The histidines of the bacterial ribosome : A tritium exchange study

BIOCHIMIE, 1982, 64, 363-367.

The histidines of the bacterial ribosome a tritium exchange study. Didier BONNET * *** and Evelyne BEGARD ** *** (Recu te 3-3-1982, acceptd le 24-3-1982). * Museum National d'H&toire Naturelle, Laboratoire de Physico-chimie de l'adaptation biologique, 13, rue P. et M. Curie, 75005 Path.

** U 128 1NSERM, Laboratoire de cryobiologie appliqu£e ?~ l'dtude des m~tabolismes. *** lnstitut de Biologie Physico Chimique, Service de Biospectroscopie, 13, rue P. et M. Curie, 75005 Paris.

R~sum~.

Summary.

L'accessibilit£ des histidines de la sous-unit£ 30S du ribosome d'E. c o l i a ~t~ d~termin~e par l'£tude de l'~change du proton C-2 des histidines dans l'eau triti~e ?~ 37°C. L'absence d'£change gt pH acide a permis la s@aration et l'identification des prot~ines sans perte du rnarquage sur l'histidine. Seules les deux prot~ines ribosomiques $5 et $6 sont marquees de fa~on significative. L' accessibilit~ des histidines est peu rnodifi~e par l'association avec la sous-unit£ 50S.

The accessibility of histidines in the E. coli 30S subunits was assessed by exchange of C-2 histidine protons with tritiated water at 37 ° C. The absence of exchange at acidic pH allowed the separation and identification of individual proteins without loss of histidine labelling. Only the two ribosomal proteins $5 and $6 exhibited significant exchange. No gross change of accessibility was detected in the 70S ribosome couples.

Mots-el~s : r~activit~ de rhistidine / ribosome d'E. coli / ~change de tritium.

Key-words : histidine reactivity / E. coli ribosome / tritium exchange.

Introduction.

While novel progress has been made, the detailed structure of the ribosome and its physiological fluctuations have yet to be resolved. In addition to sophisticated physical techniques that have been used for structural determination, chemical modification has yielded important information on the reactivity and environment of some specific groups within the ribosome. The greatest success has been obtained with the modification of cysteine residues [1-3] ; less progress has been made with lysines [1] and arginines [5]. Correspondence should be sent to Didier Bonnet, Service de biospectroscopie, I.B.P.C., 13, rue P. et M. Curie, 75005 Paris, France.

A method has appeared recently permitting selective modification of histidine residues in a non destructive manner. The method is based on the slow rate of exchange of the C-2 proton of histidine with protons of water [6-7]. To-date the method has been applied only to large quantities of proteins [8]. In this article we show that specific histidine residues in structures as complicated as the bacterial ribosome can easily be studied ; the quantities necessary are in the nanomolar range. Our results show that under several different experimental conditions, only two 30S proteins, $5 and $6 show slow exchange with water protons.

364

D. B o n n e t and E. Begard.

Material

and

Methods.

P.C.S. scintillation cocktail was purchased from Amersham France. 3H-H_.O (specific activity 2 Ci m1-1) was purchased from C.E.A. France. E. coli MRE600 were grown as described [9], in twenty liter batches. All the buffers used were prepared from sterilized stock solutions. 70 S <> ribosomes and ribosomal subunits were purified according to Hershey [10]. Light scattering experiments performed on ribosomal subunits according Io Hui Bon H o a et al. [11] show an association of 92 per cent at 10 mM Mg2 + with a half association magnesium concer~tration of 3.3 mM as can be seen in figure 1.

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513

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radioactive counting and pooled. The above manipulations were per[ormed in a glove-box vented to the external air. Subunit separation of the 70S sample was performed overnight at 40C in a SW27 Beckman rotor (16 hours at 21 000 rpm). The sample was adjusted to a NaC1 concentration of 0.4 M and loaded into a linear 10 per cent30 per cent sucrose gradient in the following buffer: Tris-HC1 pH = 7.5 10 m M ; NH~C1 50 m M ; NaC1 400 mM ; Mg "-+ acetate 10 mM. The 30S peak was pooled after collecting the gradient in 500 ~1 fractions. Protein preparation from the two resulting 30S samples was then performed essentially as described elsewbere [121. 2-D etectrophoresis was peffo~rmed using the acidic system of Madjar [131 with the following minor modifications : the sample buffer was a mixture of anode buffer - 8 M Urea (I : 10 v/v) with 100 m M ~-mercaptoethanol. The first dimension was performed in tubes of 5 mm internal diameter and the second in 2 mm thick, slab gels. Proteins were detected with 0.3 per cent Coomass~e blue G250 in acetic acid-ethanol-water (7:50:43) by staining for 30 min ; the gels were destained wiLth two liters of acetic acid-eth,a'nol-water (5 : 14 : 80). Proteins from E. coli MRE600 ribosomes migrate similarl3r ~ those from K12 except lhat proteins S9-Sll cannot be resolved whereas S15, S16 and S17 are well separated. Protein spots were cut out and extracted twice with 500 M acetic acid 66 per cent [14] ; and the extracts were counted in 10 ml PCS cocktail. Analytical centrifugatiorrs were performed in an SW56 rotor at 4°C. 100 Ixl ribosomes samples (concentration 7.10-7M) were loaded onto linear 10 per c e n t - 3 0 per cen,t sucrose gradients in 10 mM Mg 2+ incubation buffer and centrifuged 105 rain at 45 000 rpm. Gradients were then read at 260 tun on an Aminco spectrometer with a continuous flow cuvette (optic path 5 mm).

10 Results.

FIG. 1. - - Percentage of association ol ribosomal subunits prepared as described in the methods as a ]unction of Mg ,~-+concentration. The curve was obtained from light scattering experiments performed as in [11], assuming a ratio cf scattered intensities from 70S over 30S -b 50S equal to 1.8.

Ribosomal proteins were tabelled with 3H as follows : equal volumes of 30S or 70S <~tight ~ ribosomes and aH-H_~O (specific activity : 2 Ci-m1-1) were mixed at 0°C. Final buffer concentrations were 10 mM Tris-HC1, pH = 8, 50 mM NH~C1 and 7 mM ~-mercaptoethanol ; Mg 2÷ acetate was 2.5 mM or 10 r a M ; the ribosome concentrati~on was 1.5 × 10 -s M. After sealing and mbeing the samples were placed in a solid bath thermostated at 37°C - - a water bath must be avoided because of possible exchange with tritiated water. After 48 hours ribosomes were separated from excess ZH-H20 on small G.25 fine Sephadex columns (70 m m × 6 ram) equilibrated with a buffer containing 10 mM Mg 2+ (Tris-HC1 pH = 7.4 10 mM, NH,C1 50 mM Mg 2+ acetate 10 raM, ~-mercaptoethanol 7 mM). 30S or 70S peaks were detected by BIOCHIMIE, 1982, 64, n ° 5.

Associative capacity. R i b o s o m e s a n d 3 0 S s u b u n i t s f r o m E. coli s t r a i n p r e p a r e d as d e s c r i b e d a b o v e w e r e i n c u b a t e d at 3 7 ° C u n d e r c o n d i t i o n s i d e n t i c a l to t h o s e u s e d f o r t h e t r i t i a t i o n r e a c t i o n b u t in n o n - r a d i o a c t i v e w a t e r . A f t e r 24 a n d 48 h o u r s , a l i q u o t s w e r e w i t h d r a w n a n d t h e i r a s s o c i a t i o n c a p a c i t y tested. T h e y w e r e s u b j e c t e d to a n a l y t i c a l c e n t r i f u g a t i o n s at 10 m M M g -~+. E s s e n t i a l l y n o c h a n g e in the a s s o c i a t i o n p a t t e r n s of 7 0 S o r 30S ( + 5 0 S n o t i n c u b a t e d ) was f o u n d e v e n a f t e r t h e 4 8 h o u r s of i n c u b a t i o n ( d a t a n o t s h o w n ) . T h i s l a t t e r t i m e was s u b s e q u e n t l y c h o s e n f o r the t r i t i a t i o n r e a c t i o n . R a d i o a c t i v e labelling of 3 0 S proteins. T h e p H p r o f i l e f o r C - 2 t r i t i a t i o n is s i m i l a r in s h a p e to the t i t r a t i o n c u r v e of t h e N 1 p r o t o n of t h e i m i d a z o l e r i n g [15]. T h e l a b e l l i n g i n c u b a t i o n s w e r e p e r f o r m e d at p H 8 in o r d e r to f a v o u r t h e

365

Tritium exchange of ribosomal proteins.

TABLE I. N u m b e r o[ histidine residues per 3 0 S protein.

s1 $2 $3 $4 $5 $6 $7 $8 $9 S10

Not known 6 (20) 4 (21) 3 (22) 4 (23) 6 (24) 3 (25) 0 (26) 1 (27) 3 (28)

S11 S12 S13 S14 S15 S16 S17 S18 S19 $20 S2!

3 3 3 not known 4 2 3 1 5 3 1

(29) (30) (31) (32) (33) (34) (35) (36) (37) (38)

The tritiation pattern of individual proteins is shown in figure 2. Table I shows the number of histidine residues present in the ribosomal proteins when known. The chemical specificity of the labelling can be inferred from the absence of radioactivity in protein $8 which has no histidine in its primary structure. Figure 2 shows that the labelling patterns found were similar under the two sets of incubation conditions: 30S and 70S at 10 m M Mg e÷. Only the two proteins $5 and $6 which possess respectively four and six hisfidines were highly

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80¢

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60¢

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400

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3O0

200

200

100

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the 30S

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1

Proteins of the 30S

subunits

F1G. 2. ~ Radioactivity o] separated 30S proteins after 48 hours of incubation in 8H-H~O (final specific activity : 1 Ci ml-O. Buffer used : Tris, HC1 pH = 8 10 m M ; NH~C1 50 mM ; /~-mercaptoethar~oI 7 m M ; Mg~÷ acetate as indicated. (A) 30S su'buni~s alone at 10 or 2.5 mM Mg~°+. (B) 70S complexes at 10 mM Mg2+.

exchange of histidine protons while preserving the ribosomal activity. Afterwards, each step of the protein separation is performed at acidic p H to slow down the loss of tritium caused by reverse exchange. In particular, the proteins are electrophoresed in the 2-D acidic system described by Madjar [13] with the minor changes noted in the methods. BIOCHIM1E, 1982, 64, n ° 5.

labelled. However, small but highly reproducible differences were seen that we consider significant : proteins $6 and S17 were more labelled in 70S ribosomes than isolated 30S subunits. A third incubation condition was also tested: 30S subunits were allowed to exchange at 2.5 m M Mg 2+, a magnesium concentration known to preserve the 30S integrity while not sufficient to per-

366

D. B o n n e t a n d E. Begard.

mit the association with 50S to occur. The labelling pattern under these conditions was superimposable on that at 10 m M Mg ~+ for the 30S ribosomal subunit alone.

Discussion. While nearly all 30S proteins possess one or several histidine residues (see table I) only the two proteins $5 and $6 are labelled under all our experimental conditions. They possess respectively four and six histidines but the radioactivity level attained by these proteins after 48 hours of incubation can well be explained by the exchange of only one accessible residue since the complete exchange of one histidine corresponds to about 1 500 - 2 000 cpm and the half time of exchange of N-acetylhistidine at p H 8 is about 40 hours [6]. This indicates that very few histidines are accessible to water within the 30S structure. Their precise location could be obtained by protein degradation but it does not at present seem useful to proceed to the peptide level since our knowledge of ribosome structure is only at the level of resolution of protein molecules. Incubation of 30S at 2.5 m M Mg "~+ was performed to evaluate the proximity of the water accessible histidines to 16S R N A : 16S neighbouring histidines are under R N A polyelectrolytic influence and undergo pK~ changes by variations of divalent cation concentration. The histidine C-2 exchange being p H sensitive will be in turn modulated by changing [Mg 2÷] [16]. The lack of change observed when the magnesium concentration was varied suggests that no labelled histidine lies in the close vicinity of the 16S R N A . The lack of profound modification in the labelling pattern when the 30S subunits were associated with 50S subunits can be partly explained by the location of the two labelled proteins $5 and $6 obtained by immunoelectron-microscopy [17]: they lie on the opposite sides of the subunit from the contact area between the two subunits and so are probably not affected directly by their association. Because of the chemical mechanism of the exchange reaction the small enhancement of labelling of $6 and S17 may be due to acidic p K , shifts as well as to enhancement of accessibility of the histidine residues in the 70S complex. It should be noted that the same ambiguity exists in the chemical modification experiments of cysteine with N-ethylmaleimide ( N E M ) : only the cysteinate form (RS-) is able to react within the labelling times used because the basic form reacts about 10 ~ times faster than the thiol form (RSH) [18]. BIOCHIM1E, 1982, 64, n ° 5.

No matter which mechanism is involved, it is clear that the changes observed in the labelling reveal some modification of the environment of the residues concerned. In conclusion, C-2 histidine proton exchange endows the 30S subunit with two environmentally sensitive probes on the proteins $5 and $6 that to our knowledge have never been labelled in a selective manner. Since $5 was found labelled in affinity labelling experiments performed with m R N A analogs [19], further studies will be performed with 30S and 70S complexe with messenger and transfer R N A to test the involvement of the histidines in these binding processes.

Acknowledgements. We are indebted to Pr. P. Fromageot (Service de Biochimie C.E.N. ; Saclay) for giving us insights into histidine proton exchange. We wish to thank Pr. P. Douzou, M. Grunberg-Manago and Dr. Hui Bon Hoa [or stimulating discussions and encouragement, and Pr. J. Kornblatt and Dr. R. H. Buckingham ]or critical revision o] the manuscript and Mademoiselle O'Donovan for typing. This research was supported by research grants from the Ddl~gation Gdn~rale ~t Ia Recherche Scientifique et Technique (contrat n ° 81.E.0418) and the lnstitut National de la Santd et de la Recherche Mddicale (Unit~ 128).

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