Eur. J. Biochem. 52, 231-238 (1975)

Structural Studies on the Coat Protein of Alfalfa Mosaic Virus Isolation and Characterization of the Tryptic Peptides and the Alignment of the Cyanogen-Bromide Fragments Gerard M. A. VAN BEYNUM, Barend KRAAL, J. Martien DE GRAAF, and Leendert BOSCH Department of Biochemistry, State University, Leiden (Received November 22, 1974)

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.

Alfalfa mosaic virus is a plant virus with a multipartite genome [l] which is composed of at least four different nucleoproteins. The four nucleoproteins all contain the same species of protein molecules [2]. The coat protein with a molecular weight of about 25000 [3] is the only gene product which can readily be obtained in large amounts. The elucidation of its primary structure is of interest for a number of reasons [4].It will permit a closer comparison between the authentic coat protein and the biosynthetic polypeptides, which are formed in a cell-free system directed by RNA derived from the viral top component a [ 5 ] . Moreover, the remarkable biological activity of the coat protein in the infection process [6] and the singular interaction of this protein with the viral RNA [7] led to an increasing interest in its primary structure. This is also demonstrated by a recent publication concerning limited tryptic digestion of intact alfalfa mosaic virus particles [8]. Under these conditions only a short N-terminal part of the coat protein chain is degraded, accompanied by a remarkable change, however, in its structural and biological functions. The preceding paper in this series appeared in this journal [4]. Abbreviations. AMV, alfalfa mosaic virus; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;CNBr fragments, peptides obtained after cyanogen bromide cleavage of S-carboxymethylated coat protein from alfalfa mosaic virus. Enzymes. Trypsin (EC 3.4.21.4); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3).

Eur. J. Biochem. 52 (1975)

In this paper we describe the isolation and alignment of the four fragments obtained after cyanogen-bromide cleavage of the reduced and carboxymethylated protein. We also describe the isolation and characterization of the tryptic peptides obtained from the intact protein chain as well as from the four CNBr fragments. Amino acid sequence studies and the isolation of overlapping peptides to determine the alignment of all the tryptic peptides are in progress.

EXPERIMENTAL PROCEDURE Materials

Carboxypeptidases A and B (both treated with diisopropylphosphorofluoridate)were products from Sigma. Trypsin (treated with L-1-tosylamido-2-phenylethylchloromethylketone) was from Serva. Polyamide thin-layer sheets (F 1700) were purchased from Schleicher & Schull. Dowex 50WX2 (200-400 mesh) was obtained from Serva and Sephadex from Pharmacia. Whatman 3MM paper was used for electrophoresis and chromatography. Dansyl chloride was from Koch-Light. The pyridine used for the ionexchange buffers was distilled after refluxing with ninhydrin. Hydroxyapatite (DNA grade, Bio-Gel HTP, catalog number 1300530) was from Bio-Rad Laboratories.

AMV Coat Protein: Tryptic Peptides and Alignment of CNBr Fragments

232

Table 1. Amino acid compositions of the tryptic peptides from CNBr fragment I b Compositions are presented as residues per molecule (with the nearest integers in brackets). Purification procedures are indicated as follows: gelfiltration on Sephadex G-25 ( S ) ; ion-exchange chromatography on Dowex 50x2 (D); salt precipitation (P); paper chromatography (C) and highAmino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Akdnine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Tryptophan Lysine Histidine Arginine Number of residues N-terminal residue C-terminal residue

Ti

T1 a

-T2

T2 a

T3

T3a

0.9 (1)

1.0 (1)

T3b

T4 1.0 (1)

2.7 (3) 1.2 (1)

2.9 (3) 1.1 (1)

0.9 (1) 1.1 (1)

2.1 (2) 1.0 (1)

2.1 (2) 1.0 (1)

1.2 (1) 1.3 (1) 0.9 (1)

0.9 (1) 1.4 (1) 1 .o (1)

2.0 (2)

1.3 (1) 0.8 (1)

1.0 (1)

1.7 (2)

3.9 (2)

1.0 (2)

1.8 (2)

1.9 (2)

0.8 (1) 5 blocked LYS

6

blocked n.d.

5 Lys n.d.

4 Ala LYS

7 Ala Arg

6 Ala LYS

1.0 (1)

0.9 (1)

1

8

-

Ser '4%

~

Purification (yield %)

Zsolation,Modzj?cationand Cyanogen-BromideCleavage of Alfalfa Mosaic Virus Coat Protein

The alfalfa mosaic virus strain used was AMV 425 [9]. Preparation, reduction, carboxymethylation and cyanogen-bromide cleavage of the coat protein were performed as described previously [4]. Purification of the CNBr Fragments

The CNBr fragments were separated by Sephadex column chromatography as described previously [4]. In the case of CNBr fragment I1 additional purification was achieved by chromatography on a column of hydroxyapatite (6.5 x 2.5 cm) equilibrated with 0.01 M NazHP04and developed with a linear gradient up to 0.3 M NazHP04. Subsequently a simpler purification method was developed. About 20 mg impure CNBr fragment I1 was suspended in 0.15 ml acetic acid and dissolved in 10 ml HzO. An equal volume of 3 M NaCl was added. The resulting precipitate was thoroughly washed with 1.5 M NaCl, dissolved in 50% (v/v) acetic acid and desalted on a Sephadex G-25 column. The void volume fraction was lyophilized.

Tryptic Digestion

Tryptic digestion was carried out in 0.1 M ammonium bicarbonate buffer, pH 8.9. The protein concentration was 2 mg/ml. Two equal portions of 0.5 (w/v) trypsin solution in 1 mM HCI were added at zero time and after 1.5 h to a final enzyme/substrate ratio of 1 : 100 (w/v). After 3-h reaction at 37 " C , when the solution had become clear, the reaction was stopped by freeze-drying. Occasionally a third portion of enzyme was added after 3 h and the reaction was allowed to proceed for 24 h. Peptide Separation

After each tryptic digestion an analytical peptide map on Whatman 3MM paper was made as described before [8]. For preparative purposes tryptic digests were first subjected to column chromatography. The sample (about 2.0 pmol digest) was fractionated either on Sephadex G-50 (140 x 2.5 cm) in 50 % (v/v) acetic acid or on Dowex 50x2 (60 x 1.5 cm) with a linear gradient from 0.2 M pyridine acetate, pH 3.1, to 4.0 M pyridine acetate, pH 5.6. Column fractions were analyzed by spotting aliquots on Whatman 3MM paper for high-voltage electrophoresis at pH 3.5 and by Eur. J. Biochem. 52 (1975)

G . M. A. van Beynum. B. Kraal. J. M. de Graaf, and L. Bosch

233

voltage paper electrophoresis at pH 3.5 (E). Yields are calculated based on the weight of undigested material. Cysteine was detected as S-carboxymethylcysteine; methionine was detected as homoserine and homoserine lactone, tryptophan was detected by the Ehrlich staining procedure [lo]. The "total" column is the sum of the major peptides T1-TI0 and T l l a . n.d. = not determined ~

TS

T5 a

0.9( 1 ) 3.3 (3)

1.1 (1) 3.0 (3)

1.8 (2)

2.2 (2)

T5b

T6

T7

2.1 (2) 3.8 (4) 4.1 (4) 2.2 (2) 4.0 (4) 3.8 (4) 1.0 (1) 0.8 (1) 2.9 (3)

1.0 (1)

T8

T9

T10

1.2 ( 1 )

1.2 (1)

1.0 (1)

2.1 (2) 0.9 (1) 1.0 (1) 1.4 (1)

2.3 (2)

2.7 (3)

1.8 (2)

5.0(5)

1.8 (2)

1.0 (1) + (1) 1.0 (1)

1.1 (1) 1.0 (1)

1.0 (1)

1.0 ( 1 ) 0.7 (1) 0.9 (1)

11

S-E (24) D-E (12)

10 Ala

1

-

35 Val Arg

Glx n.d.

LYS

S-E (12) D-E (9)

D-E

S-E (9) D-E (21)

S-E D-E-C (1)

-

9

1.7 (2) 1.9 (2) 0.9 (1)

6.3

5

5.1

9

8.9 8.3 9.9 8.4 7.9 1.0 5.1 0.7 2.7 13.1 2.5 5.1 1.o 9.9 0.7 5.1

4 1

0.7 (1)

1.0 (1)

1.0 (1)

1 11 1 5

7 Phe Lys

6 Asx n.d.

9 Tyr n.d.

1 -

D-E (16)

D-E (32)

S-E-C D-E (7)

D-E (8)

1.1 (1)

specific staining procedures [lo]. Eluted peaks were pooled and purified further using preparative highvoltage paper electrophoresis and/or descending paper chromatography. Peptides were located by staining guide strips and were eluted with loo/, (v/v) acetic acid.

CB I b

6

3 12 3 5

2.2 (2) 1.o (1) 1.8 (2)

1.0 (1)

LYS LYS

Total

9 10 9 8 1

1.1 (1) 1.1 (1)

1 .o (1)

2.1 (2)

Tlla

103

101.7 Ac-Ser Met

Amino Acid Analysis

Amino acid compositions of peptides were determined as described previously [4]. Hydrolyses were performed on 20 to 100nmol peptides in 0.2ml 6 N HCl at 110°C for 24 h. Nomenclature of Pep tides

Determination of End Groups

The amino-terminal residues of peptides were determined by the dansyl chloride method as described by Gray and Smith [ll]. Dansyl amino acids were identified by thin-layer chromatography on polyamide sheets (5 x 5 cm) [12] using the solvent systems described by Hartley [13]. Carboxy-terminal amino acids were identified on the amino acid analyzer after hydrolysis with carboxypeptidases A and B according to the method described by Ambler [14]. After incubation at 37°C for 5 h the reaction was stopped by adding an equal volume of 14% (w/v) trichloroacetic acid containing 0.2 M sodium acetate. The centrifuged supernatant was directly applied to the amino acid analyzer. Eur. J. Biochem. 52 (1975)

The cyanogen-bromide fragments were indicated by CB and numbered according to the order of elution after gel filtration. The tryptic peptides from the coat protein are preceded by T and numbered according to their positions in the complete chain (to be published). Peptides derived by further cleavage are indicated by a subscript to the parent peptide. RESULTS .The Products of CNBr Cleavage After CNBr cleavage four major fragments could be isolated with chain lengths of 100, 65, 32 and 28 residues [4]. They were numbered Ib, 11, IIIa and 111b. Generally, fragment I1 still contained some unidentified other peptide material, as was deduced

AMV Coat Protein: Tryptic Peptides and Alignment of CNBr Fragments

234

Table 2. Amino acid compositions of the tryptic peptides from CNBr fragments IIIa and IIIb The explanation of purification symbols and other details can be found in the legend to Table 1. < Glu = pyroglutamic acid. See text for further experimental details Amino acid

Tllb

T12a

Aspartic acid Threonine Serine Glutamic acid Proline GIycine Alanine Cysteine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine

3.1 (3) 4.9 (5) 2.0 (2) 1.1 (1) 0.8 (1)

1.0 (1)

0.5 (1) 0.8 (1)

0.8 (1)

Number of residues N-terminal residue C-terminal residue Purification Yield ( %)

24

8 Ala Met

2.2 (2) 1.0 (1) 1.0 (1) 2.0 (2)

Total 4

5 2 1.0 (1) 1.1 (1) 1.9 (2) 1.0 (1) 0.8 (1)

1.0 (1)

1.0 (1)

1 2

3 3

3 1 1 1

1.8 (2)

CB IIIa 4.0 4.8

2.0 1.1 2.0 3.1 3.1 1.0 3.1

T13a

Total

1.1 (1)

3

CB I l I b 1.1 2.0

2.0 (2)

2

1.0 (1) 1.0 (1)

4 2 3 4

4.1

0.9 (1) 0.8 (1)

1

1

1.1 0.8

1.0 (1)

1

1.1

1.9 (2)

3.3 (3)

S

4.9

0.9 (1) 1.7 (2)

1.0 (1)

1 3

1.o 2.8

28

21 I < Glu Met

3.0 (3) 0.8 (1) 1.0 (1) 2.0 (2)

1.o 1.o

1.1 2.0

2

T12b

1

2.0 1 .o

32

32.4

13

Val Met

< Glu

2.0 (2) 2.0 (2)

1.9 3.0 3.9

~~

Val Arg E 19

E 50

from amino acid analyses and preliminary tryptic peptide maps. Further purification on a column of hydroxyapatite eluted with a sodium phosphate buffer gradient resulted in two peptide peaks (see Annex, Fig. 1). A minor peak emerged at the void volume of the column. Its amino acid composition appeared to be identical with the sum of amino acids from IIIa and 111b. This peptide, therefore, represents an incomplete cleavage product with an internal homoserine residue, having about the same chain length as fragment 11. A major peak emerged at about 0.12 M Na,HPO, giving the amino acid composition of highly purified CNBr fragment 11. Peptide recovery was about 40 %. During concentration of pooled fractions of the latter peak, peptide material precipitated. In this way a new and simpler purification method was worked out (see Experimental Procedure) according to the salting-out principle. The same high quality of fragment I1 was obtained with a yield of about 65

x.

Tryptic Peptides of CNBr Fragment I b

Using the two-dimensional fingerprint method all peptides could be identified separately (Annex, Fig. 2B). To fractionate larger amounts of the tryptic digest Sephadex column chromatography followed by high-voltage paper electrophoresis was used. In this

LYs C 30

1s Phe Met P 30

way most of the peptides were obtained in good yields (Annex, Fig. 2A). However, the peptides T1, T4, T8, T9 and T10 could only be obtained in a pure form using paper chromatography as a third step. Because of the low yields of this three-step procedure the tryptic peptides were separated alternatively by ion-exchange chromatography on a Dowex 50 column followed by high-voltage electrophoresis (Annex, Fig. 2C). The only tryptic peptide that could not be eluted from the Dowex 50 column (even at 4.0 M pyridine buffer) is the strongly basic peptide T3. The amino acid compositions of the tryptic peptides, the isolation procedures used, the N-terminal and C-terminal residues and the yields are given in Table 1. Besides eight peptides and three free amino acids, four couples of individual peptides T1-T1a, T2-T2a, T3-T3a and T5-T5a can be seen there. In each couple the difference between both peptides is only 1 amino acid residue : C-terminal and N-terminal amino acid determination (Table 1) strongly suggest that both peptides of each couple are derived from the same part of the chain. An obvious prediction is the occurrence of a Lys-Lys, an Arg-Lys and a Lys-Arg sequence in these parts of the amino acid chain (see Discussion). Taking these couples into account the sum of the individual amino acid compositions of the tryptic peptides agrees within the experimental error with the previously published chain composition of Eur. J. Biochem. 52 (1975)

G. M. A. van Beynum, B. Kraal, J. M. de Graaf, and L. Bosch

235

Table 3. Amino acid compositions of the tryptic peptides from CNBr fragment 11 The explanation of purification symbols and other details can be found in the legend to Table 1. The “total” column is the sum of the major peptides T13b, T14, T15, T16, T17 and T18 Amino acid

T13b

T14

T14b

T15

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine

2.1 (2)

3.1 (3)

1.0 (1)

1.2 (1)

3.2(3) 2.1 (2) 2.2 (2) 1.3 (1) 2.8 (3) 0.9 (1) 1.0 (1)

1.1 ( I )

~

~~

2.2 (2)

0.9 (1) 0.9 (1)

1.1 (1) 0.5 (1) 0.9 (1) .

~

Number of residues N-terminal residue C-terminal residue Purification Yield (%)

1.9 (2) 0.8 (1) 1.9 (2)

6

< Glu LYS S-E 17

24 His Arg

S 48

~

T16a

T16b

T17

2.2 (2) 0.9 (1)

1.0 (1)

1.2 (1)

1.1 (1)

0.6 (1)

1.1 (1) 1.1 (1) 0.7 (1)

0.9 (1)

1.1 (1) _

5 Asx Arg S-E 6

_

0.7 (1) + (1)

0.9 (1)

0.9 (1)

1.1 (1)

. _

0.8 (1)

Tryptic Peptides of CNBr Fragment IIIa

The tryptic peptides were separated by means of high-voltage paper electrophoresis (see Annex, Fig. 3). In this way the two expected fragments could be isolated in good yields. The results from amino acid analyses and endgroup determinations of these peptides are presented in Table 2. They are in complete accordance with the data known for CNBr fragment IIIa [4]. From the terminal amino acids T11 b can easily be recognized as the N-terminal peptide and T12a as the C-terminal one. Tryptic Peptides of CNBr Fragment IIIb

After tryptic digestion the incubation mixture was concentrated by rotary evaporation. During this procedure a crystalline peptide fraction precipitated. In one experiment the total mixture was

4.3 (4) 1.o (1) 0.6 (1) 1.2 (1)

10 1 4 7 4 3 6 1 4

9.6 0.9 4.2 6.8 4.2 3.1 6.1 1.1 4.2

1 6 1 6 1 3 2

0.8 6.1 1.1 5.6 0.8 3.2 1.5

5

5.2

1.1 (1) 3.4 (3)

5 LYS Arg S-E 8

0.8 (1)

0.8 (1) 1.0 (1) 1.0 (1) 1 S-E 9

4 Ala Arg S-E-C 5

0.8 (1) 0.8 (1)

0.9 (1) ~.

-.

~~

8 Phe Arg S-E 26

Total

1.0 (1) 1.2 (1)

5 Ala Arg

.

S-E-C 10

CB I1

T18

1.3 (1) 1.1 (1)

2.1 (2)

CNBr fragment I b [4].End-group determination by the dansyl chloride method failed in only one case: T1 (and T1 a). Therefore, this peptide is most probably the N-terminal one blocked with N-acetylserine [4]. The isolation of free arginine and free lysine supports the conclusion that some clusters of basic residues are present. The isolation of free homoserine suggests that the penultimate residue is arginine or lysine.

Eur. J. Biochem. 52 (1975)

T16

~

17 Ser His S-E 25

- __ 65 64.5 < Glu His P 65

completely evaporated. In another experiment the crystalline fraction was isolated, washed with 0.3 M ammonium bicarbonate buffer and analyzed. Preliminary attempts to separate the digest mixture by high-voltage electrophoresis did not succeed owing to the slow mobility and poor solubility of the peptides in the system employed. However, satisfactory results were obtained using descending paper chromatography (see Annex, Fig. 4). Some minor spots were also isolated, but were too small for unequivocal amino acid analyses. The crystalline fraction appeared to be chromatographically pure and identical with T13 a. The results from amino acid analyses and end-group determinations of the two fragments are presented in Table 2. They are in complete accordance with the data known for CNBr fragment I11b [4]. As was the case with CNBr fragment I11b the N-terminus of T12b could not be detected, probably due to a blockage by intramolecular cyclization of an N-terminal glutamine. In a renewed attempt, T12b was therefore subjected to a limited alkaline treatment (1 N NaOH at 100“Cfor 10 min) for selectivecleavage of pyrolidone rings in pyroglutamyl peptides [15]. This time after dansylation dansyl-glutamic acid was detected together with dansyl-glycine. Although nonspecific cleavage during the alkaline treatment cannot be excluded completely, glutamine is tentatively positioned at the N-terminus of T12b.

AMV Coat Protein: Tryptic Peptides and Alignment of CNBr Fragments

236

Table 4. Amino acid compositions of the methionine-containing tryptic peptides from AMV coat protein The compositions of methionine-containing tryptic peptides are compared with the best-fitting couples of terminal tryptic peptides derived from the CNBr fragments. The explanation of purification symbols can be found in the legend to Table 1. For further experimental see text Amino acid

T11

Compare with T l l a Ti1 b

T12

Aspartic acid Threonine Serine Glutamic acid Proline GIycine Alanine Cysteine Valine Mcthionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine

3.2 5.0 2.2 1.3 0.9 2.4 1.2 0.5 2.0 1.o 1.2 1.1

3 5 2

2.0

2

2.4 1.x

2 2

1

3.2 2.2 2.1 3.8

3 2 2 4

3.2 1.1 2.2 2.3

3 1

1.0 1.o

1 1

0.7 0.8

1 1

1.3

1

2.0

2

0.4 0.7

1 1

Number of residues Purification Yield (%) Overlapping peptide for :

25.1

+

1 2 1 1 2 1 3 1

25 S-E-C 4

CB Ib-Cb IIIa

Compare with T12a + T12b

T13

Compare with T13a T13b

+

2 2

1.9

2

3.6

4

1.o 2.9

1 3

1.1 0.5

1 1

21.1

21 S-E 10

21.0

CB IIIa-CB IIIb

In conclusion, one can predict that an overlapping tryptic peptide between CNBr fragment I11 a and CNBr fragment I11 b will either contain 21 residues or 30 residues depending on the mutual sequence of these two CNBr fragments. Tryptic Peptides qf CNBr Fragment 11

An analytical peptide map of CNBr fragment IT is drawn in Annex, Fig. 5 B. Peptides were fractionated on a preparative scale by the use of Sephadex column chromatography (see Annex, Fig. 5 A). The pooled fractions were subjected to preparative high-voltage paper electrophoresis and in this way nearly all the peptides could be isolated in a pure form. Peptides T16b and TI7 have about the same molecular size and electrophoretic mobility. They could be separated, however, by preparative paper chromatography using the system isoamyl alcohol pyridine - water (35 :35 :30, by vol.). Details about yields, amino acid compositions and end groups can be found in Table 3. The sum of amino acids from T13b and the other major peptides T14-Tl8 is in complete accordance with the amino acid composition of CNBr fragment 11. The minor peptides T14b,

21 S-E-C 5

CB IIIb-CB 11

T16a and TI6 b should therefore be accommodated in some of these major ones. T14b can only be located in the C-terminal part of T14. A corresponding N-terminal T14a subfragment could not be isolated in sufficient yield and purity to allow for its entering in Table 3. The composition of T16b is very similar to that of T16, one lysine being absent. This Nterminal lysine can be found as the free amino acid T16a. Peptide T13b was the only peptide that did not yield an N-terminal dansyl-amino acid; one could not be found for CNBr fragment IT either. Most probably this will be due to an N-terminal pyroglutamic acid residue. Because of C-terminal histidine peptide T18 can easily be recognized as the C-terminal tryptic peptide. Methionine- Containing Tryptic Pep tides of the Carboxymethylated A M V Coat Protein

A tryptic digest of carboxymethylated coat protein was separated on a Sephadex G-50 column (see Annex, Fig.6). Fractions were pooled and analyzed as indicated. All methionine-containing peptides appeared to be present in fraction Ill. A fingerprint of the pepEur. J. Biochem. 52 (1975)

G. M. A. van Beynum, B. Kraal, J. M. de Graaf, and L. Bosch

237

.

Tllb

=

T11-

Tlla -I

.

T1

- T10

T l Z a T12b

-

-_

112-

T13a T 1 3 b -.

T13-T

1 4 - T18-

Fig. 1 . Alignment of’ the CNBr fragments and distribution of’ the trypticpeptideles along the protein chuin

tides from this fraction is shown in Annex, Fig. 7. Peptides T11, T12 and T13 do contain methionine, as appears from the amino acid compositions in Table 4. After acid hydrolysis the amino acid analyses of peptides T12’ and T12” turned out to be identical with T12. Conversion of methionine to its sulfoxide/. sulfone, or partial deamidation may be responsible for this phenomenon. The Alignment of the CNBr Fragments of AMV Coat Protein

Fragment CB I b is located at the N-terminus and fragment CB I1 at the C-terminus of the protein chain [4]. Considering the amino acid compositions of peptide T l l a (at the C-terminus of CB Ib) and T13b (at the N-terminus of CB 11) together with the four tryptic peptides derived from CB IIIa and CB lIIb, two possible sets of overlapping methionine-containing peptides can be conceived depending on the order of CB IIIa and CB IIIb. As is obvious from Table 4 only one possibility fits the values found. Therefore the alignment of the CNBr fragments can be written as CB Ib-CB IIIa-CB IIIb-CB 11. In Fig.1 the location of the CNBr fragments is visualized together with all the tryptic peptides described.

DISCUSSION In this paper the alignment of the four CNBr fragments of AMV coat protein is presented (Fig. 1) together with the amino acid compositions and end groups of all the tryptic peptides (Tables 1-4). The data given are generally in good accordance with the known specificity of trypsin. Tryptic digestion of CNBr fragment I b yielded six peptides containing two or three basic residues together with free arginine and free lysine. The presence of two lysyl-residues in the peptides T3 a and T5 a may be explained by a trypsin-resistent Lys-Pro sequence. Since the peptides T1, T2a, T3 and T5 do not contain a prolyl-residue their existence must be explained by a Lys-Lys or a Lys-Arg sequence. The Eur. J. Biochem. 52 (1975)

isolation of free arginine (T3 b) and free lysine (T5 b) supports the occurrence of these sequences. Bol et al. [8] observed the release of eight tryptic peptides from the N-terminal side of the coat protein after limited tryptic digestion of intact alfalfa mosaic virus particles. Tryptic peptide maps from native and modified AMV coat protein were compared showing T1, T l a , T2, T2a, T3, T3a, T3b and T4 to be the released peptides. Comparison of the amino acid compositions of both proteins showed a removal of about 27 amino acids. These two observations can only be brought into agreement if some Lys-Lys or Lys-Arg sequences are present in this part of the protein chain. The clusters also explain the couples of peptides differing in only one amino acid and the presence of free arginine and free lysine. They underline once more the strongly basic character of the N-terminal part of the coat protein which may play an important role in the protein-RNA interaction as mentioned before [8]. The isolation of basic peptides by ion-exchange chromatography on Dowex 50 only can be rather misleading since T3 is not eluted (Annex, Fig. 2C). This peptide contains one arginyl and two lysyl residues and evidently binds too tightly to the column to be eluted even by a 4 M pyridine buffer. As can be seen from the Tables2 and 3 the Nterminal residues from both CB IIIb and CB I1 are most likely pyroglutamic acid. The two Met-Gln sequencesin the protein chain must have been modified to homoserine lactone and pyroglutamic acid by the strong acid conditions of the cyanogen bromide reaction [4]. Together the tryptic peptides account for 228 residues. Within experimental error this is in good agreement with earlier reports from this laboratory [3,41ANNEX The following documents have been deposited at the Archives Originales du Centre de Documentation du C.N.R.S. (F-75971 Paris-Cedex-20, France) where they may be ordered as microfiche or photocopies.

238

G . M. A. van Beynum et al.: AMV Coat Protein: Tryptic Peptides and Alignment of CNBr Fragments

Reference No. : A. 0. 542. Annex, Fig. 1. Final purijication of a CNBr fragment IIpreparation on a column of hydroxyapatite. Annex, Fig. 2. Separation of the tryptic peptides from CNBr ,fragment Ih. In a first step separation was obtained by: (A) Sephadex column chromatography or (B) descending paper chromatography or (C) ion-exchange column chromatography. In each case the second step was high-voltage paper electrophoresis. Annex, Fig. 3. Separation of the tryptic peptides ,from CNBr fragment IIIa using high-voltage paper electrophoresis at p H 3.5. Annex, Fig.4. Separation of the tryptic peptides from CNBr fragment I I I b using descending paper chromatography. Annex, Fig. 5. Separation of the tryptic peptides ,from CNBr fragment II. In a first step separation was obtained by: (A) descending paper chromatography or (B) Sephadex column chromatography. In both cases the second step was high-voltage paper electrophoresis. Annex, Fig. 6. Separation of the methionine-containing tryptic peptides from carboxymethylated A M V coat protein on a Sephadex G-50 column. Annex, Fig. 7. Peptide map of the tryptic peptides from fraction III in Annex, Fig. 6. We wish to thank Dr E. M. J. Jaspars and coworkers for their interest and for providing us with purified virus preparations and Mr P. Moonen for technical assistance.

REFERENCES 1. VanVloten-Doting, L., Kruseman, J. & Jaspars, E. M. J. (1968) Virology, 34,728 - 737. 2. Moed, J. R. & Veldstra, H. (1968) Virology, 36,459-466. 3. Kruseman, J., Kraal, B., Jaspars, E. M. J., Bol, J. F., Brederode, F. Th. & Veldstra, H. (1971) Biochemistry, 10, 447454. 4. Kraal, B., De Graaf, J . M., Bakker, T. A,, Van Beynum, G. M. A., Goedhart, M. & Bosch, L. (1972) Eur. J . Biochem. 28,20-29. 5 . Van Ravenswaay Claasen, J.C., Van Leeuwen, A. B. J., Duijts, G. A. H. & Bosch, L. (1967) J . Mol. Biol. 23, 535-544. 6 . Bol, J. F., Van Vloten-Doting, L. & Jaspars, E. M. J. (1971) Virology, 46,73-85. 7. Van Vloten-Doting, L. & Jaspars, E. M. J. (1972) Virology, 48,699 - 708. 8. Bol, J . F., Kraal, B. & Brederode, F. Th. (1974) Virology, 58, 101 - 110. 9. Hagedorn, D. J. & Hanson, E. W. (1963) Phytopathology, 53. 188- 192. 10. Easly, C. W., Zegers, B. J. M. & De-Vijlder, M. (1969) Biochim. Biophys. Acta, 175, 211-213. 11. Gray, W. R. & Smith, J. F. (1970) Anal. Biochem. 33, 36-42. 12. Woods, K. R. & Wang, K. T.(1967) Biochim. Biophys. Acta, 133, 369- 370. 13. Hartley, B. S. (1970) Biochem. J. 119,805-822. 14. Ambler, R. P. (1972) Methods Enzymol. 25, 262-272. 15. Erickson, R. P. & Steers, E. (1969) Biochem. Biophys. Res. Commun. 37.736- 743.

G. M. A. van Beynum, B. Kraal, J . M. de Graaf, and L. Bosch, Biochemisch Laboratorium, Rijksuniversiteit Leiden, Wassenaarseweg 64, Leiden, The Netherlands

Eur. J. Biochem. 52 (1975)