Protein Engineering Through in vivo Incorporation of Phenylalanine Analogs Thesis by Isaac Sheridan Carrico Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry California Institute of Technology, Pasadena, CA 91125 2004 (defended September 2, 2003) ii „ 2004 Isaac Sheridan Carrico All rights reserved iii For Lizzy and my family iv ACKNOWLEDGMENTS My time here at Caltech has been very interesting and rewarding and it will be hard for me to capture my gratitude for those who have shared my life here. I think I will start with my last two advisors, David Tirrell and Barbara Imperiali. Incredibly different, but both wonderful people who were crucial for my development. Barbara brought an immense amount of energy and personality to the lab, but perhaps more importantly she immediately treated me as an equal, someone of value. Dave makes a good run at being the perfect advisor. The inability to convincingly complain about him is the biggest fault that I have been able to find. Dave's breadth of knowledge is staggering, but it is the patience he displays in attempting to impart this knowledge that is truly impressive. My circuitous path through the labs of Caltech has caused most of my friends to become labmates at one point or another, so I will forgo the traditional format. Soojin Kwon, Mike Farwell, Doan Nguyen, Jason Belitsky, Peter Hackley, Carlos Bosques, Rob Dempski and Niki Zacharias comprise the classmates whom I met upon arriving at Caltech and hope never to grow far apart from. I want to thank Akif Tezcan and Derrick Debe, housemates and teammates who had a wonderful enthusiasm for science and perhaps only surpassed by their eagerness for cutting loose. Gabriel Brandt provided a quiet, calming, centering force, both on the field and in life. I also need to thank all of the wonderful people with whom I shared the fields with at Caltech; I lament that I may never find such a friendly atmosphere for sweating away the frustrations of life. I must thank Pin Wang and Kent Kirshenbaum for their positive outlook and perceptive manners that always kept us moving in the right direction. The rest of the Tirrell group has been wonderful to v work with; particularly I need express my gratitude towards David Flanagan for his lack of pretension and sharp wit, Yi Tang for the friendly aggressiveness, Jill Sakata for her generosity of spirit, Marissa Mock always a positive correcting force, and Sarah Heilshorn who was always willing to help. My family cannot be left out. My parents have always been unwavering in their support and love. I will never forget when, upon arriving at a new school, my mother demanded that I be put in the most advanced classes, her love for her boys has always been so strong as to preclude reason. My father also provided a continuous source of encouragement and love, thrilled that I would choose to go into the sciences for which he is probably better suited. Trey's encouragements started when I was a little boy in Austin and have never faltered. Zach who is following in science, but will without a doubt soon pass me. I also want to thank the new additions to my family, the Boons, who have welcomed me into their lives and were always kind about asking "so…when are you going to finish?" Lastly, but most importantly, I need to thank my wife who is my best friend and made every day at Caltech a joy. She is my constant companion and keeps my feet moving in the right direction. Without her I would be truly lost. vi ABSTRACT Proteins mediate the bulk of biochemical functions within the cell. These biopolymers control processes utilizing specific arrangements of the natural twenty amino acids. Expanding the set of amino acids available could both aid in the study of these macromolecules as well as significantly increase their functional capabilities. A set of enzymes known as aminoacyl tRNA-synthetases lies at the heart of the fidelity of translation, the process by which genetic information gets decoded into proteins. These synthetases accurately charge a specific tRNA with its cognate amino acid in the presence of the other nineteen natural amino acids. Interestingly these enzymes demonstrate a much higher level of promiscuity with unnatural amino acids. However, acceptable amino acids are limited to those that bear steric and electronic resemblance to the natural analog. Our efforts to expand the substrate set of phenylalanyl-tRNA (PheRS) synthetase are described in Chapters 2-4. We redesigned the catalytic site of PheRS computationally. These results combined with an already known mutant allowed us to rationally create a third mutant. All three mutants were characterized for their ability to activate a large panel of unnatural amino acids in vitro. Further, we were able to confirm the in vivo incorporation of a number of these analogs. In vitro and in vivo results were consistent and defined an expanded substrate set for the described mutants. This substrate set includes a number of analogs that are dramatically different from phenylalanine both sterically and electronically, as well as a number which contain chemical moieties valuable to protein engineering efforts. vii One example is para-azidophenylalanine (pN Phe), which provides access to 3 photochemistry as well as modified Staudinger ligations and copper mediated electrocyclizations. In Chapter 5 we describe utilization pN Phe as a 3 photocrosslinking reagent. Our aim was to create photochemically crosslinkable artificial extracellular matrix proteins for the production of synthetic vascular grafts. These proteins, produced in E. coli, were diblocks of endothelial cell binding domains and structural domains including the pN Phe site. Photochemical crosslinking of 3 these constructs provided moduli well within the range presented by the natural vascular wall. Chapter 6 describes our ability to photopattern films composed of the above protein. Photopatterning provided a means to spatially array endothelial cells, based upon a number of controllable processing parameters of such films. The final chapter details the utilization of incorporated unnatural amino acids, particularly para-iodophenylalanine, para-acetylphenylalanine and homopropargylglycine, to access Pd(0) catalyzed cross-coupling chemistry. We demonstrated this chemistry exhibits the characteristics necessary for chemoselective ligations. Futher, we demonstrated the selective modification of proteins incorporating all of the above analogs. viii TABLE OF CONTENTS CHAPTER 1. Unnatural Amino Acids in Biomaterials and Protein Engineering Introduction 2 References 16 CHAPTER 2. Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogs Introduction 24 Materials and Methods 26 Results and Discussion 29 PAGE analysis of the effects PheRS* on analog incorporation into mDHFR 29 Quantitative analysis of analog incorporation by amino acid analysis 29 Confirmation of analog incorporation by MALDI-TOF analysis 29 Large-scale expression of mDHFR containing unnatural analogs 32 Analysis of mDFHR containing analogs demonstrates new UV signatures 32 Conclusion 36 References 37 CHAPTER 3. A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in vivo Incorporation of Aryl Ketone Functionality into Proteins Introduction 42 Materials and Methods 45 Results and Discussion 51 Mutational predictions based on ORBIT calculations 51 PAGE analysis of mutant synthetase effects on incorporation of 2 into mDHFR 51 MALDI-TOF analysis of incorporation of 2 55 ix Selective modification of DHFR-2 with biotin hydrazide 55 Conclusion 59 References 60 CHAPTER 4. Engineering Relaxed Substrate Specificity into E. coli Phenylalanyl- tRNA Synthetase to Incorporate a Diverse Set of Non-natural Amino Acids Introduction 64 Materials and Methods 69 Results and Discussion 74 Expression and purification of PheRS variants 74 Activation of analogs by variant enzymes in vitro 74 In vivo evaluation by DHFR tryptic peptide analysis 78 Conclusion 85 References 86 CHAPTER 5. Efficient Photocrosslinking of an Artificial Extracellular Matrix Protein via in vivo Incorporation of Arylazide Functionality Introduction 93 Materials and Methods 97 Results and Discussion 100 Protein expression and purification 100 Infrared spectroscopy of CS5-ELF-N 100 3 Film production 105 Mechanical testing 105 Conclusion 109 References 76 CHAPTER 6. Patterning and Cell Binding Properties of a Protein Photoresist Produced in E. coli Introduction 115 x Materials and Methods 119 Results and Discussion 124 Phase contrast imaging of protein patterns 124 Infrared detection of azide photolysis kinetics 126 Cell attachment to CS5-ELF-N3 constructs 126 Cell patterning based on cell preference to non-irradiated CS5-ELF-N3 surfaces 130 Cell patterning on stripped surfaces 130 Conclusion 134 References 136 CHAPTER 7. Chemoselective Ligations via Pd(0) Chemistry on Unnatural Amino Acids Incorporated into Proteins Introduction 144 Materials and Methods 149 Results and Discussion 155 Optimization of Heck and Sonagashira couplings in aqueous conditions 155 Demonstration of tolerance to protein functionality 157 Modification of mDHFR (pCCHPhe, HAG and HPG) with pIF-FLAG tag 157 Selective fluorescent modification of Barstar-pIF with lissamine rhodamine propargylsulfonamide 158 Conclusion 165 References 166