Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 Design, ligand binding and folding of ankyrin and armadillo repeat proteins studied by solution NMR spectroscopy Ewald, Christina Abstract: Natural repeat proteins fulfil a plethora of important functions in cell biology like molecular recognition, cell adhesion and transport. Well known representatives of this protein class include ar- madillo, ankyrin, HEAT and tetratricopeptide repeat proteins. Proteins of this class constitute almost 20 % of proteins encoded in the human genome and contain tandem arrays of small, highly similar struc- tural units. Several of these units stack against each other forming non-globular, elongated structures with long hydrophobic cores and extensive solvent exposed surfaces, determining topology and function of these proteins. Repeat proteins differ from globular proteins in several important characteristics. Firstly, they commonly display an extended solenoid fold. Secondly, they are mainly stabilized by short-range interactions between residues close in sequence, whereas the importance of long-range interactions for protein stability is greatly diminished compared to globular proteins. Two repeat protein families were investigated during this PhD project – ankyrin repeat protein and armadillo repeat proteins. The natural ankyrin repeat is a very common type of motif and can be found in all three kingdoms spanning a wide range of functions, with the underlying theme being their ability to mediate protein-protein interactions by binding to three-dimensional epitopes. Armadillo repeat proteins are commonly involved in protein- protein or protein-peptide interactions, binding to peptides or unfolded parts of proteins. Importantly, their extended binding surface can bind peptides in extended conformation. Protein engineering efforts aim at developing useful proteins with new or enhanced functions. The Plückthun group has undertaken an extensive design effort to create a highly stable designed consensus ankyrin repeat protein (DARPin) scaffold. These studies have cumulated in an optimized design, in which surface residues can bemutated to achieve binding to a desired target without compromising scaffold stability. Repeat proteins in general, and DARPins in particular, are an interesting subject upon which to study protein folding in order to understand the molecular base of their unusual stability. Their low contact order and modularity rep- resents an intriguing background against which to study the mechanisms of protein folding and protein stability in a uniform environment. In this work, we investigated the stability and folding behaviour of full-consensus designed ankyrin repeat proteins (DARPins) using a range of NMR, biophysical and computational experiments. The sequence background of identical repeats used for our study can be seen as a generalised example for the study of AR protein folding and enables the investigation of folding as a function of repeat number. Using proton-exchange methods in the presence and absence of chemical denaturation, we evaluated the stability of this ankyrin scaffold in a residue-resolved manner. In order to achieve this we had to first assign the backbone resonances of each repeat and the N- and C-terminal capping repeats – a problem which becomes progressively more difficult as additional internal repeat are added. Paramagnetic spin labels attached to either end of the proteins were successfully used to decrease ambiguitiy and allowed complete backbone resonance assignments. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-164331 Dissertation Published Version Originally published at: Ewald, Christina. Design, ligand binding and folding of ankyrin and armadillo repeat proteins studied by solution NMR spectroscopy. 2013, University of Zurich, Faculty of Science. 2 Design, Ligand Binding and Folding of Ankyrin and Armadillo Repeat Proteins Studied by Solution NMR Spectroscopy Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Christina Ewald aus Deutschland Promotionskomitee Prof. Dr. Oliver Zerbe (Leitung der Dissertation) Prof. Dr. Andreas Plückthun Prof. Dr. Stephan Grzesiek Prof. Dr. Frédéric Allain Zürich, 2013 Elementary, My Dear Watson. Erklärung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe im Sinne der Promotionsordnung vom 03. Februar 2005 angefertigt. Bei der Abfassung der Dissertation wurden keine anderen als die darin angegebenen Hilfsmittel benutzt. Zürich, August 2013 Christina Ewald iii Acknowledgements The completion of a PhD thesis is rarely a singular effort, and this work has been no exception to this rule. It is impossible to spend five years working on a collaborative project without interacting and working with a large number of people. A number of individuals, groups, and organisations have contributed to the success of this work and I would like to thank these people for their valuable contributions. Firstly, I would like to thank Prof. Dr. Oliver Zerbe and Prof. Dr. Andreas Plückthun for giving me this opportunity and their continued support and guidance throughout this thesis. I am also greatful to the other members of my thesis committee Prof. Dr. Stephan Grzesiek and Prof. Dr. Frédéric Allain for their time and helpful suggestions. I am thankful for funding and financial support from the SNF (Sinergia Project Grant No. CRSI00_122686) and the Hartmann-Müller-Hahn Foundation. To the members of the Zerbe, Plückthun, Robinson and Caflisch groups who have contributed scientific input, guidance and help, I am greatly thankful, especially for your openness and unreserved generosity with respect to the sharing of equipment and expertise in times of need. Specifically I would like to thank Randall Watson and Martin Poms for their tireless dedication to produce the best coffee on the Irchel campus, for their friendship, fruitful discussions and support in the “downstairs” office and Jacopo Marino for his balance, charisma and music. Christian Reichen for his constant scientific support, enthusiasm and motivation unheard of before in the history of science, Reto Walser for his always patient explanations of NMR experiments, Fabian Bumbak for his questions and his very special self, the Italian fraction from the Caflisch and various other labs for interdisciplinary and entertaining lunch meetings and general party disorganisation, and Martin Christen for last minute custom fitting of excessive amounts of data. I would also like to thank Simon Jurt and Nadja Bross for their continuous efforts to keep the arsenal of fickle technical equipment running and Salomé Fässler and Susanna Bachmann without whose guidance and efficient paperwork no student at the OCI and MLS Programme could have survived. Ralph Bollag from the mechanical workshop has contributed invaluably to the workings of our lab and his leaving is a great loss to the entire institute. Finally, I want to thank my family for their constant long-distance support and trust to let me find my own way. Without you I would never have made it this far. Let’s see what’s next… iv Summary Natural repeat proteins fulfil a plethora of important functions in cell biology like molecular recognition, cell adhesion and transport. Well known representatives of this protein class include armadillo, ankyrin, HEAT and tetratricopeptide repeat proteins. Proteins of this class constitute almost 20 % of proteins encoded in the human genome and contain tandem arrays of small, highly similar structural units. Several of these units stack against each other forming non-globular, elongated structures with long hydrophobic cores and extensive solvent exposed surfaces, determining topology and function of these proteins. Repeat proteins differ from globular proteins in several important characteristics. Firstly, they commonly display an extended solenoid fold. Secondly, they are mainly stabilized by short-range interactions between residues close in sequence, whereas the importance of long-range interactions for protein stability is greatly diminished compared to globular proteins. Two repeat protein families were investigated during this PhD project – ankyrin repeat protein and armadillo repeat proteins. The natural ankyrin repeat is a very common type of motif and can be found in all three kingdoms spanning a wide range of functions, with the underlying theme being their ability to mediate protein-protein interactions by binding to three-dimensional epitopes. Armadillo repeat proteins are commonly involved in protein-protein or protein-peptide interactions, binding to peptides or unfolded parts of proteins. Importantly, their extended binding surface can bind peptides in extended conformation. Protein engineering efforts aim at developing useful proteins with new or enhanced functions. The Plückthun group has undertaken an extensive design effort to create a highly stable designed consensus ankyrin repeat protein (DARPin) scaffold. These studies have cumulated in an optimized design, in which surface residues can be mutated to achieve binding to a desired target without compromising scaffold stability. Repeat proteins in general, and DARPins in particular, are an interesting subject upon which to study protein folding in order to understand the molecular base of their unusual stability. Their