Exploring the molecular grammar of IDP assembly and condensation at ultra-high throughput

Introduction

The last years have seen unprecedented breakthroughs in protein structural biology, with the resolution revolution in cryo-electron microscopy and the release of AlphaFold. The combination of advanced experimental structural biology, machine learning algorithms, and molecular simulations has put the fully quantitative description of how the structure and interactions of folded proteins are defined by their amino acid sequence within close reach.

The Challenge of Intrinsically Disordered Proteins

This leaves us with a final frontier in protein science, namely to achieve a similar level of understanding for intrinsically disordered proteins (IDPs). The energy landscapes of IDPs often comprise a multitude of nearly isoenergetic states that include assembled forms, such as amyloid fibrils and liquid condensate droplets.

Current Understanding and Limitations

Much effort has been spent in order to achieve an understanding of the molecular grammar of IDP assembly and condensation, i.e., how amino acid sequence defines both kinetics and thermodynamics of these processes. Current state of the art is to evaluate a few dozens of sequence perturbations quantitatively in vitro.

Proposed Approach: EMMA

In EMMA, I propose to develop a fundamentally new approach that will ultimately allow us to improve on current methods by more than 8 orders of magnitude. This ground-breaking improvement will be achieved by exploiting the power of mRNA display, in which the biophysical behavior of each individual sequence within large libraries of protein-mRNA constructs can be evaluated in a “one pot” reaction.

Methodology

We will combine quantitative screening of the energetics of liquid condensate droplet formation of up to 10^10 sequences by mRNA display with a multiparametric biophysical toolbox that allows the key thermodynamic and kinetic parameters of binding and assembly to be evaluated for thousands of selected sequence variants.

Impact of EMMA

EMMA will transform our ability to probe the mechanisms and interrelationships of the interactions and assembly processes that define IDP function and disease-related malfunction.