(SEMINAR) César A. Ramírez-Sarmiento Institute for Biological and Medical Engineering Pontificia Universidad Católica de Chile and ANID Millennium Science Initiative Program, Millennium Institute for Integrative Biology, Santiago, Chile.

Institute for Biological and Medical Engineering Pontificia Universidad Católica de Chile and ANID Millennium Science Initiative Program, Millennium Institute for Integrative Biology, Santiago, Chile.

A recently described class of metamorphic proteins is able to fold-switch between two structurally dissimilar native states to encode or regulate different biological functions. The bacterial transcription factor RfaH is a quintessential example of such proteins, by exhibiting an all-α to all-β fold-switch of a whole protein domain to regulate its function. These proteins constitute a challenge for biophysical characterizations, identification of key residues driving the fold-switch and state-of-the-art protein structure predictions methods, such as AlphaFold2 (AF2).

Using RfaH as a case study, we will showcase how the use of simplified structure-based models allows to understand the refolding pathways of RfaH both in isolation and when bound to RNA polymerase. We will also demonstrate how the use of local energetic frustration in MD simulations and across protein sequences aids in identifying key residues involved in its fold-switch behavior, some of which we have experimentally validated. Finally, inspired by these results and by other works using subsampling of multiple sequence alignments (MSA) for protein structure prediction, we will show how a strategy of single position masking of the MSA in AlphaFold2 successfully explores the conformational landscape of RfaH.