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NMR, Spectroscopic, and Biochemical Studies of Calmodulin Variants University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 2000 NMR, spectroscopic, and biochemical studies of calmodulin variants Brokx, Richard D. Brokx, R. D. (2000). NMR, spectroscopic, and biochemical studies of calmodulin variants (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/12816 http://hdl.handle.net/1880/40536 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca NMR, Spectroscopic, and Biochemical Studies of Calmodulin Variants Richard D. Brokx A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREEOFDOCTOROFPHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA m,2000 O Richard Brokx 2000 National Library Bibliotheque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AON4 OttawaON KIA ON4 Canada Canada Your 6h9 Vorm reference Our fib Natre relerenco The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, loan, distribute or sell reproduire, preter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts from it Ni la thQe ni des extraits substantiels may be printed or othewise de celle-ci ne doivent Stre imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Calmodulin (CaM1, the ubiquitous eukaryotic Ca2+-bindingprotein, is well-known and well characterized and can be produced in good yields in a reliable bacterial expression system. In this thesis, the proline analogs 3.4-dehydroproline (Dhp) and azetidine-2-carboxylic acid (Azc) have been biosynthetically incorporated into CaM and calbindin D,, in order to evaluate these analogs as probes of prolines in proteins. The Dhp- substituted proteins were shown to have interesting NMR properties. Additionally, in NMR studies of tripeptides, Dhp and Azc had opposite effects on the thermodynamics and kinetics of prolyl cis-trans isomerization, which demonstrate the potential for the use of these analogs in work on proteins where prolyl cis-trans isomerization is important. The fluorinated amino acids cis-4-fluoroproline, 5,5,5- trifluoroleucine and S-trifluoromethylhomocysteine (trifluoromethionine) have also been incorporated into CaM and studied by fluorine-19 NMR in an examination of the properties of fluorinated aliphatic, as opposed to aromatic amino acids. The Ca"-dependent conformational change in CaM is seen in 19F-NMR spectra of trifluoroleucine- and trifluoromethionine- substituted CaMs. CaM has also been altered in other ways to study its properties. CaM was cleaved with the protease thrombin to create two fragments, TM1 (1-106) and TM2 (197-1481, which were examined by multinuclear NMR, circular dichroism, and gel bandshift assays. TM1 and TM2 can associate in the presence of metal ions to form a structure with enhanced metal ion affinity and more a-helical structures. In the presence of CaM-target peptides, TM1 and TM2 can form an even tighter complex with properties very similar to CaM-peptide complexes. In another study, CaM was dissolved in a 358 2,2,2-trifluoroethd (TFE) solution. Nitrogen15 NMR relaxation studies of CaM in 35% TFE showed that the central linker portion of the protein was more stable and ordered than in CaM in water, but the presence of alcohols is not sufficient to explain why this region of the molecule is a-helical in the crystal structure. The binding of target peptides to CaM was also studied by isothermal titration calorimetry; the enthalpy of binding and the heat capacity change upon binding varies among the peptides studied, which relate to the type of interactions involved in CaM-peptide binding. Acknowledgements There are a great many people who have given me support and encouragement during my time at the Vogel lab. There is no doubt that this list is incomplete. First, I would especially like to thank my supervisor, Dr. Hans J. Vogel. He has always been incredibly supportive and understanding during the ups and downs of my time as a graduate student. He always gave me the distance to try my own things, but also had the insight to realize when I was on to something interesting. The help from all the other members of the Vogel lab is also acknowledged, including Dr. Tao Yuan, who first taught me the ropes of the calmodulin system when I was starting out, Dr. Elke Lohmeier-Vogel, for her sense of humor and enthusiasm, Dr. Deane McIntyre for his tireless help in equipment maintenance and NMR experimentation, Kirsten Bagh for keeping the lab in order and well-stocked with reagents, Dr. Hui Ouyang for working side-by-side with me, giving me something to measure myself by, Craig Shepherd for putting up with me as an office mate, Dave Schibli and Teresa Clarke for putting up with me as a roommate, Peter Hwang for his help with NMR data analysis, and all the other members of the Vogel lab, both past and present, including Dr. Jim Aramini, Dr. Rob Penner, Jill Saponja, Alexis David (current Vogel Tankard record holder), Aalirn Weljie, Phoebe Franco, Dr. Vladimir Leontiev (Simpsons Trivia runner-up), Dr. Ning Zhou, Dr. Tung Hoang (trout zen master), and Dr. Ryan McKay. Andriyka Papish is especially acknowledged for her help with the work of Chapter 4, and Ahmad Azarnousch is also acknowledged for his help with the work on the incorporation of fluorotyrosines into schistosomal glutathione-s- transferase, although that work is not presented in great detail in this thesis. People in other groups in the department, including Dr. Gene Huber, Dr. Les Tari, Dr. Ray Turner, and Dr. Barry Phipps are also thanked for sharing ideas and equipment. The guidance of my PhD advisory committee is also acknowledged. Like my supervisor, they too gave me the space to do my own thing but also grabbed on when they found something interesting. Dr. Robert Edwards and Dr. Morley Hollenberg have been with me since the beginning, Dr. Gene Huber and Dr. Raghav Yamdagni are thanked for going easy on me during my PhD candidacy exam. Dr. Brian Keay is acknowledged for being on my PhD oral examination committee, and I would especially like to thank Dr. Cheryl Arrowsmith (University of Toronto) for being my external examiner; perhaps we can work together on a project in the future. For financial support, I would like to thank the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research for providing me with studentships. Dr. John Honek (University of Waterloo) is thanked for the gift of trifluorornethionine, and Dr. Ileane McIntyre is thanked for the synthesis of cis-4-fluoroproline used in Chapter 4. Dustin Lippert (University of Victoria) and Dr. Gilles Lajoie (University of Waterloo) are thanked for mass spectrometry analysis of protein samples, and Dr. Don McKay (Department of Medical Biochemistry) is thanked for amino acid analysis. Dr. Ruud Scheek (University of Groningen, the Netherlands) and his group are thanked for the 600 MHz NMR spectrometer time and initial help with the work in Chapter 6. Dr. George Makhatadze (Hershey Medical Center, Penn State University) and his group are thanked for the use of their isothermal titration calorimeter and their collaboration with the work in Chapter 7. There is also space here for people who had nothing directly to do with my project. My soccer team (Crispin Jordan, perennial captain) kept me in shape in the summer, and all present and past members of the University of Calgary Curling Club are thanked for the good times in the winter. My buddies Matt, Chad, Kent, and Gord were always fun to be with on weekends. As far as other distractions go, I guess I should thank all of the people who ever poured a beer for me, and the developers of all of the video games I ever played while I was here, but I'm sure if I tried to comprise a list of either of these it would soon get very long and terribly embarrassing. Lastly, of course, I would like to thank my family. My parents have provided me with all kinds of support from afar, both emotional and financial. It has also made me feel really good to know they are so proud. My brother, Walter, made me realize that life should be enjoyed to the fullest, and it's important to define yourself not only by what you do for a living, but also what you do outside of work. My identical twin brother, Stephen, has always been like a best friend, someone to be with and talk to. Talking to him was helpful because even though we do almost exactly the same thing we still found plenty of time to talk about things other than science. To him and everyone else on this list I wish a very bright future. vii Table
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