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Study of the Desmoplakin Protein Using Nuclear Magnetic Resonance Spectroscopy and Its Interaction with Gold Nanoclusters Using Atomic Force Microscopy

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Study of the Desmoplakin Protein Using Nuclear Magnetic Resonance Spectroscopy and Its Interaction with Gold Nanoclusters Using Atomic Force Microscopy Study of the Desmoplakin Protein using Nuclear Magnetic Resonance Spectroscopy and its Interaction with Gold Nanoclusters using Atomic Force Microscopy Pen´elope Rodr´ıguezZamora A thesis submitted for the degree of Doctor of Philosophy Nanoscale Physics Research Laboratory School of Physics and Astronomy THE UNIVERSITY OF BIRMINGHAM October 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Study of the Desmoplakin Protein using Nuclear Magnetic Resonance Spectroscopy and its Interaction with Gold Nanoclusters using Atomic Force Micrsocopy by Pen´elope Rodr´ıguez Zamora Nanoscale Physics Research Laboratory School of Physics and Astronomy THE UNIVERSITY OF BIRMINGHAM Abstract Desmoplakin is a cytolinker protein that establishes connections with the cell cytoskele- ton and to anchoring junctions at the cell membrane, providing the adaptable structural rigidity within the skin and heart cells required to accommodate shear forces and main- tain tissue integrity. While the C terminal of desmoplakin, composed of three plakin repeat domains and a linker domain, is in charge of intermediate filament binding, the N terminal end, consisting of a plakin domain (PD), is responsible for plaque attachment and interaction with other desmosomal components. Such critical interactions have been studied but important structural and dynamical aspects of desmoplakin have remained unknown. In this thesis a research study is presented that combines three physical techniques for the study of desmoplakin, namely Nuclear Magnetic Resonance Spectroscopy and Small Angle X-Ray Scattering (for investigating the desmoplakin C terminal end) and Atomic Force Microscopy (for studying the interaction of the desmoplakin N terminal end with size-selected clusters on surfaces). Regarding the desmoplakin C terminal end, the structure of the linker domain - that is conserved in six plakin family members - has been resolved by nuclear magnetic resonance spectroscopy, exhibiting an unprecedented fold that contains a pair of regular and irregular subdomains, with the latter offering a pair of basic residues that recognise acidic residues on helical intermediate filament proteins such as desmin and vimentin. Its monomeric state is evident by small angle X-ray scattering, and, in combination with desmoplakin plakin repeat domains, it enhances the desmoplakin interaction with the ii intermediate filaments. This ensures the adaptable meshwork of contacts that helps to prevent irreversible damage to desmosomes and the cell cytoskeleton upon exposure to mechanical stress. Regarding the desmoplakin N terminal, the plakin domain immobilisation on a surface of graphite decorated with size-selected gold clusters was performed to address the in- teraction of this protein with cluster-functionalised surfaces towards the study of its mechanical and other physical properties. The gold clusters were selected to contain 55 or 147 atoms so that their dimensions were smaller than those of single plakin domain molecules, and also in order to achieve maximum stability since these sizes represent magic numbers for gold clusters. Images from atomic force microscopy operating in contact mode have revealed that, despite having been tagged with two cysteine residues, the plakin domain does not establish a covalent bond with the gold nanoclusters. On the other hand, tapping mode atomic force microscopy provides evidence of enhanced weak adsorption of desmoplakin plakin domain to the clusters, increasing the interaction between the protein and the graphite surface. This mode allowed the characterization of desmoplakin plakin domain protein molecules in the liquid phase. The protein di- mensions are compared with predicted values based on the small angle X-ray scattering and crystallographic studies, identifying the height of the protein features with the low- est dimension of the crystallised segment of desmoplakin plakin domain that includes SR3-SR6. Images of the protein are obtained with sub-nanometer vertical resolution demonstrating the use of the AFM as a tool for protein imaging and measurement of single isolated protein molecules, as well as protein complex formation, in this case the growth of quasi-2D protein islands. With the aim of improving the technique of immobilisation of single biological molecules with metal nanoparticles, a new technique of cluster immobilisation has been developed using small metal clusters (Au20) to create channels in a graphite substrate. These channels function as defects on the surface of the graphite to anchor soft-landed clusters with the potential to bind the biomolecules. The method is tested with atomic force mi- croscopy and compared with previous clusters immobilisation techniques using argon ion defects. A full characterisation of soft-landed gold clusters with 923, 561, 309 and 147 atoms has been completed with high-resolution, non-contact atomic force microscopy. Contact mode atomic force microscopy experiments have demonstrated stronger immo- bilisation of the gold clusters on the support by using this new technique than by the use of its predecessors. Acknowledgements First of all, I would like to thank my supervisor Prof. Richard E. Palmer for giving me the opportunity to undertake a PhD at the University of Birmingham as a member of the NPRL group, and for all his ideas, guidance and support. I would also like to thank my co-supervisors Dr. Martyn Chidgey and Prof. Michael Overduin for opening the doors to the Laboratory of Structural Biology and NMR at the School of Cancer Sciences, and for their teaching and patience. I am also grateful to CONACyT for funding my PhD. I would like to thank everyone at the Nanoscale Physics Research Laboratory, with special thanks to: Dr. Gueorgui Barreto and Dr. Tian-Luo Pan for their training in SPM techniques; Dr. Feng Yin, Dr. Vahideh Habibpour and Ahmed Abdela for producing the clusters for my experiments. Also, my PhD experience would not have been the same without the presence of my fellow students, past and present nano-friends with whom I spent nice times: Mim, Javi and Rich (the bio-team!); Alina, Dongsheng, Martin, Ivy and Max (my generation), Dave, Chris, Thibault, James and Tom (the experienced), Karl, Ruth, Ray, Charlotte, Lu, Wei, Dongxu, Will, Scott, Dogan, Caroline and Nan (the freshers). My thanks are also extended to everyone at the Laboratory of Structural Biology and NMR, expressly to Dr. Caezar Al-Jassar for initiating the collaboration of this project and for providing most of the structural biology laboratory instruction; Dr. Mark Jeeves for NMR spectra acquisition help and NMR teaching; Dr. Timothy Knowles for NMR data analysis assistance; and Dr. Claudia Fogl for NMR titration experiments assistance and production of protein mutants in collaboration with Arvinder Malli. Everyone in G64 was very welcoming and helpful when I started in Cancer Sciences, so I am also very grateful to my colleagues and friends Piv and Nazia; and Pooja, Jas and Sandya, the best technicians I know. Also thanks to James Bowen from Chemical Engineering for all his AFM advice. I wish to thank Daniel, for his constant support, for being by my side in the good and bad times and for making the colours of my life more vivid. My heartfelt thanks to my Dad and all my family, Carlos and specially my beloved siblings Yael, Charlie and Karlita, who, despite the distance, always put a smile on my face. Finally, this thesis is dedicated to my Mother and my Grandma, to whom I owe every- thing, and who gave me the love and courage to achieve my goals in life. iii Contents Abstract i Acknowledgements iii Abbreviations ix 1 Introduction 1 1.1 Physics Meets Biology in Understanding Protein Structure and Function . 1 1.2 About this Work . 3 1.3 NMR Spectroscopy and SAXS for Protein Structure Determination and Protein-Protein Interactions Studies . 4 1.3.1 Atomic-level Protein Structure and Interactions by NMR . 4 1.3.1.1 Protein structure determination . 5 1.3.1.2 Protein-protein interactions . 6 1.3.2 Scope of SAXS in Ascertaining Protein Morphology . 6 1.3.2.1 X-ray scattering at small angles in molecules . 7 1.4 AFM for Studies of Protein Interaction with Surfaces . 8 1.4.1 Imaging Proteins with AFM . 8 1.4.2 Protein Immobilisation Methods . 10 1.4.3 Immobilisation of Proteins with Size-Selected Clusters . 11 1.4.3.1 Size-selected metal clusters . 12 1.4.4 AFM Characterisation of Size-Selected Metal Clusters . 14 1.5 Desmoplakin . 15 1.5.1 The Plakin Family . 15 1.5.2 The Role of Desmoplakin in Desmosomes . 17 1.5.2.1 Cell junctions . 17 1.5.2.2 Desmosomes . 18 1.5.3 Desmoplakin Structure and Function . 20 1.5.3.1 The N-terminal end of desmoplakin . 22 1.5.3.2 The C-terminal end of desmoplakin . 23 1.5.4 Interactions with Intermediate Filaments . 24 1.5.4.1 Vimentin and desmin . 25 1.5.5 ARVC and other Diseases Related to Desmoplakin Mutations . 26 1.6 Global and Specific Aims of this Work . 27 2 Materials and Methods 29 iv Contents v 2.1 Constructs . 29 2.2 Protein Expression . 30 2.2.1 Transformation of BL21(DE3) Cells . 30 2.2.2 Overnight Growth of Starter Cultures and Large Scale Expression 30 2.3 Protein Purification . 31 2.3.1 Harvesting Cells and Mechanical Cell Lysing . 31 2.3.2 Purification of GST-tagged Proteins including Removal of the GST Tag . 31 2.3.3 Purification of His-tagged Proteins .
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