In the focus of our research is the phenomenon of structural disorder of proteins. It has been recently recognized that regions of proteins or even full-length proteins exist and function without well-defined 3D structures, which challenged the classical structure-function paradigm and called for studies aiming at understanding this phenomenon in detail. These studies have shown that structural disorder is prevalent in eukaryotic proteomes and disordered proteins carry out unique functions. Due to their frequent involvement in regulatory and signaling functions, structural disorder also plays important roles in serious diseases, such as cancer and neurodegeneration.
The study of structural disorder has already progressed way beyond simply establishing the disordered status of a protein. The current idea is that detailed experimental and theoretical characterization of the structural ensemble of disordered proteins in isolation, their structure in complex with their physiological partner(s), and the thermodynamics and kinetics of their interactions with their partners, hold the key to understanding these proteins and extending the structure –function paradigm to the disordered state. In this spirit, we undertake three different lines of research to push the frontiers of the field of disorder.
Our first project aims to extend the paradigm of disordered chaperones to cellular conditions. We suggested some time ago that fully disordered proteins or disordered regions of classical chaperones can have chaperone function on their own. We underscored this tenet by observations on a plant dehydrin, ERD14. Here we seek to elucidate details of the underlying mechanism by making: i) proteomic studies to determine the physiological partners of this disordered chaperone, ii) in-cell NMR studies to see the structure and interactions of ERD14 in live plant cells, and iii) detailed structure-function studies to see which sequence elements are involved in transient binding to the partner and how the interplay of induced folding and disorder in the bound state contribute to chaperone activity.
We also aim to understand the structure-function relationship of very large (“oversized”) proteins, practically neglected from a structural point of view thus far. Structural biology has traditionally addressed the structure of small folded proteins, whereas the field of structural disorder has focused on either fully disordered proteins/regions or short disordered elements that undergo induced folding in the presence of their partner. Here we would like to probe into the structure of the very large transcriptional co-activator CREB-binding protein (CBP), by addressing its structure by means traditionally applied in the case of protein complexes. CBP has about seven domains and disordered linker regions connecting them, the topology of which will be outlined by a combination of high-resolution (NMR, X-ray) and low-resolution (MS, EM, AFM) techniques.
In the third project we want to demonstrate that our knowledge on the structure and function of IDPs has already progressed to a state where we can describe their function in terms of binding motifs and linker elements. To this end, we will systematically alter, replace and mutate binding motifs and connecting regions of calpastatin, the disordered inhibitor of the enzyme calpain, to explore how much we can diverge from the wild-type sequence without compromising function. Functional effects of such extensive mutations will be approached by in vitro binding and inhibitory assays, by solving the structure of the enzyme-inhibitor complex and also by in vivo assays of the cellular effects of mutations.