Here a brief description of these work performed along chemREPEAT:
- Site-Specific Isotopic Labelling (SSIL): The key methodological development of chemREPEAT has been the possibility to introduce isotopically enriched atoms in specific protein positions. We have shown that, by combining the tRNA suppression strategy and Cell-Free (CF), these samples could be produced and studied by NMR. This approach was initially developed for glutamines (Urbanek et al. Angewan. Chem. 2018) and subsequently for prolines (Urbanek et al. JACS 2020). During the last year, we have also developed SSIL for Alanine. Technical details of the methodology have been described in (Morató et al., Biomolecules 2020) and are at the centre of a review (Urbanek et al., ChemBioChem 2020).
- Structural bases of the pathological threshold in HD: Using SSIL, we have investigated non-pathogenic, HttQ16 (Urbanek et al., Structure 2020) and pathogenic, HttQ46 and Htt66 (ms in revision), versions of the protein. By the comparison of their structures a novel perspective of the pathological threshold was derived. Concretely, we assigned a prominent role to the structure in the aggregation process. We have demonstrated that the stability of the helix is the main feature governing the aggregation kinetics and the structure of the resulting fibrils.
- Reduced cis population in poly-Proline (Poly-P): Using SSIL, we addressed the study of the cis/trans populations of individual prolines in Poly-P using Htt as example (Urbanek et al. JACS 2020). Our results unambiguously show that prolines placed in the inner position of Poly-P have a reduced population of the cis conformation with respect to isolated ones.
- Site-specific incorporation of fluorinated amino acids: We have demonstrated that aminoacyl tRNA synthetases (aaRS) used in SSIL to load tRNA also recognize non-natural amino acids with small modifications. We validated this feature by substituting hydrogen atoms by fluorine, which enables 19F-NMR experiments. We have applied this approach for Fluoro-glutamine and three different fluoro-proines (ms in preparation).
- Segmental labelling and SANS experiments. Profiting the power of CF to control the nature of the amino acids in a protein, we have endeavoured the SANS study of Htt using segmentally deuterated samples in which we specifically deuterate or protonate Q and P.
- Bioinformatic analyses of homorepeat-containing proteins: In collaboration with the group of Miguel Andrade, we have studied the role of flanking regions in glutamine-rich proteins and their evolution (Mier et al., Comp Struct Biotech J. 2020). While we identified that leucine is highly enriched in the position preceding Poly-Q, prolines were highly abundant in the C-flanking regions. We have also performed similar analyses for alanine-rich proteins (ms in revision).
- Robotics-inspired algorithms to study disordered proteins. Together with Juan Cortés, we have applied robotics-inspired algorithms in biomolecules. The main tool we use is a large database of tripeptide fragments derived from high-resolution structures. By concatenating these tripeptides, we built realistic ensembles of proteins that were in excellent agreement with experimental data (Estaña et al, Structure 2019). Moreover, we identified partially structured regions in disordered regions (Estaña et al., J Mol Biol 2020). By parallelizing the building algorithm (Estaña et al. Parall. Comput 2018), we also used the database to decipher the folding pathway of small protein (Estaña et al, Molecules 2019). We have also applied tailored statistical methods to robustly explore whether the conformation of a residue is influenced by its neighbours (ms in revision).
