Design, ligand binding and folding of ankyrin and armadillo repeat proteins studied by solution NMR spectroscopy

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 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 Pluckthun 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 low contact order and modularity represents 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.