N-TERMINAL MODIFICATION AND CODON REASSIGNMENT WITH NON-CANONICAL AMINO ACIDS IN PROTEINS
Thesis by Rebecca E. Connor
In Partial Fulfillment of the Requirements for the degree of
Doctor of Philosophy in Chemistry
CALIFORNIA INSTITUTE OF TECHNOLOGY
Pasadena, California 2008
(Defended February 19, 2008)
2008 Rebecca E. Connor All Rights Reserved
ACKNOWLEDGEMENTS
Nothing in this thesis was possible without the advice and support of my advisor David Tirrell. My collaborators Kostya Piatkov and Professor Alex Varshavsky were also vital to the work presented here. Kostya, in particular, has been invaluable and is an inspiration to me. My collaborators within the Tirrell lab, Jamie Link, John Ngo, and Alison Glazier have also been great to work with.
I could not have survived Caltech without the loving support of my husband, Jason, and my son, Dylan. My parents have also been staunch supporters of my scientific career, particularly my mother, who dropped everything and stayed with me for a year to take care of Dylan. The friends who got me through my first year of grad school (and my first two trimesters) and have remained close through the years are Susanna Widicus-Weaver, Jeremy Weaver, Andy Waltman and Andrew Udit. Stacey Maskarinec has been a constant source of fun times, emotional support, and good advice. Jamie Link and Jay Woodward are like brothers to me. John Ngo has been a sympathetic ear, an exercise partner, and a source for many stimulating conversations. Meg Schmitt introduced me to the wonders of yoga and helps me maintain my sanity in many ways. The Askew family, Sally, Chris, Marielle, and Evelyn, have provided me with a happy home and surrogate family for my last six months in Pasadena for which I will always be grateful.
Everyone in the Tirrell group has been kind, helpful and knowledgeable. In particular, group members Shelly Tzlil, Kimberly Beatty, Marissa Mock, Mandy Vink, Caglar Tanrikulu, Tae Hyeon Yoo, Sarah Heilshorn, Jin Montclare, Inchan Kwon and Dave Flanagan have always been tremendously generous in their time and knowledge.
ABSTRACT
Proteins are ubiquitous macromolecules that effect and control all the processes of life from reproduction to respiration to physical motion. These diverse molecules also provide physical structure and defensive mechanisms. The twenty canonical amino acids can be found in virtually every protein; however, in some organisms, the set of endogenous amino acids also contains residues outside the “canon,” such as pyrrolysine, selenocysteine, and formylmethionine. Although a range of chemistries is available through natural side- chain diversity, some functionalities such as halogens, ketones, azides, alkenes, and alkynes are not found in nature. The introduction of a broader range of chemical functionality into proteins and protein-based materials through the use of non-canonical amino acids represents a challenging goal for protein engineering. The persistence of all the amino acids throughout protein sequences also presents a challenge for biochemical modification at a particular location. The insertion of a non-natural amino acid at a single location on a protein can allow specific modification without further affecting the natural protein sequence. The focus of this thesis is on a new method for the post-translational site-specific introduction of non-canonical amino acids to the N-terminus of proteins in vitro and progress made towards developing a complementary in vivo method using the E. coli L,F- transferase. Addition of non-proteinogenic functionality to the coat proteins of M13 bacteriophage using non-canonical amino acids is also explored.
TABLE OF CONTENTS
Acknowledgements... iii
Abstract...iv
Table of Contents...v
List of Figures, Schemes and Tables... vii
Chapter 1: Introduction ...1
1.1 Protein biosynthesis...2
1.2 Methods for the introduction of non-natural amino acids into proteins...3
1.3 Protein degradation and the N-end rule pathway...8
1.4 Outline of thesis ...12
1.5 References ...13
Chapter 2: Non-canonical Amino Acid Substrates of the L,F-transferase ...18
Abstract ...19
2.1 Introduction...20
2.2 Materials and Methods... 24
2.3 Results and Discussion ...26
2.4 Conclusion ...42
2.5 References...43
Chapter 3: Enzymatic N-terminal Addition of Non-canonical Amino Acids to Proteins...48
Abstract ...49
3.1 Introduction...50
3.2 Materials and Methods...55
3.3 Results and Discussion ...59
3.4 Conclusions...66
3.5 References...67
Chapter 4: Progress Towards an L,F-transferase Selective for a Non-canonical Amino Acid...71
Abstract ...72
4.1 Introduction...73
4.1.1 Selection design ...74
4.1.2 Design of mutant L,F-transferase library...78
4.2 Materials and Methods...80
4.3 Results ...87
4.4 Conclusions...89
4.5 References...90
Chapter 5: Incorporation of Non-canonical Amino Acids into Filamentous
M13 Bacteriophage...93
Abstract...94
5.1 Introduction...95
5.2 Materials and methods ...99
5.3 Results and discussion...103
5.4 Conclusions...110
5.5 References...111
Appendix I: Mass Spectrometry Data ...114
Appendix II: Plasmid Maps...139
LIST OF FIGURES AND TABLES
Page Chapter 1. Introduction
1-1. The central dogma of biology...2 1-2. Diagram of the N-end rule pathway...11
Chapter 2.
2-1. Illustration of the dipeptide coumarin assay for aminoacyl-tRNA substrates of the L,F-transferase ...22
Scheme 2-1. Non-canonical amino acids tested as substrates for
the L,F-transferase ...23 2-2. HPLC analysis of the transfer of leucine analogs 1, 2, and 3 to
the dipepetide substrate...27 2-3. HPLC analysis of the transfer of phenylalanine analogs 4, 5, 6
and 7 to the dipeptide substrate...29 2-4. HPLC analysis of the transfer of phenylalanine analogs 8, 9, 10 and 11 to the dipeptide substrate...30 2-5 HPLC analysis of the transfer of phenylalanine analogs 12, 13, and 14 to the dipeptide substrate...32 2-6 HPLC analysis of the transfer of tryptophan analogs 15, 16, and and 17 to the dipeptide substrate after charging by the E. coli T251/A294G PheRS...33 2-7 HPLC analysis of the transfer of tryptophan analogs 18, 19,
and 20 to the dipeptide substrate by the double mutant E. coli PheRS...34 2-8 HPLC analysis of the transfer of methionine analogs 21, 22, 23, and 24 to the dipeptide substrate...36
2-9 HPLC analysis of the transfer of tryptophan analogs 12, 14, and 11 delivered to the transferase by a mutant yeast PheRS ...38 2-10 HPLC analysis of the transfer of tryptophan analogs 15, 16, and 17 delivered to the transferase by a mutant yeast PheRS ...40 2-11 HPLC analysis of the transfer of tryptophan analogs 18, 19, and 20 delivered to the transferase by a mutant yeast PheRS ...41
Chapter 3. Enzymatic N-terminal addition of non-canonical amino acids to proteins
Scheme 3-1 Chemical structures of the amino acids and affinity tags used to modify E. coli dihydrofolate reductase...52 3-1 N-terminal modification of eDHFR...54 3-2 Western analysis of R-eDHFR modified with non-canonical
amino acids and ligated to affinity tags ...62 3-3 Chromatographic analysis of the first residue of Etf-R-DHFR by Edman degradation ...63 Table 3-1 The percent of modified protein Etf-F-DHFR after increasing reaction length as determined by comparison of the molar amounts of arginine found by Edman degradation...64 3-4 Pegylation of Etf-eDHFR with azide-PEG-5000-fluorescein...65
Chapter 4. Progress towards an L,F-transferase selective for a non-canonical amino acid
4-1 Design strategy for the selection of Etf-specific transferase
mutant ...75 4-2 Close-up of the binding pocket of the E. coli L,F-transferase with puromycin bound...79 Table 4-1 Primers for the assembly of the yeast tRNA gene cassette ....83 Table 4-2 Primers for the synthesis of the transferase library...84 Table 4-3 Primers for the assembly of MetGp-Ub-GFP constructs ...85
4-3 PCR assembly of MetGp-Ub-R-GFP and the transferase library...89
Chapter 5. Incorporation of non-canonical amino acids into filamentous M13 bacteriophage
5-1 The lifecycle of the M13 bacteriophage...96 5-2 Western analysis of bacteriophage produced with methionine, azidohomoalanine, or no amino acid during induction of pIII expression. ...104 5-3 Comparison of catalysts for the azide-alkyne ligation of propargyl- PEO-biotin to Aha-enhanced bacteriophage proteins...107 5-4 Infectious titer of bacteriophage produced with methionine,
azidohomoalanine or no amino acid during induction of pIII expression. ...108 5-5 Bacteriophage modified with cyclooctyne-biotin remain
infectious...109
Appendix I. Mass Spectrometry data on the tripeptide products of the transferase reaction
Figure A-1. Analysis of the transfer reaction of dehydroleucine to
KA-AMC ...115 Figure A-2. Analysis of the transfer reaction of trifluoroleucine to KA-AMC ...116 Figure A-3. Analysis of the transfer reaction of oxonorvaline to
KA-AMC ...117 Figure A-4. Analysis of the transfer reaction of p-azidophenylalanine to KA-AMC ...118 Figure A-5. Analysis of the transfer reaction of p-ethynylphenylalanine to KA-AMC ...119 Figure A-6. Analysis of the transfer reaction of p-cyanophenylalanine
to KA-AMC ...120 Figure A-7. Analysis of the transfer reaction of p-iodophenylalanine to KA-AMC ...121 Figure A-8. Analysis of the transfer reaction of p-bromophenylalanine to KA-AMC ...122 Figure A-9. Analysis of the transfer reaction of 2,3-difluorophenylalanine to KA-AMC ...123 Figure A-10. Analysis of the transfer reaction of 3, 4, 5 –
trifluorophenylalanine to KA-AMC...124 Figure A-11. Analysis of the transfer reaction of 2,5-bromothienylalanine to KA-AMC ...125 Figure A-12. Analysis of the transfer reaction of
p-trifluoromethylphenylalanine to KA-AMC ...126 Figure A-13. Analysis of the transfer reaction of p-acetylphenylalanine to KA-AMC ...127 Figure A-14. Analysis of the transfer reaction of benzothienylalanine to KA-AMC ...128 Figure A-15. Analysis of the transfer reaction of 5-bromotryptophan to KA-AMC ...129 Figure A-16. Analysis of the transfer reaction of 5-chlorotryptophan to KA-AMC ...130 Figure A-17. Analysis of the transfer reaction of 5-methylryptophan to KA-AMC ...131 Figure A-18. Analysis of the transfer reaction of 6-bromotryptophan to KA-AMC ...132 Figure A-19. Analysis of the transfer reaction of 6-chlorotryptophan to KA-AMC ...133 Figure A-20. Analysis of the transfer reaction of 6-methyltryptophan to KA-AMC ...134 Figure A-21. Analysis of the transfer reaction of azidohomoalanine
to KA-AMC ...135
Figure A-22. Analysis of the transfer reaction of homopropargylglycine to KA-AMC ...136
Figure A-23. Analysis of the transfer reaction of homoallylglycine to KA-AMC ...137
Figure A-24. Analysis of the transfer reaction of azidonorleucine to KA-AMC ...138
Appendix II. Plasmid Maps 1. pQE9_GFP6_lacI_yFRS_tRNAwt...140
2. pQE9_GFP_yFRS_tRNAwt ...141
3. pQE9_MetGp_Aat_yFRS_tRNAwt ...142
4. pQE70_MetGp_Aat...143
5. pQE30_Aat ...144
6. pQE70_MetGp_Ub_R_GFP ...145
