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Characterization of a new defective strain of TMV
VIROLOGY
37,203-208
(1969)
Characterization
of a New
Defective
V. HARIHARASUBRAMANIAX Department
of Agricullural
Biochemistry,
AND
University
Strain
ALBERT
of Arizona,
of TMV’
SIEGEL Tucson, Arizona
85781
Accepted September 30, 1968 A new nitrous acid-induced defective strain of tobacco mosaic virus (TMV) designated PM5 was isolated and some of its characteristics were studied. The coat protein of this defective strain showed a change from arginine to cysteine at position 112 in the amino acid sequence of the protein. The protein aggregated into rodlike structures simila,r to aggregated TMV protein but the subunits seem to be more loosely packed with a tendency to stacked disk configuration. MATERIALS
INTRODUCTION
The isolation
of TMV has been reported earlier (Siegel et al., 1962). The coat proteins synthesized in plants infectred by these strains are defective and hence unable to copolymerize with viral nucleic acid and form stable nucleoprotein rods. This defect seems to result from a change or changes at a crucial region in the amino acid sequence of the protein subunit. Thus, in the defective strain PM2 there is a change from threonine to isoleucine and glutamic to aspartic acid at positions 28 and 95, respectively (Wittmann, 1965), resulting in a protein which packs in open helices rather than the tightly closed helices of the type strain of TMV (Zaitlin and Ferris, 1964; Siegel et al., 1966). Using similar techniques, we have isolated a new defective strain (PN5) and have established its defective nature as before (Siegel et al., 1962). This paper describes the purification, electron microscopy and chemical characterization of the coat protein of this strain. A preliminary report has been published earlier (Hariharasubramanian and Siegel, 1967). induced
AND
METHODS
Purification of the coat protein. The infected leaf tissue w-as deribbed and ground in an Omnimixer with 3 volumes of M/15 phosphate buffer containing 0.01 M EDTA (ethylenediaminetetraacetic acid) and 0.001 at pH 7.0. The M mercaptoethanol grindate was strained through cheesecloth and centrifuged at 12,000 9 for 10 min in a Sorvall refrigerated centrifuge. The supernatant was centrifuged for 60 min at 105,000 9 in a Spino L2 ultracentrifuge. The pellet was discarded and the supernatant was adjusted t’o pH 5.1 and kept cold overnight. It was then clarified at 12,000 y and the supernat,ant was centrifuged at 105,000 g as before. The resultant pellet was suspended in a solution containing 0.001 dd each of EDTA and DIECA (diethyldithiocarbamate) adjusted to pH 7.5 with NaOH. After clarification at 105,000 g the supernatant was brought to pH 5.0, subjected to low speed centrifugation at 12,000 n for 10 min, followed by ultracentrifugation at 105,000 g for 60 min. The high speed pellet was resuspended, clarified, and sedimented at pH 5.0, and the process was repeated until a clear pale yellow pellet was obtained. This pellet was resuspended in suitable solutions and used as purified virus protein. Electron microscopy. All electron microscopic observations were made with a Philips EM200 microscope at 80 kV and using a
of defective strains by nitrous acid treatment
1 This work was supported in part by grants from the National Science Foundation and by Atomic Energy Commission Contract AT(ll-l)873. University of Arizona Agricultural Experiment Station Technical Paper No. 1376. 203
204
HARIHARASUBRAMANIAN
liquid nitrogen-cooled cold stage. The microscope was calibrated by taking a series of pictures of a carbon grating of known mesh size at different magnifications. Carboncoated copper grids were prepared by floating off a thin film of carbon deposited on cleaved mica and picking up the resultant film on copper grids of 400-mesh size. The specimens for electron microscopy were prepared by placing a drop of freshly prepared uranyl formate (Leberman, 1965) on the grid followed by a drop of the protein solution placed on top of the stain. The excess liquid was blotted off and the specimen grids were dried at room temperaturebefore examination. The protein solutions for electron microscopy were usually adjusted to pH 5.0 before being placed on the stain. Sometimes uranyl acetate was used instead of uranyl formate to stain the preparation. Electron microscopic observations were also carried out on material deposited from epidermal strips (Hitchborn and Hills, 1965) in order to look for structures similar to those described by Siegel et al. (1966) in PM2-infected plants. Amino acid composition. The purified protein was hydrolyzed in vacua in sealed tubes at 108” in 6 N HCl for 24 hours and 72 hours, and the amino acid composition determined as described by Zait,lin and McCaughey (1965). Tryptophan was determined by the calorimetric procedure of Spies and Chambers (1948). Tryptic diqestion of protein. Trypsin prepared in the presence of the chymotryptic inhibitor diphenyl carbamyl chloride (Erlanger et al., 1966) was obtained from Seravac Laboratories, Berkshire, England. Digestion was carried out for 3 hours at 37” at a pH of 7.8 and a trypsin to protein ratio of 1: 100. At the end of the digestion period the pH of the reaction mixture was adjusted to 4.6 with 1 N acetic acid. The mixture was allowed to stand for 30 min in an ice bath, and was then centrifuged at 12,000 q. The precipitate was dissolved in water at pH 7.0 and purified further by repeated isoelectric precipitation (Tsugita et al., 1960). The portion soluble at pH 4.6 was lyophilized and used for peptide mapping by ion exchange chromatography and paper chromatography.
AND
SIEGEL
Ion exchange chromatography. The procedure was similar to that of Funatsu (1964). The pH 4.6 insoluble fraction of the protein tryptic digest was purified by repeated isoelectric precipitation and was used as such for determination of amino acid composition. This constitutes the “I” peptide of the protein. The pH 4.6 soluble fraction after lyophilization was taken up in a small volume of pH 8.8 pyridine-collidine-acetic acid buffer and centrifuged at 12,000 g. The insoluble pellet proved to consist essentially of peptide “10” when it was purified by gel filtration in Sephadex G-25 (Pharmacia Ltd.). The supernatant was subjected to ion exchange chromatography on a Dowex 1 X 2 column (0.9 X 150 cm) in the acetat’e form as described by Funatsu (1964). The flow rate was maintained at 40 ml an hour by using a minipump (Buchler instruments), and 3.3 ml of the eluate were collected per tube. From each tube 0.2 ml was dried down in test tubes in a vacuum oven at 50” and analyzed for peptides by the Folin-Lowry method (Lowry et al., 1951). The contents of the tubes under each peak were pooled and analyzed directly for amino acid composition or were further purified by paper chromatography before analysis. Paper chromatography. Peptides to be purified further were chromatographed on Whatman No. 3 MM paper by descending chromatography in n butanol-acet,ic acidwater-pyridine : 30 : 6 : 24 : 20 v/v (Waley and Watson, 1953). This procedure was also employed when the pH 4.6 soluble peptides were directly applied on paper and chromatographed without resorting t’o prior ion exchange chromatography. The peptides were located by cutting matching strips and developing with ninhydrin or hypochloritestarch-potassium iodide (Pan and Dutcher, 1958). The peptides from the matching, unstained portions were eluted from the paper with 1.0 % ammonium hydroxide or 0.2 N acet,ic acid (Funatsu and Funatsu, 1967). Aminoethylation of PM5 protein. The method was essentially that of Tsung and Fraenkel-Conrat (1966). 45-50 mg of protein were taken up in 6 ml of a solution containing 8 Al urea, 0.025 M mercaptoethanol,
NEW DEFECTIVE
FIG. 1. Electron micrograph
205
OF TMV
of aggregated PM5 protein stained with uranyl formate.
and 0.4 M Tris [tris(hydroxymethyl)aminomethane]-HCl buffer at pH 8.6. The reaction mixture was maintained at 23-25” under nitrogen for 2 hours. Ethylenimine (Rfatheson, Coleman and Bell) (0.05 ml) was added 10 times at 5-min intervals while the pH was maintained at 8.69.0 by dropwise addition of 2 N acetic acid in 8 M urea. The react,ion was allowed to proceed for 1 hour after the final addition of ethylenimine. The reaction mixture was diluted with an equal volume of water and dialyzed for 2 days in the cold (4”) with repeated changes of distilled water. The aminoethylated protein which precipitated on dialysis was sedimented at 12,000 g for 10 min and mashed with distilled water and resedimented again. This was repeated twice, and the final pellet was suspended in 0.001 N NaOH for tryptic digestion. A similar amino-
0.01 M EDTA,
STRAIN
ethylation was carried out using the protein of the common strain of TMV. Tryptic digestionof aminoethylated protein. The time of enzymatic hydrolysis by trypsin was extended to 6 hours since digestion of aminoethylated cysteine is rather slow (Tsung and Fraenkel-Conrat, 1966). During digestion, the insoluble aminoethylated protein dissolved almost completely. The pH of the hydrolyzate was adjusted to 4.6, and the insoluble fraction was sedimented and purified as before. The soluble fraction was lyophilized and the peptides separated by techniques described earlier. RESULTS
An electron micrograph of the PM5 coat protein aggregated at pH 5.1 is shown in Fig. 1. The aggregated protein seems to somewhat resemble aggregated TMV pro-
206
HARIHARASUBRAMANIAN TABLE
AMINO
ACID
Amino acid residue ASP Thr Ser Glu Pro GUY Ala CYS Val Ile Leu Tyr Try Phe Arg LYS
I
COMPOSITION
OF PM5
PROTEIN”
Moles amino acid per mole protein 24.hour hydrolysis
72.hour hydrolysis
18.00 15.1 14.8 16.0 7.8 6.0 13.6 1.6d 12.8 7.8 12.0 3.9
18.1 14.1 12.7 16.0 8.1 6.4 13.8 14.1 8.7 12.1 3.4
8.4 9.9 2.0
8.0 10.2 2.3
I;t$gal
TMV
18 16” 16b 16” 8 6 14 2 14 9 12 4 3e 8 10 2
18 16 16 16 8 6 14 1 14 9 12 4 3 8 11 2
158
158
a The protein values represent average of six runs on three preparations for each hydrolysis time. b Obtained by extrapolation to zero time of hydrolysis. c Other values calculated on basis of 16 for Glu. d Value from separate analyses of performic acid oxidized protein. c Determined calorimetrically.
tein, but it would appear that the packing of the subunits is not as tight-suggested by a number of breaks or nicks along the particle. We have observed this to be the case in all the preparations that we have examined, and hence imperfect packing seems to be a general characteristic of the aggregation form of this protein. There is a suggestion that the protein may aggregate into a stacked disklike configuration in regions, but the stacking is not in pairs as is characteristic of the TMV protein stacked-disk configuration. Our analyses of the protein of the common strain of TMV are in agreement with the accepted composition (Tsugita el al., 1960). Using similar techniques the composition of the PM5 protein was determined and is listed in Table 1. The protein has one extra cysteine residue at the expense of one arginine residue. The composition of the purified “I” peptide is the same as that of the common strain.
AND
SIEGEL
Chromatography of the pH 4.6 soluble tryptic hydrolyzate as shown in Fig. 2 reveals a difference from that of the common strain in that peptides 8 and 9 are missing. The composition of the other peptides agreed with published figures for these peptides. This suggested that the arginine residue at position 112 whose carboxyl group is liberated to form peptides 8 and 9 is the one involved in the amino acid replacement. Since the exchange is from arginine to cysteine this locus is no longer available for tryptic digestion. Although we were unable to isolate peptide 8 and 9 combined as a single peptide by column chromatography (Fig. 2), we were able to do this by paper chromatography of the pH 4.6 soluble peptides on Whatman No. 3 MM paper. The peptide remained at the origin and could be detected easily by the hypochlorite-starchiodide test and reacted only very slowly with ninhydrin. The amino acid composition of this peptide was the same as that of the combined values of peptides 8 and 9 of the common strain except for a cysteine replacing one of the arginine residues. Column chromatography of the soluble peptides of the aminoethylated protein yielded peptides 8 and 9 which were recovered individually as expected since aminoethylcysteine is susceptible to trypsin (Fig. 3). Peptide 8 released by trypsin treatment of the aminoethylated PM5 protein had the same composition as that expected, but instead of a residue of arginine, a new amino acid eluting in the region of lysine was found. It is known that aminoethylcysteine behaves in this fashion (Tsung and Fraenkel-Conrat, 1966) Peptide 9 was found to he unaltered. DISCUSSION
Electron micrographs of the aggregated PM5 protein show clearly that, although the quaternary structure appears to be somewhat similar to that of the common strain, the packing of the subunits is loose, with a tendency for stacked disk configuration. Disorderly aggregation of coat protein subunits is known to stem from certain mutations which lead to alteration of selected amino acid residues (Zaitlin and McCaughey, 1965; Siegel et al., 1966; Jockusch, 1964, 1966). Thus, in the present instance the defective aggregation behavior is caused
NEW
DEFECTIIZ
by a single amino acid replacement, arginine to cysteine at position 112. The PM5 strain was isolated after nitrous acid treatment of the common strain, and the arginine to cysteine exchange can be accounted for by the demonstrated mode of action of nitrous acid involving deamination of cytosine to uracil,
STRAIN
207
OF TM\’
and adenine to guanine. Thus, in the present instance a codon shift from CGC to UGC or CGU to UGU involving a single nucleotide in the codon could explain the mutation. It has been inferred from a number of studies that the phosphates of the RNA are attached to basic groups of the protein by establishing 6
0.7 Ill 0.6
0.1,
1 \ti
& A
I
160
200 TUBE
300
400
500
NUMBER
FIG. 2. Chromatography of the pH 4.6 soluble PM5 protein tryptic digest on Dowex 1 X 2 acetate column (0.9 X 150 cm). Peptide “10” was removed prior to chromatography as described in Materials and Methods. 3.3 ml of the eluate was collected per tube, 0.2 ml from each tube was dried down, and the color obtained by the Folin-Lowry reagent was read at 750 rnp using a spectrophotometer. Flow rate 40 ml/hour at 37”. Peptides are numbered sequentially as they appear in the coat protein of the common strain of TMV. Note the absence of peptides 8 and 9.
lb
TUBE
8lllMBER
FIG. 3. Chromatography of the pH 4.6 soluble tryptic peptides from amino-ethylated PM5 protein on Dowex 1 X 2 acetate column (0.9 X 150 cm). Peptide “10” was not removed prior to chromatography. All conditions of chromatography and other details same as described in Fig. 2. Peptide lb is a product of the tryptic break at position 27 of the aminoethylated cysteine in the “I” peptide and represents sequence from 27 to 41. Note the reappearance of peptides 8 and 9 in the chromatogram.
208
HARIHARASUBRAMANIAN
salt links (Caspar, 1963). Hence it is conceivable that the arginine at position 112 is directly involved in such an attachment with RNA and any change here could lead to a defective coat protein. Thus, in PM5 the conversion of this basic polar amino acid at position 112 to the nonpolar cysteine would render the protein nonfunctional. Whether this or some other explanation proves to be correct, will probably be properly deduced only when the tertiary structure of the TMV coat protein is deciphered. It may, however, be mentioned here that no chemically induced mutant with functional coat protein has been obtained with changes in the region represent’ing positions 105-121 in the amino acid chain. ACKNOWLEDGMENTS We wish to thank Dr. Milton Zaitlin for his suggestions and Dr. Wayne Ferris for placing the facilities of his electron microscope laboratory at our disposal. The competent technical assistance of Ruth Smith, Helen Dorman, and Gayle Spratt is appreciated. REFERENCES CASPAR, D. L. D. (1963). Assembly Stability of the Tobacco Mosaic Torus Particle. Advan. Protein Chem. 18, 37-121: ERLBNGER, B. F., COOPER, A. G., and COHEN, W. (1966). The inactivation of chymotrypsin by diphenylcarbamyl chloride and its reactivation by nucleophilic agents. Biochemistry 5, 190-196. FUNATSU, G. (1964). Separation of tryptic peptides of tobacco mosaic virus and strain proteins by an improved method of column chromatography. Biochemistry 3, 1351-1355. FUNATSU, G., and FUNATSU, M. (1967). Studies on the amino acid sequence of Holmes Rib-Grass strain virus protein. I. Separation of pH 4.5 soluble tryptic peptides and amino acid sequences of some small peptides of Holmes RibGrass strain virus protein. Agr. Biol. Chhgm. 31, 48-53. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I-,, and SIEGEL, A. (1967). Studies on a new defective strain of tobacco mosaic virus. Phytopathology 57, 814. (Abstract.)
AND
SIEGEL
HITCHBORN, J. H., and HILLS, G. J. (1965). Electron microscopic examination of wild cucumber mosaic virus in negativelg stained crude preparations. virology 26, 756-758. JOCKUSCH, H. (1964). In vivo-und in vitro Verhalten temperatursensitiver Mutanten des Tabakmosaikvirus. Z. Vererbunqslehre 95, 379-382. JOCKUSCH, H. (1966). Relations between temperature sensitivity, amino acid replacements, and quaternary structure of mutant proteins. Biothem. Biophys. Res. Commun. 24, 577-583. L~BERMAN, R. (1965). Use of uranyl formate as a negative stain. J. Mol. Biol. 13, 606. LOIVRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. PAN, S. C., and DUTCHER, J. D. (1958). Separation of acetylated neomycins B and C by paper chromatography. Anal. Chem. 28, 836-838. SIEGEL, A., ZAITLIN, M., and SEHGAL, 0. P. (1962). The isolation of defective tobacco mosaic virus strains. Proc. Natl. Acad. Sci. U.S. 48, 1845-1851. SIEGEL, A., HILLS, G. J., and MARKH~~M, R. (1966). Zn vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein. J. Mol. Biol. 19, 140-144. SPIES, J. R., and CHBMBERS, D. C. (1949). Chemical determination of tryptophan in proteins. Anal. Chem. 21, 1249-1266. TSUGITA, A., GISH, D. T., YOUNG, J., FRAENKELCONRAT, H., KNIGHT, C. A., and STANLEY, W. M. (1960). The complete amino acid sequence of the protein of tobacco mosaic virus. Proc. Natl. Acad. Sci. U.S. 46, 1463-1469. TSUNG, C.M., and FRAENKEL-CONRAT, fI. (1966). The preparation and tryptic hydrolysis of saminoethylated tobacco mosaic virus protein. Biochemistry 5, 2061-2066. WALEY, S. G., and WATSON, J. (1953). The action of trypsin on polylysine. Biochem. J. 55.328-337. WITTMANN, H. G. (1965). Die Proteinstruktur der Defektmutante PM2 des Tabakmosaikvirus. Z. Vererbunqslehre 97, 297-304. ZAITLIN, M., and FERRIS, W. R. (1964). Unusual aggregatmn of a nonfunctional tobacco mosaic virus protein. Science 143, 1451-1452. ZBITLIN, M., and MCCAUGHEY, W. F. (1965). Amino acid composition of a nonfunctional tobacco mosaic virus protein. Virology 26, 500-503.
37,203-208
(1969)
Characterization
of a New
Defective
V. HARIHARASUBRAMANIAX Department
of Agricullural
Biochemistry,
AND
University
Strain
ALBERT
of Arizona,
of TMV’
SIEGEL Tucson, Arizona
85781
Accepted September 30, 1968 A new nitrous acid-induced defective strain of tobacco mosaic virus (TMV) designated PM5 was isolated and some of its characteristics were studied. The coat protein of this defective strain showed a change from arginine to cysteine at position 112 in the amino acid sequence of the protein. The protein aggregated into rodlike structures simila,r to aggregated TMV protein but the subunits seem to be more loosely packed with a tendency to stacked disk configuration. MATERIALS
INTRODUCTION
The isolation
of TMV has been reported earlier (Siegel et al., 1962). The coat proteins synthesized in plants infectred by these strains are defective and hence unable to copolymerize with viral nucleic acid and form stable nucleoprotein rods. This defect seems to result from a change or changes at a crucial region in the amino acid sequence of the protein subunit. Thus, in the defective strain PM2 there is a change from threonine to isoleucine and glutamic to aspartic acid at positions 28 and 95, respectively (Wittmann, 1965), resulting in a protein which packs in open helices rather than the tightly closed helices of the type strain of TMV (Zaitlin and Ferris, 1964; Siegel et al., 1966). Using similar techniques, we have isolated a new defective strain (PN5) and have established its defective nature as before (Siegel et al., 1962). This paper describes the purification, electron microscopy and chemical characterization of the coat protein of this strain. A preliminary report has been published earlier (Hariharasubramanian and Siegel, 1967). induced
AND
METHODS
Purification of the coat protein. The infected leaf tissue w-as deribbed and ground in an Omnimixer with 3 volumes of M/15 phosphate buffer containing 0.01 M EDTA (ethylenediaminetetraacetic acid) and 0.001 at pH 7.0. The M mercaptoethanol grindate was strained through cheesecloth and centrifuged at 12,000 9 for 10 min in a Sorvall refrigerated centrifuge. The supernatant was centrifuged for 60 min at 105,000 9 in a Spino L2 ultracentrifuge. The pellet was discarded and the supernatant was adjusted t’o pH 5.1 and kept cold overnight. It was then clarified at 12,000 y and the supernat,ant was centrifuged at 105,000 g as before. The resultant pellet was suspended in a solution containing 0.001 dd each of EDTA and DIECA (diethyldithiocarbamate) adjusted to pH 7.5 with NaOH. After clarification at 105,000 g the supernatant was brought to pH 5.0, subjected to low speed centrifugation at 12,000 n for 10 min, followed by ultracentrifugation at 105,000 g for 60 min. The high speed pellet was resuspended, clarified, and sedimented at pH 5.0, and the process was repeated until a clear pale yellow pellet was obtained. This pellet was resuspended in suitable solutions and used as purified virus protein. Electron microscopy. All electron microscopic observations were made with a Philips EM200 microscope at 80 kV and using a
of defective strains by nitrous acid treatment
1 This work was supported in part by grants from the National Science Foundation and by Atomic Energy Commission Contract AT(ll-l)873. University of Arizona Agricultural Experiment Station Technical Paper No. 1376. 203
204
HARIHARASUBRAMANIAN
liquid nitrogen-cooled cold stage. The microscope was calibrated by taking a series of pictures of a carbon grating of known mesh size at different magnifications. Carboncoated copper grids were prepared by floating off a thin film of carbon deposited on cleaved mica and picking up the resultant film on copper grids of 400-mesh size. The specimens for electron microscopy were prepared by placing a drop of freshly prepared uranyl formate (Leberman, 1965) on the grid followed by a drop of the protein solution placed on top of the stain. The excess liquid was blotted off and the specimen grids were dried at room temperaturebefore examination. The protein solutions for electron microscopy were usually adjusted to pH 5.0 before being placed on the stain. Sometimes uranyl acetate was used instead of uranyl formate to stain the preparation. Electron microscopic observations were also carried out on material deposited from epidermal strips (Hitchborn and Hills, 1965) in order to look for structures similar to those described by Siegel et al. (1966) in PM2-infected plants. Amino acid composition. The purified protein was hydrolyzed in vacua in sealed tubes at 108” in 6 N HCl for 24 hours and 72 hours, and the amino acid composition determined as described by Zait,lin and McCaughey (1965). Tryptophan was determined by the calorimetric procedure of Spies and Chambers (1948). Tryptic diqestion of protein. Trypsin prepared in the presence of the chymotryptic inhibitor diphenyl carbamyl chloride (Erlanger et al., 1966) was obtained from Seravac Laboratories, Berkshire, England. Digestion was carried out for 3 hours at 37” at a pH of 7.8 and a trypsin to protein ratio of 1: 100. At the end of the digestion period the pH of the reaction mixture was adjusted to 4.6 with 1 N acetic acid. The mixture was allowed to stand for 30 min in an ice bath, and was then centrifuged at 12,000 q. The precipitate was dissolved in water at pH 7.0 and purified further by repeated isoelectric precipitation (Tsugita et al., 1960). The portion soluble at pH 4.6 was lyophilized and used for peptide mapping by ion exchange chromatography and paper chromatography.
AND
SIEGEL
Ion exchange chromatography. The procedure was similar to that of Funatsu (1964). The pH 4.6 insoluble fraction of the protein tryptic digest was purified by repeated isoelectric precipitation and was used as such for determination of amino acid composition. This constitutes the “I” peptide of the protein. The pH 4.6 soluble fraction after lyophilization was taken up in a small volume of pH 8.8 pyridine-collidine-acetic acid buffer and centrifuged at 12,000 g. The insoluble pellet proved to consist essentially of peptide “10” when it was purified by gel filtration in Sephadex G-25 (Pharmacia Ltd.). The supernatant was subjected to ion exchange chromatography on a Dowex 1 X 2 column (0.9 X 150 cm) in the acetat’e form as described by Funatsu (1964). The flow rate was maintained at 40 ml an hour by using a minipump (Buchler instruments), and 3.3 ml of the eluate were collected per tube. From each tube 0.2 ml was dried down in test tubes in a vacuum oven at 50” and analyzed for peptides by the Folin-Lowry method (Lowry et al., 1951). The contents of the tubes under each peak were pooled and analyzed directly for amino acid composition or were further purified by paper chromatography before analysis. Paper chromatography. Peptides to be purified further were chromatographed on Whatman No. 3 MM paper by descending chromatography in n butanol-acet,ic acidwater-pyridine : 30 : 6 : 24 : 20 v/v (Waley and Watson, 1953). This procedure was also employed when the pH 4.6 soluble peptides were directly applied on paper and chromatographed without resorting t’o prior ion exchange chromatography. The peptides were located by cutting matching strips and developing with ninhydrin or hypochloritestarch-potassium iodide (Pan and Dutcher, 1958). The peptides from the matching, unstained portions were eluted from the paper with 1.0 % ammonium hydroxide or 0.2 N acet,ic acid (Funatsu and Funatsu, 1967). Aminoethylation of PM5 protein. The method was essentially that of Tsung and Fraenkel-Conrat (1966). 45-50 mg of protein were taken up in 6 ml of a solution containing 8 Al urea, 0.025 M mercaptoethanol,
NEW DEFECTIVE
FIG. 1. Electron micrograph
205
OF TMV
of aggregated PM5 protein stained with uranyl formate.
and 0.4 M Tris [tris(hydroxymethyl)aminomethane]-HCl buffer at pH 8.6. The reaction mixture was maintained at 23-25” under nitrogen for 2 hours. Ethylenimine (Rfatheson, Coleman and Bell) (0.05 ml) was added 10 times at 5-min intervals while the pH was maintained at 8.69.0 by dropwise addition of 2 N acetic acid in 8 M urea. The react,ion was allowed to proceed for 1 hour after the final addition of ethylenimine. The reaction mixture was diluted with an equal volume of water and dialyzed for 2 days in the cold (4”) with repeated changes of distilled water. The aminoethylated protein which precipitated on dialysis was sedimented at 12,000 g for 10 min and mashed with distilled water and resedimented again. This was repeated twice, and the final pellet was suspended in 0.001 N NaOH for tryptic digestion. A similar amino-
0.01 M EDTA,
STRAIN
ethylation was carried out using the protein of the common strain of TMV. Tryptic digestionof aminoethylated protein. The time of enzymatic hydrolysis by trypsin was extended to 6 hours since digestion of aminoethylated cysteine is rather slow (Tsung and Fraenkel-Conrat, 1966). During digestion, the insoluble aminoethylated protein dissolved almost completely. The pH of the hydrolyzate was adjusted to 4.6, and the insoluble fraction was sedimented and purified as before. The soluble fraction was lyophilized and the peptides separated by techniques described earlier. RESULTS
An electron micrograph of the PM5 coat protein aggregated at pH 5.1 is shown in Fig. 1. The aggregated protein seems to somewhat resemble aggregated TMV pro-
206
HARIHARASUBRAMANIAN TABLE
AMINO
ACID
Amino acid residue ASP Thr Ser Glu Pro GUY Ala CYS Val Ile Leu Tyr Try Phe Arg LYS
I
COMPOSITION
OF PM5
PROTEIN”
Moles amino acid per mole protein 24.hour hydrolysis
72.hour hydrolysis
18.00 15.1 14.8 16.0 7.8 6.0 13.6 1.6d 12.8 7.8 12.0 3.9
18.1 14.1 12.7 16.0 8.1 6.4 13.8 14.1 8.7 12.1 3.4
8.4 9.9 2.0
8.0 10.2 2.3
I;t$gal
TMV
18 16” 16b 16” 8 6 14 2 14 9 12 4 3e 8 10 2
18 16 16 16 8 6 14 1 14 9 12 4 3 8 11 2
158
158
a The protein values represent average of six runs on three preparations for each hydrolysis time. b Obtained by extrapolation to zero time of hydrolysis. c Other values calculated on basis of 16 for Glu. d Value from separate analyses of performic acid oxidized protein. c Determined calorimetrically.
tein, but it would appear that the packing of the subunits is not as tight-suggested by a number of breaks or nicks along the particle. We have observed this to be the case in all the preparations that we have examined, and hence imperfect packing seems to be a general characteristic of the aggregation form of this protein. There is a suggestion that the protein may aggregate into a stacked disklike configuration in regions, but the stacking is not in pairs as is characteristic of the TMV protein stacked-disk configuration. Our analyses of the protein of the common strain of TMV are in agreement with the accepted composition (Tsugita el al., 1960). Using similar techniques the composition of the PM5 protein was determined and is listed in Table 1. The protein has one extra cysteine residue at the expense of one arginine residue. The composition of the purified “I” peptide is the same as that of the common strain.
AND
SIEGEL
Chromatography of the pH 4.6 soluble tryptic hydrolyzate as shown in Fig. 2 reveals a difference from that of the common strain in that peptides 8 and 9 are missing. The composition of the other peptides agreed with published figures for these peptides. This suggested that the arginine residue at position 112 whose carboxyl group is liberated to form peptides 8 and 9 is the one involved in the amino acid replacement. Since the exchange is from arginine to cysteine this locus is no longer available for tryptic digestion. Although we were unable to isolate peptide 8 and 9 combined as a single peptide by column chromatography (Fig. 2), we were able to do this by paper chromatography of the pH 4.6 soluble peptides on Whatman No. 3 MM paper. The peptide remained at the origin and could be detected easily by the hypochlorite-starchiodide test and reacted only very slowly with ninhydrin. The amino acid composition of this peptide was the same as that of the combined values of peptides 8 and 9 of the common strain except for a cysteine replacing one of the arginine residues. Column chromatography of the soluble peptides of the aminoethylated protein yielded peptides 8 and 9 which were recovered individually as expected since aminoethylcysteine is susceptible to trypsin (Fig. 3). Peptide 8 released by trypsin treatment of the aminoethylated PM5 protein had the same composition as that expected, but instead of a residue of arginine, a new amino acid eluting in the region of lysine was found. It is known that aminoethylcysteine behaves in this fashion (Tsung and Fraenkel-Conrat, 1966) Peptide 9 was found to he unaltered. DISCUSSION
Electron micrographs of the aggregated PM5 protein show clearly that, although the quaternary structure appears to be somewhat similar to that of the common strain, the packing of the subunits is loose, with a tendency for stacked disk configuration. Disorderly aggregation of coat protein subunits is known to stem from certain mutations which lead to alteration of selected amino acid residues (Zaitlin and McCaughey, 1965; Siegel et al., 1966; Jockusch, 1964, 1966). Thus, in the present instance the defective aggregation behavior is caused
NEW
DEFECTIIZ
by a single amino acid replacement, arginine to cysteine at position 112. The PM5 strain was isolated after nitrous acid treatment of the common strain, and the arginine to cysteine exchange can be accounted for by the demonstrated mode of action of nitrous acid involving deamination of cytosine to uracil,
STRAIN
207
OF TM\’
and adenine to guanine. Thus, in the present instance a codon shift from CGC to UGC or CGU to UGU involving a single nucleotide in the codon could explain the mutation. It has been inferred from a number of studies that the phosphates of the RNA are attached to basic groups of the protein by establishing 6
0.7 Ill 0.6
0.1,
1 \ti
& A
I
160
200 TUBE
300
400
500
NUMBER
FIG. 2. Chromatography of the pH 4.6 soluble PM5 protein tryptic digest on Dowex 1 X 2 acetate column (0.9 X 150 cm). Peptide “10” was removed prior to chromatography as described in Materials and Methods. 3.3 ml of the eluate was collected per tube, 0.2 ml from each tube was dried down, and the color obtained by the Folin-Lowry reagent was read at 750 rnp using a spectrophotometer. Flow rate 40 ml/hour at 37”. Peptides are numbered sequentially as they appear in the coat protein of the common strain of TMV. Note the absence of peptides 8 and 9.
lb
TUBE
8lllMBER
FIG. 3. Chromatography of the pH 4.6 soluble tryptic peptides from amino-ethylated PM5 protein on Dowex 1 X 2 acetate column (0.9 X 150 cm). Peptide “10” was not removed prior to chromatography. All conditions of chromatography and other details same as described in Fig. 2. Peptide lb is a product of the tryptic break at position 27 of the aminoethylated cysteine in the “I” peptide and represents sequence from 27 to 41. Note the reappearance of peptides 8 and 9 in the chromatogram.
208
HARIHARASUBRAMANIAN
salt links (Caspar, 1963). Hence it is conceivable that the arginine at position 112 is directly involved in such an attachment with RNA and any change here could lead to a defective coat protein. Thus, in PM5 the conversion of this basic polar amino acid at position 112 to the nonpolar cysteine would render the protein nonfunctional. Whether this or some other explanation proves to be correct, will probably be properly deduced only when the tertiary structure of the TMV coat protein is deciphered. It may, however, be mentioned here that no chemically induced mutant with functional coat protein has been obtained with changes in the region represent’ing positions 105-121 in the amino acid chain. ACKNOWLEDGMENTS We wish to thank Dr. Milton Zaitlin for his suggestions and Dr. Wayne Ferris for placing the facilities of his electron microscope laboratory at our disposal. The competent technical assistance of Ruth Smith, Helen Dorman, and Gayle Spratt is appreciated. REFERENCES CASPAR, D. L. D. (1963). Assembly Stability of the Tobacco Mosaic Torus Particle. Advan. Protein Chem. 18, 37-121: ERLBNGER, B. F., COOPER, A. G., and COHEN, W. (1966). The inactivation of chymotrypsin by diphenylcarbamyl chloride and its reactivation by nucleophilic agents. Biochemistry 5, 190-196. FUNATSU, G. (1964). Separation of tryptic peptides of tobacco mosaic virus and strain proteins by an improved method of column chromatography. Biochemistry 3, 1351-1355. FUNATSU, G., and FUNATSU, M. (1967). Studies on the amino acid sequence of Holmes Rib-Grass strain virus protein. I. Separation of pH 4.5 soluble tryptic peptides and amino acid sequences of some small peptides of Holmes RibGrass strain virus protein. Agr. Biol. Chhgm. 31, 48-53. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I-,, and SIEGEL, A. (1967). Studies on a new defective strain of tobacco mosaic virus. Phytopathology 57, 814. (Abstract.)
AND
SIEGEL
HITCHBORN, J. H., and HILLS, G. J. (1965). Electron microscopic examination of wild cucumber mosaic virus in negativelg stained crude preparations. virology 26, 756-758. JOCKUSCH, H. (1964). In vivo-und in vitro Verhalten temperatursensitiver Mutanten des Tabakmosaikvirus. Z. Vererbunqslehre 95, 379-382. JOCKUSCH, H. (1966). Relations between temperature sensitivity, amino acid replacements, and quaternary structure of mutant proteins. Biothem. Biophys. Res. Commun. 24, 577-583. L~BERMAN, R. (1965). Use of uranyl formate as a negative stain. J. Mol. Biol. 13, 606. LOIVRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. PAN, S. C., and DUTCHER, J. D. (1958). Separation of acetylated neomycins B and C by paper chromatography. Anal. Chem. 28, 836-838. SIEGEL, A., ZAITLIN, M., and SEHGAL, 0. P. (1962). The isolation of defective tobacco mosaic virus strains. Proc. Natl. Acad. Sci. U.S. 48, 1845-1851. SIEGEL, A., HILLS, G. J., and MARKH~~M, R. (1966). Zn vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein. J. Mol. Biol. 19, 140-144. SPIES, J. R., and CHBMBERS, D. C. (1949). Chemical determination of tryptophan in proteins. Anal. Chem. 21, 1249-1266. TSUGITA, A., GISH, D. T., YOUNG, J., FRAENKELCONRAT, H., KNIGHT, C. A., and STANLEY, W. M. (1960). The complete amino acid sequence of the protein of tobacco mosaic virus. Proc. Natl. Acad. Sci. U.S. 46, 1463-1469. TSUNG, C.M., and FRAENKEL-CONRAT, fI. (1966). The preparation and tryptic hydrolysis of saminoethylated tobacco mosaic virus protein. Biochemistry 5, 2061-2066. WALEY, S. G., and WATSON, J. (1953). The action of trypsin on polylysine. Biochem. J. 55.328-337. WITTMANN, H. G. (1965). Die Proteinstruktur der Defektmutante PM2 des Tabakmosaikvirus. Z. Vererbunqslehre 97, 297-304. ZAITLIN, M., and FERRIS, W. R. (1964). Unusual aggregatmn of a nonfunctional tobacco mosaic virus protein. Science 143, 1451-1452. ZBITLIN, M., and MCCAUGHEY, W. F. (1965). Amino acid composition of a nonfunctional tobacco mosaic virus protein. Virology 26, 500-503.