Volume 60, number 1

FEBS LETTERS

THE PRIMARY STRUCTURE OF PROTEIN L30 FROM ESCHERICHIA

December 1975

COLI RIBOSOMES

Eberhard RITTER and Brigitte WlTTMANN-LIEBOLD Max-Planck-lnstitut fiir Molekulare Genetik, Berlin-Dahlem, Germany

Received 16 October 1975

1. Introduction

Protein L30 from the 50S ribosomal subunit of E. coli is located near the GTPase-center of the

particle, as shown by studies with a GDP photoaffinity label [1] and by the presence of L30, although in Small amount, in the 5S RNA-protein complex [2], which has GTPase and ATPase activity [3]. A knowledge of the primary structure may allow further insight into the role of this protein.

2. Materials and methods The protein L30 was isolated from E. coli according to procedures previously described [4]. About 2/amol of protein were digested by trypsin. The trypsin peptides were separated either on a SE-cellulose column [5] followed by chromatography ori a column (0.2 X 5 cm) of Dowex M71 [6] or on Sephadex G-15 followed by a small SE-cellulose column (0.3 × 30 cm). Amino acid analyses were performed using either a Beckman Multichrom 4255 or a Durrum D-500 analyzer. The intact protein was sequenced according to Edman and Begg [7] using an improved Beckman protein sequencer [8]. The sequences of the tryptic peptides were determined by the Dansyl-Edman technique [9]. It was possible to discriminate between Asp/Asn and Glu/Gln by electrophoresis of the tryptic peptides [10] or by thin-layer chromatography of the PTH-amino acids. In order to obtain overlapping peptides the lysine residues were blocked by ETPA (exo-cis-3,6-endoxo4-tetra-hydrophthalic acid anhydride) [ 11 ] and the modified protein was digested by trypsin, in which case cleavage occurred after the arginine residues only. North-Holland Publishing Company - Amsterdam

After deblocking, these peptides were purified by chromatography on Dowex M71 or by the fmgerprint technique. In addition, the protein was cleaved by thermolysin, chymotrypsin, Staphylococcus protease [12] and with CNBr [13]. The resulting peptides were separated by the fingerprint technique. Full details will be given later.

3. Results and discussion

The primary structure of protein L30 is shown in fig. 1. It has the composition Asp~ ASnl Thr6 Ser2 Glu4 Glnl Pro2 Glys Alas Val4 Met2 Ile6 Leus Phel His2 Lyss Arg6. This is in very good agreement with the results of the total hydrolysis. Cysteine and tryptophan are absent, which was confirmed by the appropriate methods for their detection [ 14,15]. Protein L30 contains 58 amino acids and its mol. wt is 6410. The sequence up to position 23 was determined by the sequenator and was completed by isolation and analysis of all tryptic peptides. Cleavage with trypsin resulted in 14 peptides, two of which (T1 and T2) contained no arginine or lysine. The determination of the sequences of the peptides T2 and T11 showed that they were part of the peptides T3. The cleavage after phenylalanine in position 52 was occasionally observed after digestion with trypsin or with Staphylococcus protease. The tryptic peptides were aligned using the information obtained by the analyses of the peptides isolated after cleavage with other enzymes and with CNBr (fig. 1). All the peptides found are in complete agreement with the given sequence. The sequence of the C-terminal region was confirmed by sequencing 153

Volume 60, number 1

FEBS LETTERS 5

December 1975

10

15

N H 2 - A I o - L y s - T h r - I te - L y s - I l e - T h r - G I n - T h r - A r g - S e r - A I o - I Tryp

I-~T12.,.-,,~

ETPA

I,

Ther

I

=

T10

I l,

T7

I I

E1 Thl

I

h2.~l

T8

I

SP

=

CNBr

J

I I

I

"r,,,.o T h 3

L

T9

i [

"" I I

Chl

Lys -

I ~T1;--.4

I

Th&b

Chym

20

le- Gly-Arg-Leu-Pro-Lys-His-

Th5b

ThSc

4 I

Ch2 Ch3 SP1

N 1 - -

Sequ.

25

30

35

,'0

A l o - T h r - L e u - L e u - G l y - L e u - G l y - L e u - A r g - A r g - I le - G l y - H i s - T h r - V a l - Glu -Arg-Glu - A s p - T h r Tryp

I

ETPA

~

Ther

T5 E

I ~-T6-~ ~,

2

T13

4

4

E3

~--Th6--~ I

1

Chym

I I

Th7

Th8

I

I I

Thg

'

Ch5

-

-

,I

SP

CNBr Sequ.

/.5

50

55

P r o - A I o - I l e - A r g - G l y - M e t - I l e - A s n - A I o -VQ I - S e r - P b e - M e t - V g l - L y s - Vo I - Glu - Glu - COO H Tryp

--'Tt,

~

ETPA

4

Ther

J t

T2

I =

Thl0

Tll

I

T1

I

E&

F

I t

Th11

T h 12Cl - - - ' ~ I I ~Th12--~ ~Th13--~

I~Thl/*.~l

T h IL, o I

Th15

/

/

I

Chym

Ch6

I I

Ch7

I

SP2o

$P

Thl&b.

SP2

CNBr

I I

N2

I

I

N3

Fig.1. Amino acid sequence o f protein L30 from E. coli ribosomes. Tryp = tryptic peptides. Ther = thermolysin peptides. Chym = chymotryptic peptides. ETPA = tryptic peptides after blocking the lysines with ETPA. SP = peptides from digestion with Staphylococcus protease. CNBr = peptides from cleavage with CNBr. Sequ = sequence region elucidated by the sequenator.

L30

~8 20 ~ Lys-His-Lys-AIo ~- Thr-Leu-Leu 70

35

77

39 &2 ~- Lys-His-Lys-A,o+

Fig.2. Comparison o f homologous regions of the primary structures o f proteins L7/L12, LI 3 and L30. 154

41 Arg-Glu-Asp-ThrT ~ ~ 91

~7

VO,- Arg-Gly-AIo -Thr+ Gly - Leu-Gly- LeuJj-Lys-Glu -AIQ-Lys -Asp-L e u ~

LT/L12 L13

25

I Gly-Leu-Gly-Leu+ Arg-Arg-lle - Gly- His-Thr ~

Ser-AIo

Volume 60, number 1

FEBS LETTERS

the overlapping peptide E4. After modification of the lysine residues by ETPA, only partial cleavage after arginine in position 37 with trypsin was observed, in contrast to tryptic digestion o f the native protein in which cleavage at this point was complete. There are some similarities in the primary structure of the proteins L7/L12, L13 en L30 from the 50S subunit as illustrated in fig.2. Several homologous regions among E. coli ribosomal proteins occur much more frequently than can be expected on a random basis [16]. Helical and/3-sheet regions were predicted according to the method o f Chou and Fasman [17,18]. The protein. is proposed to contain 31% a-helix (in the regions 1 7 - 2 6 and 3 3 - 4 0 ) and 43%/3-sheet (from the Nterminus to position 10 and position 4 2 - 5 6 . However, a-helix and/3-sheet structure cannot be unequivocally assigned to certain regions o f the protein, because they contain both high a-helix and/3-sheet forming potential.

December 1975

References [1] Maassen, J. A., MiSller,W. (1974) Proc. Nat. Acad. Sci. USA 71, 1277. [2] Home, J. R., Erdmann, V. A. (1972) Mol. gen. Genet. 119, 337. [3] Home, J. R., Erdmann, V. A. (1973) Proc. Nat. Acad. Sci. USA 70, 2870. [4] Hindennach, J., Kaltschmidt, E., Wittmann, H. G. (1971) Eur. J. Biochem. 23, 12. [5] Wittmann-Liebold, B. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352, 1705. [6] Chen, R., unpublished. [7] Edman, P., Begg, G. (1967) Eur. J. Biochem. 1, 80 [8] Wittmann-Liebold, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1415. [9] Gray, W. R., Hartley, B. S. (1963) Biochem. J. 89, 379. [10] Offord, R. E. (1966) Nature 211,591. [11] Riley, M., Perham, R. N. (1970) Biochem. J. 118, 733. [12] Hommard, J., Drapeau, G. R. (1972) Proc. Nat. Acad. Sci. USA 69, 3506. [13] Gross, E., Witkop, B. (1962) J. Biol. Chem. 237, 1856. [14] Hits, C. H. W. (1967) in: Methods in Enzymology vol. 11 (C; H. W. Hirs, ed.), p. 59. [15] Liu, T. Y., Chang, Y. H. (1971) J. Biol. Chem. 246, 2842. [16] Wittmann-Liebold, B., Dzionara, M., manuscript in preparation. [17] Chou, P. Y., Fasman, G. D. (1974) Biochem. 13, 211. [18] Chou, P. Y., Fasman, G. D. (1974) Biochem. 13, 222.

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