The amino acid sequence of the tryptic peptides of the f1 bacteriophage coat protein : a thesis submitted to Massey University of the Manawatu in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry

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THE AMINO ACID SEQUENCE OF THE

TRYPTIC PEPTIDES OF THE fl

BACTERIOPHAGE COAT PROTEIN.

by

Sarry Charles Richardson

A thesis submitted to Massey University of the Manawatu in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry.

July, 1970

i.

ABSTRACT

Five uajor peptides were isolated by paper

electrophoresis froo a tryptic digestion of purified fl bacteriophage coat protein. The aoino acid cocposition of the peptides was deteroined and shown to be:-

I' 1 Ala 2 , Glu1, Asp

2, Pro 1, Gly

1, Lys 1,

Tz

^Ala₁^{, Ser}₁^•

T3 Phe,, Thr

1, Ser

1, Lys 1•

l

T4 ^Leu₁^{, Phe}₁^{, Lys}₁^•

T5

^Lys1•

Sequential degradation of the intact fl coat

protein using the Ednan technique showed the N-teroinal sequence to be:-

Ala - Glu - Gly - Asp - Asp -

The sequence of the tryptic peptide

T1

^indicated

i t was derived from the N-terninal of the protein and was assigned the sequence:-

Ala - Glu - Gly - Asp - Asp - {Pro1, Ala_{1 ) -} Lys.

After two cycles of the Edaan degradation reaction the sequence of T2 was shown to be:-

Ala - Ser

Digestion of the intact fl coat protein with carboxypeptidase A indicated T

2 was the C-terrainal peptide since carboxypeptidase A showed the C-teroinal sequence to be:-

- Lys - Ala - Ser

Thio peptide was shown to have the sequence:-

Phe - Thr - Ser - Lys

This peptide was assigned the sequence

Leu - Phe - Lys

r

5 was shown to be a free Lys residue.

i i .

i i i . ACKNOWLEDGEMENTS

I ac grateful to oy supervisor, Dr G.G. Midwinter for his continual help and guidance throughout this study and also to Professor G.B. Petersen, Dept. of Biochecistry, University of Otago Medical School, Dunedin for the supply of the fl phage.

I would like to thank my future wife, Joy for her continual assistance during the preparation of this canuscript, and for typing the draft copy. I also wish to thank Miss Wendy Nash of the N.Z. Dairy Research Institute for typing the final aanuccript.

iv.

CONTENTS

CHAPTER 1 INTRODUCTION

Page 1 Bacteriophages and the study of their coat

proteins.

CHAPTER 2 MATERIALS AND METHODS (a) Purification of solvents for sequence

analysis.

10

17

(b) Chromatography. 18

(c) Purification of the fl phage coat protein. 19 (d) The aoino acid analysis of the fl phage

coat protein. 20

(e) Tryptophan analysis of the fl coat protein. 21 (f) Tryptic digestion of the coat protein. 21 (g) Separation of tryptic peptides. 22 (h) Separation of core a~d protein resulting

froa tryptic digestion of the fl coat protein.25 (i) Araino acid cooposition of the tryptic

peptides.

25

(j) Edr.lan degradation of the ft coat protein. 26 (k) Carboxypeptidase digestion of the coat

protein. 27

(1) Ech:aan degradation of the tryptic peptides. 28

CHAPTER 3 RESULTS

(a) Characterization of the fl phage coat protein.

(b) (c) (d)

Aoino acid analysis of the coat protein.

Tryptophan analysis.

Tryptic digestion of the coat protein.

(e) Separation of the tryptic peptides.

(f) Separation of the tryptic core and fl coat protein.

v.

30 30 30

31 32

33

36

(g) Araino acid conposition of the tryptic peptides. 36 (h) N-Tercinal sequence of coat protein.

38

(i) Digestion of the coat protein with carboxy-

peptidase A. 40

{j)

The Edraan degradation of the tryptic peptides. ~O

CHAPTER

4

DISCUSSION

44

(a) Purification of the fl coat protein.

44

(b) Aaino acid analysis of the fl coat protein. 46

(c} Tryptophan analysis.

51

(d) Tryptic digestion of coat protein and separation

of the tryptic peptides.

52

(e) Edoan degradation of the fl coat protein and

the tryptic peptides. 58

(f) C-Terminal sequence of the coat protein. 63

(g) General summary

65

References

69

vi

TABLES Between pages

1. Acino Acid Cooposition of the Phage Coat Protein.

2. Values of S Calculated froo the Spectra of Known Mixtures of Tyrosine and Tryptophan. 3.

s.

The Electrophoretic Mobility (E~) of the Tryptic Peptides at pH

6 . 4

in Pyridine Acetate buffer.

Anino Acid Coaposition of the Tryptic Peptides of the fl Phage Coat Protein. Ecklan Degradation of the fl Coat Prote, n. 6. Aaino Acids Released During the Carboxy-

30-31

30- 31

34- 35

36- 37 38- 39

peptidase A Dieestion of the fl Coat Protein~38-39

7.

Edr~an Degradation of the Tryptic Peptides. 41- 42

8.

Aoino Acid Cooposition of the fl, fd, M13

and ZJ- 2 Coat Proteins.

48-49

9. Comparison of the C-Tercinal Sequences of fl,

fd, TMV TUlgare and TMV dahleoense.

64-65

vii

FIGURES Between pages

1 Aoino acid analysis of the fl coat protein

after hydrolysis in 6N HCl for 24 hours. 30-31 2a Ultra-violet spectra of the fl coat protein

obtained at pH 13.0. 31-32

2b Ultra-violet spectra of the fl coat protein obtained at pH 12.5.

3 Value of Sas a function of R for the standard aoino acid oixtures used for estiaating the tryptophan content of the protein.

4

5

6

7

Tryptic digestion of the fl coat protein.

High voltage paper electrophoresis of the tryptic peptides, using pyridine: acetic acid: water buffer, pH

6. 4 .

Thin layer finger print aap of a tryptic digestion after hydrolysis for 24 hours. Separation ofthe tryptic peptides of the fl coat protein on a AG 50W-X2 colw:m. 8 Separation of the core and coat protein by

chromatography on Sephadex G-50.

9 Edman degradation of the fl coat protein and identification of the PTH-amino acids forced using solvent E.

31-32

31-32 32-33

34-35 35-36

35-36

36-37

38-39

10 Ultra-violet spectra of the N-teroinal PTH-Ala

11 12

of the f1 coat protein in absolute ~ethanol.

Edoan degradation of the tryptic peptides. Co~parison of the anino acid sequence of the f l and fd coat proteins.

viii.

38 - 39

41-42

&1- 62

Ala Asp

Dansylchloride DNA

DNFB

DNP-a:.::.1ino acid ONS-peptide DPTU

Glu Gly Ile Leu Lys Met

MPTU

Phe

PITC

Pro

PTC-derivative PTH-deri vati ve Ser

Thr Trp Tyr Val

ix ABBREVIATIONS

Alanine

Aspartic acid

1-dioethylaminonapthalene-5-sulphonyl chloride

Deoxyribonucleic acid

2,4-Dinitro-1-fluorobenzene Dinitrophenyl-amino acid Dans yl - peptide

Diphenylthiourea Glutamic acid Glycine

Isoleucine Leucine Lysine Methionine

Monophenythiourea Phenylalanine

Phenylisothiocyanate Praline

Phenylthiocarbanyl-derivative

3-Phenyl-2-thiohydantoin-derivative Serine

Threonine Tryptophan Tyrosine Valine