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The Interaction of A-Actinin-2 with ZASP and Titin

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The Interaction of A-Actinin-2 with ZASP and Titin The Interaction of a-Actinin-2 with ZASP and Titin By Yunghan Au January 2004 Thesis for the degree of Doctor of Philosophy from University College London Supervisor: Dr. Annalisa Pastore Division of Molecular Structure National Institute for Medical Research The Ridgeway, Mill Hill London, NW7 1AA 1 UMI Number: U602696 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602696 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 To my parents Perhaps the most valuable result o f all education is the ability to make yourself do the thing you have to do, when it ought to be done, whether you like it or not; it is the first lesson that ought to be learned; and however early a man's training begins, it is probably the last lesson that he learns thoroughly. Thomas Henry Huxley (1825-1895) 3 Abstract Z-band Alternately Spliced PDZ-containing protein (ZASP) is a sarcomeric Z- disk protein expressed in human cardiac and skeletal muscle that is involved in a dominant familial dilated cardiomyopathy. ZASP interacts with the last 150 amino acids of a-actinin-2, the major component of the Z-disk, via an N-terminal PDZ domain. ZASP and a-actinin-2 are thought to play an important structural role, probably by forming a ternary complex with titin Z-repeats. We have determined the structure of ZASP PDZ and characterised its interaction with a-actinin-2. We show that ZASP PDZ is a classical class 1 PDZ domain that recognises with micromolar affinity the carboxy- terminal sequence of an a-actinin-2 calmodulin-like domain. We also characterised the ternary complex ZASP/a-actinin-2/titin and the role of each component, showing that the a-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that involved in the recognition of the titin Z repeats. Finally, we used the ZASP PDZ structure to model other members of the enigma family by homology and to predict their abilities to bind a-actinin-2. 4 Table Of Contents A bstract..................................................................................................................................4 Table Of Contents ................................................................................................................ 5 List Of Figures .......................................................................................................................8 List Of Tables.......................................................................................................................16 Acknowledgements ............................................................................................................. 17 Abbreviations .......................................................................................................................18 Chapter 1 ..............................................................................................................................20 Introduction.........................................................................................................................20 1.1 Muscle........................................................................................................................ 20 1.2 a-Actinin-2................................................................................................................. 27 1.3 Titin.............................................................................................................................31 1.4 ZASP...........................................................................................................................33 1.5 Nuclear Magnetic Resonance ....................................................................................37 1.6 Aim s............................................................................................................................39 Chapter 2 ..............................................................................................................................40 Materials and Methods ......................................................................................................40 2.1 Bioinformatics ............................................................................................................40 2.2 Plasmid Production .................................................................................................... 40 2.3 Protein Expression ......................................................................................................41 2.3.1 Transformation of Competent Cells ..................................................................41 2.3.2 Fermentation ........................................................................................................41 2.4 Protein Purification .................................................................................................... 43 2.4.1 Cell Lysis .............................................................................................................43 2.4.2 Nickel NTA Agarose Protein Purification From Lysate .................................44 2.4.3 Tobacco-Etch Virus Cleavage ........................................................................... 44 2.4.4 Gel Filtration (FPLC) Protein Purification ....................................................... 44 2.4.5 Nickel NTA Protein Purification After TEV Cleavage ................................... 45 2.4.6 Buffer Exchange Using PD-10 Columns ......................................................... 45 2.4.7 Concentration Determination Using Absorption Spectroscopy ..................... 45 2.5 NMR............................................................................................................................47 2.5.1 Spectroscopy .......................................................................................................47 2.5.2 Spectral Processing ............................................................................................. 48 2.6 NMR Structure Determination ..................................................................................49 2.6.1 NOESY spectra ...................................................................................................50 2.6.2 Distance Calibration ........................................................................................... 50 2.6.3 Hydrogen Bonds, Dihedral Angles, and Residual Dipolar Couplings ........... 51 2.6.4 CYANA............................................................................................................... 52 2.6.5 CANDID............................................................................................................. 52 2.6.6 XPLOR................................................................. 52 2.6.7 CHARM22 Water Refinement...........................................................................53 2.7 15N-HSQC Titration ...................................................................................................54 2.8 Fluorometric Titration................................................................................................56 2.9 Peptide synthesis and purification of the Minimal Titin ZR7 peptide ...................57 5 Chapter 3 ............................................................................................................................ 58 Protein Expression and Purification ............................................................................. 58 3.1 Protein Purification of ZASP-PDZ ........................................................................... 58 3.2 Protein Purification of Act-EF34..............................................................................61 3.3 Purification of Act-EF1234 .......................................................................................64 3.4 Conclusion ................................................................................................................ 66 Chapter 4 ............................................................................................................................67 Resonance Assignment: ZASP-PDZ with and without Act-EF1234 ...................... 67 4.1 Assignment of ZASP-PDZ ........................................................................................67 4.2 Assignment of I5N-ZASP-PDZ bound to Act-EF1234 .......................................... 70 4.3 Assignment of Act-EF34. ..........................................................................................72 4.4 Assignment of Act-EF1234 .......................................................................................77 4.5 Conclusion ................................................................................................................ 79 Chapter 5 ............................................................................................................................80 NMR Structure of ZASP-PDZ .........................................................................................80 5.1 Secondary Structure Prediction of ZASP-PDZ ......................................................
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