Periodic Reporting for period 1 - EMMA (Exploring the molecular grammar of IDP assembly and condensation at ultra-high throughput)
Reporting period: 2023-06-01 to 2025-11-30
Proteins are the molecules of life. Many of them fulfill their functions in living organisms only if they take on a very well-defined shape, such that they are able to work like little machines. This is for example the case for enzymes that catalyze biochemical reactions. However, there is a large number of proteins that are not folded but intrinsically disordered. These proteins can fold into well-defined structures when they bind to their target, but in some cases they even remain disordered when they bind to other molecules. Furthermore, such intrinsically disordered proteins (IDPs) can often undergo a very wide range of different processes, some of which are functional and some of which are deleterious. Examples include the association into highly concentrated liquid-like droplets, so-called condensates, or into highly ordered fibrillar structures, so-called amyloid fibrils. The latter assemblies are hallmarks of a range of severe disorders, such as Alzheimer's and Parkinson's diseases. It is becoming increasingly clear that despite the lack of a clear structure in their native state, small changes in the amino acid sequence of an IDP can have dramatic consequences on its interactions and assembly behavior. In the EMMA project, our aim is to develop a range of new methods to study the effects of changes in amino acid sequence more widely and efficiently than has been possible until now. Notably, a systematic variation of the protein sequence for quantitative biophysical studies requires an enormous number of sequence variants to be studied. That is why new methods at higher throughput and that are automatable are required for such studies. In EMMA, we will develop further and apply our new methods to the study of the proteins alpha-synuclein and a fragment of TDP-43. The choice of these protein systems is motivated through the observation that the assembly, condensate formation and aggregation of these proteins are linked to the diseases Parkinson's and amyotrophic lateral sclerosis, respectively.
We have made good progress towards a range of the methods we aim to develop in the EMMA project. Our goal is to develop a two-pronged methodology. First, we want to bring a very high throughput methodology based on mRNA display into biophysics. In mRNA display, a large library of proteins can be studied simultaneously, because every protein is attached to its own coding mRNA, which means that it can be uniquely identified after having undergone a biophysical process. mRNA display is currently mostly used to identify new binders to molecular targets, but not for large scale biophysics. We want to change that and exploit the tremendous throughput and ability therefore to explore a truly meaningful part of amino acid sequence space. Second, we want to develop a range of new methods which are more what one might traditionally have called high throughput methods, mostly based on multiwell plates. This would allow to study tens or perhaps hundreds of amino acid sequence variants in high detail and in a variety of different scenarios. Those include condensate formation, maturation and dissolution, amyloid fibril formation kinetics and thermodynamics, binding to lipid vesicles, etc.
We have made good progress in both areas and a set of first scientific articles have been published. We have been able to firmly establish that our mRNA display-based method is indeed possible as expected and we have already obtained extensive quantitative insight into the driving forces of condensate formation by IDPs. Furthermore, we have been able to show that we can measure condensate formation and thermodynamics, as well as amyloid fibril thermodynamics in our newly devised multiwell plate-based assays. A major achievement is the application of several of our methods to the elucidation of the exact mechanism by which an amyloid fibril grows through the addition of soluble monomer to the ends. We were able to achieve this by adapting the Phi-value analysis method that had originally been developed for the study of protein folding to protein aggregation. This work was recently published in the prestigious journal Nature Chemistry.
We have made good progress in both areas and a set of first scientific articles have been published. We have been able to firmly establish that our mRNA display-based method is indeed possible as expected and we have already obtained extensive quantitative insight into the driving forces of condensate formation by IDPs. Furthermore, we have been able to show that we can measure condensate formation and thermodynamics, as well as amyloid fibril thermodynamics in our newly devised multiwell plate-based assays. A major achievement is the application of several of our methods to the elucidation of the exact mechanism by which an amyloid fibril grows through the addition of soluble monomer to the ends. We were able to achieve this by adapting the Phi-value analysis method that had originally been developed for the study of protein folding to protein aggregation. This work was recently published in the prestigious journal Nature Chemistry.
Beyond the state of the art are notably:
- our use of mRNA display for quantitative biophsyics and ultrahigh throughput
- the mechanistic study of protein aggregation through the use of Phi-value analysis
- the production, purification and detailed biophysical study of several dozens of sequence variants of alpha-synuclein in a single study
In these aspects, our group is currently at the leading edge of what is possible in these research fields, and this is in large parts thanks to the extensive resources that are available to us through the ERC CoG project EMMA.
- our use of mRNA display for quantitative biophsyics and ultrahigh throughput
- the mechanistic study of protein aggregation through the use of Phi-value analysis
- the production, purification and detailed biophysical study of several dozens of sequence variants of alpha-synuclein in a single study
In these aspects, our group is currently at the leading edge of what is possible in these research fields, and this is in large parts thanks to the extensive resources that are available to us through the ERC CoG project EMMA.