [13], using lysozyme as a standard [5].

Results

Hardy [1] used L1 and $4 as standard proteins, represented by their average molecular weights of 24 350 and 25 500, respectively, and on their basis calculated the stoichiometric values for each r-protein in the ribosome for the minimal and maximal molecular weight value according to Dzionara et al. [3]; the results are listed in his tables. Now that the real molecular weights are available, we used them in recalculating Hardy's data, confining ourselves to the well separated r-proteins, for obvious reasons. The following expressions were used throughout:

Hardy's roinimral X

]V[~OVsumdard(re~) ~lWstandard (average)

stoic.~omctr~ C

~[Wprotein i (real)

value of protein i

MWp~ot~ i (max)

revised S value of protein i

and MWstandard (real)

Hardy's maximal X stoichiometric value of protein i

MWsumdard(awrage)

revised S value of protein i

MWprotm i ( ~ ) MWp~ou~ i (mi~)

where S is the stoichiometric value. The revisions were carried out with the averages used by Hardy for the standard proteins. While most of the pairs were almost identical and their stoichiometric values were close to one, several major discrepancies emerged. The corrected stoichiometric values of L13, L15, L19, L21, L22, L27 were 1.28, 1.37, 1.40, 1.61, 1.27 and 1.43 respectively (Table 1).

253 TABLE 1

Accurate molecular weights and the revised stoichiometric values of r-proteins Dzionara et al. M W values

Gift et al. M W values

Hardy's stoichiometric values

Revised stoichiometric values

standard 0.95 0.69 0.68 0.91 1.00 1.36 0.29 0.82 0.71 1.54 1.01 0.72 1.16 0.54 0.90 0.81 1.03 1.32 0.90 0.99 0.74 1.01 0.69 1.01 0.68 0.73 0.65 0.74 0.71

1.02 0.83 0.90 1.00 1.19 1.75 0.36 0.98 0.94 1.87 1.28 1.00 1.36 0.79 1.05 1.09 1.40 1.61 1.27 1.16 1.16 1.20 1.01 1.43 1.16

0.84

1.02 0.84 0.89 1.00 1.19 1.74 0.32 0.98 0.94 1.98 1.27 0.99 1.37 0.78 1.06 1.09 1.36 1.61 1.26 1.15 1.17 1.19 0.91 1.44 1.16 1.21 1.15 1.24 1.20

0.76 0.78 standard 0.81 0.89 0.85 0.90 0.72 0.63 0.86 0.61 0.74 0.85 0.80 0.66

0.96 1.12

0.78 0.90

0.79 0.90

1.06 1.03

1.01 0.92 1.02 1.02 1.00 0.80 0.90 0.77 1.11 1.12 1.06 0.97

1.01 0.92 0.89 1.03 1.05 0.81 0.91 0.79 1.10 1.12 1.08 1.06

mill

max

L1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 30 32 33

22000 28 000 23 000 25 800 17 500 21000 13 400 19000 19000 13 200 17 800 16 200 17 000 17 900 15 000 14 300 14900 13 900 14 800 12 500 14 300 12 000 12 000 12 000 12 300 12000 10000 10 500 9 000

26 700 31500 27 000 28 500 22000 22 200 15 500 17 300 21000 19 600 15 500 20000 18 500 17 500 22 000 16 700 17 000 17000 14000 17 000 12 700 17 500 12 500 12 500 12 700 15 000 12000 11200 10 500

24 599 29 730 22 258 22087 20171 18 831 12 220 15 696 17 737 14 874 12178 16 019 13 541 14 981 15 296 14 364 12 770 13002 11565 12 227 11013 11185 10 694 9 553 8 993 8 875 7274 6411 6 315 6 255

$2 3 4 5 6 7 8 10 12 13 14 18 19 20 21

24 000 23 000 23 000 18 500 15 500 22 700 15 500 12 400 15 000 14000 14000 10 500 13100 12000 12 200

30 000 33 000 26 700 24000 18 000 26 000 17 600 18 000 19 000 14900 15 600 14 600 15000 14000 13 500

26 613 25 852 23137 17 515 15 704 19 732 13 996 11736 13 606 12968 11191 8 896 10299 9553 8 369

1.07 0.80 0.76 1.14 1.06 1.58 0.32 0.91 0.73 1.71 1.14 0.83 1.19 0.67 1.00 0.96 1.21 1.33 1.04 1.01 0.90 1.06 0.80 1.06 0.83 0.73

1.03 1.10 0.81 0.93 0.70 1.03 0.97 0.95 0.80

1.15 1.22

254

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